POLYPEPTIDES USEFUL IN DIAGNOSIS OF COCCIDIA INFECTION AND RECOMBINANT-DNA METHODS FOR MANUFACTURING SAME

Abstract: A class of polypeptides useful in an in vitro diagnosis of coccidiosis infection in chickens is disclosed. These polypeptides are also capable of inducing an immune response in chickens which were previously not exposed to Eimeria. Recombinant DNA methods for the production of these polypeptides and phage vectors useful in these methods are also disclosed.

Description: POLYPEPTIDES USEFUL IN DIAGNOSIS OF COCCIDIA INFECTION AND RECOMBINANT-DNA METHODS FOR MANUFACTURING SAME The present invention relates to a class of polypep¬ tides useful in diagnostic assays to determine the presence of antibodies to coccidia organisms in mammals and fowl and par¬ ticularly in chickens. The invention also relates to recombinant DNA methods for the manufacture of these polypep¬ tides and recombinant phage clones containing DNA sequences suitable for use in the recombinant methods. " toccidiosis in chickens is caused by a variety of Eimeria species. Eimeria is endemic in chicken flocks throughout the world with the U.S.D.A. estimating in 1965 that the loss to poultry farmers in the U.S. due to Eimeria infec¬ tions was in excess of 35 million dollars. While the various Eimeria species generally have the same life cycle, the site of invasion of gastro-intestinal epithelial cells by these organisms varies according to spe¬ cies. Normally, E. tenella is found only in the cecae of the chicken. E . necatrix inhabits the intestine while E_ mivati and E. brunetti normally parasitize the posterior intestinal tissues. E. acervulina and E. maxima tend to invade the duode¬ num. By way of example of the life cycle of Eimeria, an Eimeria tenella infection begins when a chicken ingests food or water that is contaminated with sporulated oocysts . The oocyst wall ruptures in the bird's gizzard and the activated sporozoites leave the sporocyst in the small intestine. Once in the cecae, the sporozoites enter the cells of the surface epithelium, pass through the basement membrane into the lamina propria and locate in glands of Lieberkuhn. The sporozoites invade the glandular epithelial cells of ■ the crypt, where they locate between the cell nucleus and the plasma membrane. Within the glandular epithelial cell, the sporozoite becomes a trophozoite, feeding on the host cell and enlarging to become a schizont. During schizogony, the schizont divides into about 900 first-generation merozoites . The first-generation merozoites break out into the lumen of the cecae about three days after infection, destroying the host cell. Each first-generation merozoite then enters another cecal epithelial cell and develops into a second-generation schizont. About 300 second-generation merozoites are formed by the second schizogony. These second-generation merozoites rup¬ ture the host cell and proceed into the lumen of the cecum about 5 days after infection. Some of the second-generation merozoites enter new cells to begin a third generation of schizogony that yields up to 30 third-generation merozoites. The remainder of the second-generation merozoites enter new epithelial cells and begin gametogenesis. Fertilized oocysts can be found in the feces about 8 days after infection. The new oocyst contains a single cell, the sporont. Development of the sporont into sporocysts and sporozoites is triggered by the presence of atmospheric oxygen and occurs out¬ side of the chicken. The number of oocysts produced during an infection is quite large. A single oocyst containing eight sporozoites, when ingested by a chicken, can give rise to 2,000,000 second-generation merozoites. Growth of the Eimeria in the gastro-intestinal tis¬ sues results in considerable tissue destruction, a high mortal¬ ity rate in young birds and decreased weight gain in more ma¬ ture animals. Release of merozoites and gametes from an intestinal cell destroys the cell. The cumulative effect of a coccidia infection may therefore be bloody diarrhea and sloughing of patches of epithelium which can lead to death. A mass of clotted blood may also form from the extensive he orrhaging and plug the cecal opening causing destruction of the cecum. Birds that are not killed by initial infection often fail to gain weight and are susceptible to other dis¬ eases . Because of the seriousness of an Eimeria infection, diagnostic testing methods have been sought which will indicate the presence of an infection caused by Eimeria in chickens. The present inventors have discovered a class of polypeptides useful in the diagnosis of coccidia infections. These polypeptides , when used in _in vitro diagnostic assays, indicate the presence of antibodies against Eimeria organisms in infected chicken sera. To facilitate use of these polypeptides, the present invention also relates to recombinant DNA methods for manufac¬ turing the polypeptides . These recombinant DNA methods utilize DNA sequences contained in various recombinant phage clones which are described herein. In addition, some of these phage clones are on deposit at the American Type Culture Collection in Rockville, Maryland. SUMMARY OF THE INVENTION It is an object of the present invention to provide polypeptides useful in the diagnosis of coccidial infections in chickens. It is also an object of the present invention to identify recombinant-DNA methods for the manufacture of these polypeptides . Additional objects and advantages of the present invention will be set forth in part in the description which follows, or may be learned from the practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. To achieve the objects and in accordance with the purposes of the present invention, Proteins A, B, C, D, E, F, G, H, I, J, K, L, and M have been disclosed. The DNA corre¬ sponding to these proteins is contained in various lambda phages which also are identified herein. In addition, a recombinant-DNA method for the manu¬ facture of polypeptides analogous to Eimeria polypeptides is disclosed. These proteins are capable of creating an immuno- diagnostic complex when exposed to sera from chickens infected with Eimeria tenella. This method comprises: (a) Preparation of a DNA sequence capable of directing a host microorganism to produce a polypeptide pos¬ sessing antigenic properties analogous to those possessed by a polypeptide produced by Eimeria organisms; (b) Cloning the DNA sequence into a vector capable of being transferred into and replicating in a host organism, such vector containing operational elements for the DNA se¬ quence; (c) Transferring the vector containing the DNA se¬ quence and operational elements into a host microorganism capa¬ ble of expressing the antigenic polypeptide; (d) Culturing the host microorganism under condi¬ tions appropriate for amplification of the vector and expres¬ sion of the polypeptide; and (e) In either order: (i) harvesting the polypeptides7 and (ii) causing the polypeptide to assume a struc¬ ture whereby it possesses antigenic prop¬ erties analogous to properties possessed by polypeptides produced by Eimeria organisms. It is understood that the foregoing general descrip¬ tion and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed- The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one embodiment of the invention and, together with the description, serve to explain the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a restriction map of phage lambda ET::Rbl-8. The region corresponding to a cDNA clone Rbl-8 is indicated. Fig. 2 is a restriction map of phage lambda ET::Rbl-17. The region corresponding to cDNA clone Rbl-17 is indicated. Fig. 3 is a restriction map of phage lambda ETt:CH2-13. The region corresponding to cDNA clone CH2-13 is indicated. Fig. 4 is a restriction map of phage lambda ET..CH2-15. The region corresponding to cDNA clone CH2-15 are indicated. Fig. 5 is a restriction map of phage lambda ET::Rbl-145. The region corresponding to cDNA clone Rbl-145 are indicated. Fig . 6 is a restriction map of phage lambda ET::CHAD-19. The region corresponding to cDNA clone CHAD-19 is indicated. Fig. 7 is a restriction map of phage lambda ET::Cal-3gn. The region corresponding to cDNA " clone Cal-3gn is indicated. Fig. 8 is a restriction map of phage lambda ET::Cal-4cn. The region corresponding to cDNA clone Cal-4cn is indicated. Fig. 9 is a restriction map of phage lambda ET::Rbl-24. The region corresponding to cDNA clone Rbl-24 is indicated. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the presently preferred embodiments of the invention which, together with the drawings and the following examples, serve to explain the prin¬ ciples of the invention. As noted above, the present invention relates to a class of polypeptides which are useful, inter alia, for in vitro diagnosis of coccidia infection in chickens. These sub¬ stantially purified proteins are analogous to various Eimeria tenella proteins which are believed to be capable of inducing an immune response when present in chicken tissue. Because an immune response has been mounted in infected chickens against analogous antigens, the sera of such infected chickens will contain antibodies which recognize one or more of the polypep¬ tides of the present invention. Thus, these polypeptides may serve, either in combination or individually, as the 'active ingredient in an in vitro diagnostic assay to determine the presence in chicken sera of antibodies directed toward various Eimeria species . As used herein, the term "analogous," when used in connection with a protein, antigen or polypeptide, is intended to mean a polypeptide which is capable of detecting antibodies raised in response to an infection with natural Eimeria pro¬ teins in chickens. A polypeptide possessing analogous anti¬ genic properties will thus exhibit some homology to the native Eimeria protein. It should be noted that "analogous" polypeptides , as the term is used herein, may raise an immune response which is stronger than, the same as, or weaker than the immune response raised by natural Eimeria proteins. The following proteins, in substantially pure form, have been discovered by the present inventors as being useful in such in vitro diagnostics. These include: Protein A, a 120kd protein of E. tenella; Protein B, a lOOkd protein of E. tenella; Protein C r a 200kd protein of E. tenellar Protein D, an 80kd protein of E. tenella; Protein E, an 80kd protein of E. tenella; Protein F, a 45k'd protein of E^ tenella; Protein G, a 3 " 0kd protein of E_. tenella; Protein R r a 25kd protein of E^_ tenella; Protein I, a 14kd protein of E. tenella; Protein J, a lOkd protein of E. tenella; Protein K of E. tenella; Protein L of E. tenella; and Protein M of E. tenella. It should be noted - that the molecular weights associated with the proteins dis¬ closed herein are not to be interpreted as absolute values. It is intended that these values be interpreted in light of the standard deviation of +10%. It is believed that some of the Proteins A through M are present on the surface of the Eimeria organism at some stage in its life cycle. In particular, Protein B is believed to be present on the anterior end, in the region of .the conoid, during the sporozoite stage of the organism. Protein B appears late during the sporulation of the Eimeria and is believed to be actively synthesized by the sporulating oocyst. Protein B also cross-reacts with sera pro¬ duced by animals presented with the merozoite and schizont stages, suggesting that the protein is synthesized during these stages as well. Protein B first appears approximately 44 hours into sporulation of the oocyst and persists into the sporozoite stage. Protein A is also capable of cross-reacting with sera produced in response to either the sporozoite, merozoite or schizont stages. Protein A is thus believed to be actively synthesized and most abundantly present during sporulation of the oocysts . Proteins C, D, E, F, G, H, I, J, K, L, and M are all produced at least during the development of the oocyst through the sporozoite stage. Protein D has been found to be present in sporozoites, merozoites and schizonts and is believed to be actively synthesized in each of these stages. Protein H first appears approximately 8 hours into sporulation of the oocyst and persists into the sporozoite stage. Protein G contains at least 5, preferably at least 21 and more preferably at least 45 glutamine residues. Such a preponderence of glutamine residues is a feature believed to be unique to Eimeria proteins. It is believed that these glutamine residues are in a direct sequence and that at least some additional amino acid residues may be appended to either the amino or carboxylic terminus of the polyglutamine sequence. Moreover, it is believed that each of these proteins contain one or more specific portions which may serve as anti¬ genic determinants capable of binding to at least one antibody present in sera of Eimeria infected chickens. These specific antigenic portions, either singly or in various combinations are, therefore, capable of serving as the basis for an in vitro diagnostic assay. DNA sequences encoding these proteins are contained in the EMBL phage clones identified herein. The DNA sequences •encoding for the proposed specific antigenic portions of these proteins are also contained in the EMBL phage clones. Table 1 indicates the relationship between the proteins, the EMBL phage clones containing the Eimeria DNA encoding the proteins and the insert clones encoding the proposed specific antigenic portions of the proteins. Table 1 Type of Molecular Lambda Plasmid Plasmid Insert Weight (kd) EMBL E age Insert Insert (cENA or Polypeptide in Sporozoite Clone Clone Size (bp) genαnic) A 120 ET: :Rbl-8 Rbl-8 1900 cI-NA B 100 ET: :Rbl-17 Rbl-17 1700 cI_MA C 200 ET: :CH2-13 CH2-13 2400 cENA D 80 ET: :CH2-15 CH2-15 725 CENA GRS-84 310 Genαnic CH2-6 870 cI2_A GRS-53 1600 Gencmic E 80 ET: :Rbl-145 Rbl-145 640 cE_>_A F 45 ET: : <__RS-74 GRS-74 600 Gencmic GRS-77 900 ' Gencmic G 30 ET: :CHAD-19 CHAD-19 270 CENA GRS-15 275 Gencmic GRS-72 450 Gencmic GRS-45 3000 Gencmic H 25 ET: :CAL-2a Cal-2a 280 cEΪM Cal-2cn 280 cENA I 14 'ET: :CAL-3gn Cal-3gn cI-NA Cal-3in 970 cENA J 10 ET: :C_ L-4cn Cal-4cn 2000 cENA Rbl-124 2000 cE_>TA K ET: :Rbl-19 Rbl-19 750 cENA L ETr :Rbl-24 Rbl-24 450 cDNA M ΞT: :CH2-3 CH2-3 270 cI-NA Restriction maps for certain of the EMBL clones listed in Table 1 are depicted in the Figures. In the Figures, the fol¬ lowing restriction sites are indicated by the abbreviations designated below for the corresponding restriction endonucleases : Endonuclease Abbreviation EcoRl R Sail S Hindlll H BamHI B Kpnl ' K Fig. 1 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein A. The region of the ET phage corresponding to cDNA clone Rbl-8 is depicted. Subcloning the Rbl-8 cDNA insert into an appropriate expression vector (see .Section IV) will result in synthesis of a portion of Protein A. DNA that directs synthesis of the entire Protein A polypeptide is contained within the Eimeria DNA portion of the phage and can be identified as described in Section VI . Fig. ' 2 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein B. The region of the ET phage corresponding to cDNA clone Rbl-17 is depicted. Subcloning the Rbl-17 cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein B. DNA that directs synthesis of the entire Protein B polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. Lambda phage ET::Rbl-17 has also been deposited with the American Type Culture Collection (ATCC) under Accession No. 40273. Fig. 3 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein C. The region of the ET phage corresponding to cDNA clone CH2-13 is depicted. Subcloning the CH2-13 cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein C. DNA that directs synthesis of the entire Protein C polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. Fig. 4 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein D. The region of the ET phage corresponding to cDNA clone Ch.2-15 is depicted. Subcloning the Ch.2-15 cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein D. DNA that directs synthesis of the entire Protein D polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. Fig. 5 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein E. The region of the ET phage corresponding to cDNA clone Rbl-145 is depicted. Subcloning the Rbl-145 cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein E. DN -that directs synthesis of the entire Protein E polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. Fig. 6 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein G. The region of the ET phage corresponding to cDNA clone CHAD-19 is depicted. Subcloning the CHAD-19 cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein G. DNA that directs synthesis of the entire Protein G polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VII. As noted previously, Protein G contains the "repeat" glutamine sequences. In addition to CHAD-19, other DNA se- ' quences have been found which encode repeat glutamine se¬ quences. These include GRS-15, GRS-72, GRS-45y GRS-74, GRS-77 and CH2-3. Inserts GRS-77 and CH2-3 have been described previ¬ ously. The remainder of these DNA sequences and any other Eimeria tenella DNA sequences may be isolated according to the methods set forth in Example 7. Fig. 7 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein I. The region of the ET phage corresponding to cDNA clone Cal-3gn is depicted. Subcloning the Cal-3gn cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein I. DNA that directs synthesis of the entire Protein I polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. Fig. 8 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein J. The region of the ET phage corresponding to cDNA clone Cal-4cn is depicted. Subcloning the Cal-4cn cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein J. DNA that directs synthesis of the entire Protein J polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. Fig. 9 is a restriction enzyme map of a lambda ET phage containing Eimeria DNA that encodes Protein L. The region of the ET phage corresponding to cDNA clone Rbl-24 is depicted. Subcloning the Rbl-24 cDNA insert into an appropriate expres¬ sion vector (see Section IV) will result in synthesis of a por¬ tion of Protein L. DNA that directs synthesis of the entire Protein L polypeptide is contained within the Eimeria DNA por¬ tion of the phage and can be identified as described in Section VI. A lambda ET phage containing Eimeria DNA that encodes Pro¬ tein H has been deposited with the ATCC under Accession No. 40270. The cDNA clone is called Cal-2a. A lambda ET phage containing Eimeria DNA that encodes Pro¬ tein K has been deposited" with the ATCC under Accession No. 40272. The cDNA clone is called ' Rbl-19. A lambda ET phage containing Eimeria DNA that encodes Pro¬ tein M has been deposited with the ATCC under Accession No. 40269. The cDNA clone is called CH2-3. The proteins described herein may be manufactured by the recombinant DNA methods set forth more fully below, using DNA sequences contained on the associated EMBL phage clones as set forth in Table 1. Additionally, the DNA sequences correspond¬ ing to the proposed antigenic portions of the proteins contained in the EMBL phage clones may be utilized in recombinant DNA methods to create the amino acid sequences which, it is believed, form certain of the antigenic determi¬ nants of the proteins , Various methods may be used to express the DNA encoding the proteins or the proposed antigenic determinants. In par¬ ticular, it is contemplated that the DNA contained on the EMBL phage clones may be expressed in mammalian systems. These sys¬ tems include, for example, viral vector delivery systems as set forth in Eukaryotic Viral Vectors edited by Y. Gluzman, Cold Spring Harbor Laboratory Cold Spring Harbor, New York (1982), specifically incorporated herein by reference. In an alternate preferred embodiment, it is contemplated that the DNA of interest will be excised from the EMBL phage clone and inserted, in a suitable form, into a microbial expression system. In this embodiment, the antigenic polypep¬ tides are produced by a method comprising : (a) preparation of a DNA sequence capable of directing a host microorganism to produce a polypeptide pos¬ sessing antigenic properties analogous to those pos¬ sessed by a polypeptide produced by Eimeria organ¬ isms; (b) cloning the DNA sequence into a vector capable of being transferred into and replicating in a host mi¬ croorganism, such vector containing operational ele¬ ments for the DNA sequence; (c) transferring the vector containing the DNA sequence and operational elements into a host microorganism capable of expressing the antigenic polypeptides,* (d) culturing the host microorganism under conditio'ns appropriate for amplification of the vector and expression of the polypeptide? and (e) in either order: (i) harvesting the polypeptide; and (ii) causing the polypeptide to assume a structure whereby it possesses antigenic properties analo¬ gous to properties possessed by polypeptides produced by Eimeria organisms. In this method, the DNA is prepared either by removal of any introns present in the lambda ET phage sequence encoding the polypeptide or by identifying a cDNA clone capable of hy¬ bridizing to the lambda ET phage sequence of interest. In the first method of preparing the DNA, the 5 ' and 3 ' gene limits are identified and it is determined whether any intervening se¬ quences, i.e., introns, are present. These intervening se¬ quences are readily identifiable to one of ordinary skill in the art in light of the teaching and phage maps contained here¬ in and the available literature such as Berk, A.J. and Sharp, P.A. in Cell _12 ^ : 721 (1977 ) , specifically incorporated herein by reference. For example, the presence of an intervening se¬ quence may be determined by ascertaining the nucleotide se¬ quence of the DNA encoding the protein and examining that se¬ quence to identify nucleotide sequences known to occur in introns. Any identified introns could then be excised by bridging the intron with an oligonucleotide and cleaving the intron sequence with an appropriate nuclease such as SI nuclease. Examples "of the SI nuclease procedure are set forth in Berk, A.J. and Sharp, P.A. in Cell 1_2: 721 (1977) , specifical¬ ly incorporated herein by reference. In an alternate method of preparing the DNA for expression in a microbial system, the portion of the lambda ET phage of interest is identified in light of the phage maps contained herein. This portion of the lambda phage, or a polynucleotide subset thereof is used to probe a cDNA library created from Eimeria mRNA obtained from an appropriate stage of the Eimeria life cycle. Preferably, the mRNA is obtained from sporozites . A preferred method for the creation of the Eimeria cDNA library is set forth in the Examples below. The hybridizing cDNA clone thus identified is suitable for use in the microbial expression system. The DNA prepared in accordance with the above methods is inserted into an expression vector suitable for use in the in¬ tended expression system. Embodiments of the present invention are envisioned as employing other known or currently undiscovered vectors which would contain one or more of the DNA sequences encoding antigenic polypeptides described herein. In particular, it is preferred that these vectors have some or all of the following characteristics: (1) possess a minimal number of host-organism sequences? (2) be stable in the desired host,- (3) be capable of being present in a high copy number in the desired host,- (4) possess a regulatable promoter; (5) have at least one DNA sequence coding a selectable trait present on a portion of the plasmid separate from that where the DNA se¬ quence encoding for the antigenic polypeptide will be inserted; and (6) be integrated into the vector. The following, noninclusive, list of cloning vectors and operational elements for E. coli and other organisms is believed to set forth vectors "which can easily be altered to meet the above-criteria and are therefore preferred for use in the present invention. Such alterations are easily performed by those of ordinary skill in the art in light of the available literature and the teachings herein. TABLE 2 Transcriptional Regulated Transcription mDlSP- Start Site & RS Bind Hos Prαnαt-ers Inducer Terminator Stabilization Leader Peptide .Marker Site Lac 1 , Tac 2 IPTG, rrnB αmpA Λ 8 bla amp cil lin 3 4 9 „12 Lambda pL ' increased temperature, rrnC lambda mt cmpA b tetracycl .i •ne 14 < , l 5 E. coli Trp IAA addi tion, or trp phoS 13 ch 1 oraπiphenical tryptophan depletion *alpha amylase E. coli rrn B.amy neutral Kan r 24 B.amy 21 *sι_htilisinl8 BT.T20 protease Cam r 25 i neutra Bacillus *p-4319 B.amy alpha- prote spac-126 IPTG amylase22 B.amy B.subt. alpha- subtilisin23 amylas Trp27 (E. coli) IAA addition, or phospholipase sulfonaτιide30 Trp (E. Lac (E. coli) tryptophan C28 streptατιycin30 coli ) depletion exotoxin A29 Pseulo- Tac (E. coli) IPTG iiDn s Gal 131, 1032 Glucose depletion Cyc 1 Invertase36 Ura 337 and galactose Una Acid phos- Leu 238 Yeast M 133, 1134 Glucose depletion Alpha factor phatase36 Pho 535 Phosphate depletion Sac 2 Alpha Factor His 3 Tap 139 *noι_-regul ated 1. Back-nan, K. , Ptashne, M. and Gilbert, W. Proc. Natl. Acad. Sci. USA 1' 4174-4178 (1976). 2. de Boer, H.A., Comstock, L.J., and Vasser, M. Proc. Natl. Acad. Sci. USA _80, 21-25 (1983). 3. Shimatake, H. and Rosenberg, M. Nature 292 , 128-132 (1981). 4. Derom, C. , Gheysen, D. and Fiers, W. Gene 1/7, 45-54 (1982). 5. Hallewell, R.A. and Emtage, S. Gene 9_, 27-47 (1980). 6. Brosius, J. , Dull, T.J., Sleeter, ' D.D. and Noller, H.F. J. Mol. Biol. 148, 107-127 (1981). 7. Normanly, J. , Ogden, R.C., Horvath, S.J. and Abelson, J. Nature 321, 213-219 (1986). 8. Belasco, J.G. , Nilsson, G. , von Gabain, A. and Cohen, S.N. Cell 46_, 245-251 (1986). 9. Schmeissner, U. , McKenney, K. , Rosenberg M. and Court, D. J. Mol. Biol. 176, 39-53 (1984). 10. Mott, J.E., Galloway, J.L. and Platt, T. EMBO J. 4, 1887-1891 • (1985) . 11. Koshland, D. and Botstein, D. Cell 20_, 749-760 (1980). 12. Movva, N. R. , Nakamura, K. and Inouye, M. J. Mol. Biol. 143, 317-328 (1980). 13. Surin, B.P., Jans, D.A. , Fimmel, A.L., Shaw, D.C., Cox, G.B. and Rosenberg, H. J. Bacteriol. 157, 772-778 (1984). 14. Sutcliffe, J.G. Proc. Natl. Acad. Sci. USA 21 * 3737-3741 (1978) . 15. Peden, K.W.C. Gene 22, 277-280 (1983). 16. Alton, N.K. and Vapnek, D. Nature 282 , 864-869 (1979). 17. Yang, M. , Galizzi, A., and Henner, D. Nuc. Acids Res. 11(2) , 237-248 (1983). 18. Wong, S.-L., Price, C.W. , Goldfarb, D.S., and Doi, R.H. Proc. Natl. Acad. Sci. USA 81, 1184-1188 (1984). 19. Wang, P.-Z- , and Doi, R.H. -J. Biol. Chem. 259, 8619-8625, (1984) . 20. Lin, C.-K., Quinn, L.A. Rodriquez, R.L. J. Cell Biochem. Suppl. (9B) , p.198 (1985). 21. Vasantha, N. , Thompson, L.D., Rhodes, C. , Banner, C. , Nagle, J., and Filpula, D. J. Bact. 159(3) , 811-819 (1984). 22. Palva, I., Sarvas, M. , Lehtovaara, P., Sibazkov, M. , and Kαariainen, L. Proc. Natl. Acad. Sci. USA 19_, 5582-5586 (1982) . 23. Wong. S.-L., Pricee, C.W., Goldfarb, D.S., and Doi, R.H. Proc. Natl. Acad. Sci. USA _81_, 1184-1188 (1984). 24. Sullivan, M.A., Yasbin, R.E., and Young, F.E. Gene 29, 21-46 (1984). 25. Vasantha, N. , Thompson, L.D., Rhodes, C, Banner, C. Nagle, J., and Filpula, D. J. Bact. 159(3 ) , 811-819 (1984). 26. Yansura, D.G. and Henner, D.J. PNAS j , 439-443 (1984). 27. Gray, G.L., McKeown, K.A., Jones, A.J.S., Seeburg, P.H. and Heyneker, H.L. Biotechnology, 161-165 (1984). 28. Lory, S. , and Tai, P.C. Gene _22, 95-101 (1983). 29. Liu, P.V. J. Infect. Dis. 130 (suppl), 594-599 (1974). 30. Wood, D.G., Hollinger, M.F., and Tindol, M.B. J. Bact. 145 , 1448-1451 (1981). 31. St. John,' T.P. and Davis, R.W. J. Mol. Biol. 152, 285-315 (1981) . 32. Hopper, J.E., and Rowe, L.B. J. Biol. Chem. 253, 7566-7569 (1978). 33. Denis, C.L., Ferguson, J. and Young, E.T. J. Biol. Chem. 258, 1165-1171 (1983). 34. Lutsdorf, L. and Megnet, R. Archs . Biochem. Biophys . 126, 933-944 (1968). 35. Meyhack, B. , Bajwa, N. , Rudolph, H. and Hinnen, A. EMBO. J. _6, 675-680 (1982) . 36. Watson, M.E.E. Nucleic Acid Research _12_, 5145-5164 (1984). 37. Gerband, C. and Guerineau, M. Curr. Genet. _1, 219-228 (1980) . 38. Hinnen, A., Hicks, J.B. and Fink, G.R. Proc. Natl. Acad. Sci. USA 1' 1929-1933 (1978). 39. Jabbar, M.A. , Sivasubramanian, N. and Nayak, D. P. Proc. Natl. Acad. Sci. USA 82_, 2019-2023 (1985). It is to be understood that additional cloning vectors may now exist or will be discovered which have the above-identified properties and are therefore suitable for use in the present invention. These vectors are also contemplated as being within the scope of the disclosed series of cloning vectors into which the cDNA sequences may be introduced, along with any necessary operational elements, and which altered vector is then included within the scope of the present invention and would be capable of being used in the recombinant-DNA method set forth more fully below. A. Characteristics of Preferred Vectors Certain embodiments of the present invention are envi¬ sioned which employ known or currently undiscovered vectors which would contain one or more of the cDNA sequences described herein. In particular, it is preferred that these vectors have some or all of the following characteristics: (1) possess a minimal number of host-organism sequences? (2) be stably main¬ tained and propagated in the desired host? (3) be capable of being present in a high copy number in the desired host; (4) possess a regulatable promoter positioned so as to promote transcription of the gene of interest? (5) have at least one DNA sequence coding for a selectable trait present on a portion of the plasmid separate from that where the portable DNA se¬ quence will be inserted? and (6) a DNA sequence capable of terminating transcription. In various preferred embodiments, these cloning vectors containing and capable of expressing the cDNA sequences of the present invention contain various operational elements. These "operational elements," as discussed herein, include at least one promoter, at least one Shine-Daigarno sequence and initiator codon, and at least one terminator codon. Prefer¬ ably, these "operational elements" also include at least one operator, at least one leader sequence for proteins to be exported from intracellular space, at least one gene for a reg¬ ulator protein, and any other DNA sequences necessary or pre¬ ferred for appropriate transcription and subsequent translation of the vector DNA. Certain of these operational elements may be present in each of the preferred vectors of the present invention. It is contemplated any additional operational elements which may be required may be added to these vectors using methods known to those of ordinary skill in the art, particularly in light of the teachings herein. In practice, it is possible to construct each of these vectors in a way that allows them to be easily isolated, assem¬ bled, and interchanged. This facilitates assembly of numerous functional genes from combinations of these elements and the coding region of the cDNA sequences. Further, many of these elements will be applicable in more than one host. It is addi¬ tionally contemplated that the vectors, in certain preferred embodiments, will contain DNA sequences capable of functioning as regulators ("operators"), and other DNA sequences capable of coding for regulator proteins. 1. Regulators These regulators, in one embodiment, will serve to prevent expression of the cDNA sequences in the presence of certain environmental conditions and, in the presence of other environ¬ mental conditions, will allow transcription and subsequent expression of the protein coded for by the cDNA sequence. In particular, it is preferred that regulatory segments be in¬ serted into the vector such that expression of the cDNA se¬ quence will not occur, or will occur to a greatly reduced extent, in the absence of, for example, isopropylthio- D-galac- toside. In this situation, the transformed microorganisms con¬ taining the cDNA sequence may be grown to a desired density prior to initiation of the expression of Proteins A through N. In this embodiment, expression of the desired trypsin inhibitor is induced .by addition of a substance to the microbial environ¬ ment capable of causing expression of the DNA sequence after the desired density has been achieved. 2. Promoters The expression vectors must contain promoters which can be used by the host organism for expression of its own proteins. While the lactose promoter system is used commonly, other mi¬ crobial promoters have been isolated and characterized, enabling one skilled in the art to use them for expression of the Protein A through N. 3. Transcription Terminator The transcription terminators contemplated herein serve ' to stabilize the vector. In particular, those sequences as described by Rosenberg, M. and Court, D. , in Ann. Rev. Genet. 1^:319-353 (1979), specifically incorporated herein by refer¬ ence, are contemplated for use in the present invention. 4. Non-Translated Sequence It is noted that, in the preferred embodiment, it may also be desirable to reconstruct the 3 ' or 5 ' end of the coding re¬ gion to allow incorporation of 3 ' or 5' non-translated se¬ quences into the gene transcript. Included among these non- translated sequences are those which stabilize the mRNA as they are identified by Schmeissner, U. , McKenney, K. , Rosenberg, M. and Court, D. in J. Mol. Biol. 176: 39-53 (1984), specifically incorporated herein by reference. 5. Ribosome Binding Sites The microbial expression of foreign proteins requires cer¬ tain operational elements which include, but are not limited to, ribosome binding sites. That particular element is a se¬ quence which a ribosome recognizes and binds to in the initiation of protein synthesis as set forth in Gold, L. , et al. , Ann. Rev. Microbiol. _3_! : 557-580 (1983); Marquis, D.M., e al.-, Gene 4_2: 175—183 (1986), specifically incorporated herein by reference. The operational elements as discussed herein can be rou¬ tinely selected by those of ordinary skill in the art in light of prior literature and the teachings contained herein. Gen¬ eral examples of these operational elements are set forth in B. Lewin Genes, Wiley & Sons, New York (1983), which is specifi¬ cally incorporated herein by reference. Various examples of suitable operational elements may be found on the vectors dis¬ cussed above and may be elucidated through review of the publi¬ cations discussing the basic characteristics of the aforementioned vectors. 6. Leader Sequence and Translational Coupler Additionally, 'it is preferred that DNA coding for an appropriate -secretory leader (signal) sequence be present at the 5 l end of the portable DNA sequence. The DNA for the lead¬ er sequence must be in a position which allows the production of a fusion protein in which the leader sequence is immediately adjacent to and covalently joined to the inhibitor, i.e., there must be no transcription or translation termination signals between the two DNA coding sequences. The presence of the leader sequence is desired in part for one or more of the following reasons. First, the presence of the leader sequence may facilitate host processing of Proteins A through N. In particular, the leader sequence may direct cleavage of the ini¬ tial translation product by a leader peptidase to remove the leader sequence and leave a polypeptide with the amino acid se¬ quence which has potential protein activity. Second, the pres¬ ence of the leader sequence may facilitate purification of the Protein A through N, through directing the protein out of the cell cytoplasm. In some species of host microorganisms, the presence of the appropriate leader sequence will allow trans¬ port of the completed protein into the periplasmic space, as in the case of some E. coli. In the case of certain E. coli, saccharomyces and strains of Bacillus and Pseudomonas, the appropriate leader sequence will allow transport of the protein through the cell membrane and into the extracellular medium. In this situation, the protein may be purified from extra¬ cellular protein. Thirdly, in the case of some of the proteins prepared by the present invention, the presence of the leader sequence may be necessary to locate the completed protein in an environment where it may fold to assume its active structure, which structure possesses the appropriate protein activity. 7. Translation Terminator The translation terminators contemplated herein serve to stop the translation of mRNA. They may be either natural, Kohli, J. , Mol. Gen. Genet. 182:430-439 (1981), or synthesized, Pettersson, R.F., Gene 2Λ : 15-27 (1983), both of which refer¬ ences are specifically incorporated herein by reference. 8. Selectable Marker Additionally, it is preferred that the cloning vector con¬ tain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host microorganism. In one embodiment of the present invention, the gene for ampicillin resistance is included in the vector while in other plasmids, the gene for tetracycline resistance or the gene for chloramphenicol resistance is in¬ cluded. Such a drug resistance or other selectable marker is in¬ tended in part to facilitate in the selection of transformants . Additionally, the presence of such a selectable marker on the cloning vector may be of use in keeping contaminating microor¬ ganisms from multiplying in the culture medium. In this embodiment, such a pure culture of the transformed host micro¬ organisms would be obtained by culturing the microorganisms under conditions which require the induced phenotype for sur¬ vival. B. Vector Assembly Upon synthesis and/or isolation of all necessary and desired component parts of the above-discussed cloning vectors, the vectors are assembled by methods generally known to those of ordinary skill in the art. Assembly of such vectors is believed to be within the duties and tasks performed by those with ordinary skill in the art and, as such, is capable of being performed without undue experimentation. For example, similar DNA sequences have been ligated into appropriate cloning vectors, as set forth in Maniatis, T., et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1982), which is specifically incorporated herein by ref¬ erence. C. Host Organisms The vectors- and methods disclosed here are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms. Prokaryotes are preferred for the cloning of DNA sequences and for the expression of the gene. The E. coli strains JM105, JM107 and JM109 (available from Ph.arm.acia) , as well as Bacilli, and Pseudomonas species, may be used for expression of the gene. In addition to prokaryotes, eukaryotic microbes such as Saccharomyces cerevisiae may be used for expression of the gene. In general, plasmid vectors containing operational ele¬ ments which are derived from species compatible with the host cells are used. For example, E. coli is typically transformed using pBR322, a plasmid derived from E. coli. Table 2 indi¬ cates the list of host organisms and the compatible vectors. 1. Pseudomonas Vectors In addition to the -vectors listed in Table 2, several vector plasmids which autonomously replicate in a broad range of Gram Negative bacteria are preferred for use as cloning ve¬ hicles in hosts of the genera Pseudomonas . These are described by Tait, R.C., Close, T.J., Lundquist, R.C., Hagiya, M. , Rodriguez, R.L., and Kado, C.I. in Biotechnology, May, 1983, pp. 269-275; Panopoulos, N.J. in Genetic Engineering in the Plant Sciences, Praeger Publishers, New York, New York, pp. 163-185, (1981); and Sakaguchi, K. in Current Topic in Microbiology and Immunology _96_: 31-45, (1982), each of which is specifically incorporated herein by reference. One particularly preferred construction would employ the plasmid RSFIOIO and derivatives thereof as described by Bagdasarian, M. , Bagdasarian, M.M., Coleman, S., and Timmis, K.N. in Plasmids of Medical Environmental and Commercial Impor¬ tance, Timmis, K.N. and Puhler, A. eds., Elsevier/North Holland Biomedical Press, (1979), specifically incorporated herein by reference. The advantages of RSFIOIO are that it is relatively small, high copy number plasmid which is readily transformed into and stably maintained in both E. coli and Pseudomonas spe¬ cies. In this system, it would be preferred to use the Tac expression system as described for Escherichia, since it appears that the E. coli trp promoter is readily recognized by Pseudomonas RNA polymerase as set forth by Sakaguchi, K. in Current Topics in Microbiology and Immunology _96_: 31-45 (1982) and Gray, G.L., McKeown, K.A. , Jones, A.J.S., Seeburg, P.H., and Heyneker, H.L. in Biotechnology Feb. 1984, pp. 161-165, both of which are specifically incorporated herein by refer¬ ence. Transcriptional activity may be further maximized by requiring the exchange of the promoter with, e.g., an E. coli or P. aeruginosa trp promoter. Additionally, the laclq gene of a laclq strain of E. coli would also be included in the plasmid to effect regulation. Translation may be coupled to translation initiation for any of the E. coli proteins as described in the Examples, as well as to initiation sites for any of the highly expressed proteins of the host to cause intracellular expression of the inhibitor. In those cases where restriction minus strains of a host Pseudomonas species are not available, transformation efficiency with plasmid constructs isolated from E. coli are poor. Therefore, passage of the Pseudomonas cloning vector through an r- m.- strain of another species prior to transforma¬ tion of the desired host, as set forth in Bagdasarian, M. , et al. , Plasmids of Medical, Environmental and Commercial Importance, pp. 411-422, Timmis and Puhler eds., Elsevier/North Holland Biomedical Press (1979), specifically incorporated herein by reference, is desired. 2. Bacillus Vectors Furthermore, a preferred expression system in hosts of the genera Bacillus involves using plasmid _pUB110 as the cloning vehicle. As in other host vector systems, it is possible in Bacillus to express the trypsin inhibitors of the present invention as either an intracellular or a secreted protein. The present embodiments include both systems. Shuttle vectors that replicate in both Bacillus and E. coli are available for constructing and testing various genes as described by Dubnau, D., Gryczan, T., Contente, S. , and Shivakumar, A.G. in Genetic Engineering, Vol. 2, Setlow and Hollander eds., Plenum Press, New York, New York, pp. 115-131, (1980), specifically incorpo¬ rated herein by reference. For the expression and secretion of trypsin inhibitors from B. subtilis, the signal sequence of alpha-amylase is preferably coupled to the coding region for the inhibitor. (For synthesis of intracellular inhibitor, the portable DNA sequence will be translationally coupled to the ribosome binding site of the alpha-amylase leader sequence) . Transcription of either of these constructs is preferably directed by the alpha-amylase promoter or a derivative thereof. This derivative contains the RNA polymerase recognition se¬ quence of"the- native alpha-amylase promoter but incorporates the lac operator region as well. - Similar hybrid promoters con¬ structed from the penicillinase gene promoter and the lac oper¬ ator have been shown to function in Bacillus hosts in a regu- latable fashion as set forth by Yansura, D.G. and Henner in Genetics and Biotechnology of Bacilli, Ganesan, A.T. and Hoch, J.A. , eds., Academic Press, pp. 249-263, (1984), specifically incorporated by reference. The lacl gene of a laclq strain of E. coli would also be included in the placement to effect regulation. 3. Clostridium Vectors One preferred construction for expression in Clostridium is in plasmid pJU12 described by Squires, C. H. et al in J. Bacteriol. 159: 465-471 (1984), specifically incorporated herein by reference, transformed into C. perfringens by the method of Heefner, D. L. et al . as described in J. Bacteriol. 159: 460-464 (1984), specifically incorporated herein by reference. Tran¬ scription is directed by the promoter of the tetracycline re¬ sistance gene. Translation is coupled to the Shine-Dalgarno sequences of thi .s same tetr gene i.n a manner strictly analogous to the procedures outlined above for vectors suitable for use in other hosts. 4. Yeast Vectors Maintenance of foreign DNA introduced into yeast can be effected in several ways (Botstein, D. , an Davis, R. .W. , in The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Strathern, Jones and Broach, eds., pp. 607-636 (1982). One preferred expression system for use with host organisms of the genus Saccharomyces harbors the trypsin inhibitor gene on the 2 micron plasmid. The advantages of the 2 micron circle include relatively high copy number and stabil¬ ity when introduced into cir° strains. These vectors prefer¬ ably incorporate the replication origin and at least one antibiotic resistance marker from pBR322 to allow replication and selection in E. coli. In addition, the plasmid will pref¬ erably have 2 micron sequences and the yeast LEU2 gene to serve the same purposes in LEU2 defective mutants of yeast. EXAMPLES I. CONSTRUCTION OF THE LAMBDA G 11 EXPRESSION LIBRARIES A. Construction of The "Small Fragment"- E. Tenella Genomic DNA Expression Library The rationale for construction of a "small fragment" genomic expression library was to obtain a representative clone for every antigenically active epitope that could be expressed by E. tenella. By using small genomic fragments, the probabil¬ ity of a peptide determinant folding in a way that approximates its in vivo state may be increased. A large number of clones are required to adequately represent the E. tenella genome. g Using 3 X 10 bp as the estimate of the genome size for E. tenella, it was calculated that for a 99% probability of having every 300 bp fragment in both orientations and in all three 7 reading frames, 3 x 10 individual recombmants were required. Because a large number of recombinants are required for a representative genomic expression library, it was decided to use the lambda gtll system as described by Young, R. and Davis, R. in PNAS £I0_:1194 (1983). Through use of the lambda gtll sys¬ tem, large numbers of recombinants could be obtained due to the highly efficient in vitro packaging systems that have been developed for lambda. In lambda gtll, cloned DNA fragments coding for peptides are expressed as fusion proteins when they are inserted at the unique EcoRl site near the carboxy terminus of the beta-galactosidase gene in the phage. Insertion of for¬ eign DNA in the beta-galactosidase gene inactivates the gene, thus allowing identification of recombinant phages on indicator plates . The "small fragment" E. tenella genomic expression library was obtained in four consecutive steps. First, lambda gtll DNA was purified and prepared for efficient cloning of insert DNA fragments. Second, genomic DNA was obtained from E _ tenella sporozoites. Third, "small fragments" of the E. tenella genome were prepared by cleavage with Rsal restriction endonuclease, modification with EcoRl methylase and subsequent frame adapta¬ tion with different length, oligonucleotides. Fourth, the pre¬ pared "small fragments" were ligated into the lambda gtll vector DNA and packaged in vitro. Finally, the in vitro pack¬ aged recombinant phage were amplified. 1. Purification and Preparation of Lambda gtll DNA A lysate of lambda gtll was obtained from Dr. D. Hirsh (MCD Biology Dept. , University of Colorado) . The phage was plated on the host E. coli strain KM392 which contains a lac deletion. Plate lysates of this phage were prepared using the methods described by Davis, R. r et al. in 1981. The gtll phage were concentrated from the lysate by centrifugation at 20,000 rpm, using a Beckman JA-20 rotor, for 3 hour. The concentrated phage were purified by sedimentation through Cesium Chloride (CsCl) gradients as described in Section III (part VB page 58), infra. The DNA was extracted from the phage particles by exhaustive dialysis against 50% formamide (MCB) and subsequent dialysis against DNA storage buffer lOmM Tris-HCl (pH7.5 )? lmM EDTA (TE buffer) Since there is no genetic selection for recombinant phage with the lambda gtll system, a biochemical selection was used. In order to "protect" the cohesive ends of the phage during this selection, the following reactions were done. First, the purified DNA was heated to 70°C, cooled to 42 °C, and the cohesive ends of the phage were allowed to anneal for 3 hour. The mixture was then cooled to 14°C and a 1/10 volume of 10X ligase buffer was added, followed by T4 DNA ligase (P.L. Biochemicals) . The ligation reaction was conducted for 14 hour at 14°C. After the gtll arms were sealed, the DNA was treated with an excess of EcoRI. After digestion, the EcoRI was inactivated by heating the reaction mix at 70°C for 10 minutes, then calf intestinal alkaline phosphatase (Boehringer-Mannheim) was added and the reaction mix incubated at 37°C for 15 minutes. The quantity of alkaline phosphatase was empirically determined to give a low background level after ligation and packaging. To ensure that the EcoRI ends of the vector were intact, recovery of beta-galactosidase activity in lambda gtll was monitored after treating the EcoRI ends with polynucleotide kinase (P.L. Biochemicals) and T4 DNA ligase (P.L. Biochemicals) . The alkaline phosphatase was removed from the vector DNA prepara¬ tion by three phenol extractions followed by three ether extractions. The extracted vector DNA was then made 0.3 M with sodium acetate, and 2.5 volumes of ethanol were added to pre¬ cipitate the DNA. The precipitated DNA was resuspended in a small volume of Tris-EDTA (TE) buffer (10 mM Tris-HCl, pH 7.5; 1 M EDTA). A typical phosphatase treatment of gtll phage DNA decreased the infectivity of the phage by at least two logs. The dephosphorylated molecules cannot religate to themselves, and the only way to obtain biological activity is to insert a DNA molecule between the two phage arms. 2. Preparation of Genomic DNA From Eimeria Tenella Sporozoites DNA was obtained from E. tenella using protocols such as those described by Maniatis, T. , et al. in Cell L5:687 (1978) and Blin, N. and Stafford, D.W. in Nucleic Acids Res. _3: 2303 9 (1976). Approximately 5 x 10 frozen E. tenella sporozoites were thawed on ice and transferred to a plastic tube. The vol¬ ume of cells was 2.5 ml. The original container was rinsed with 0.5 ml of TE buffer and the rinse solution was added to the sporozoi-te solution to give a final volume of 3.0 ml. Five milliliters of proteinase K solution (0.2 M Tris, pH 8.3? .01 M EDTA? 0.4 M NaCl ? 0.2% SDS? and 400 ug/ l Proteinase K (Boehringer Mannheim Biochemicals, Indianapolis, Indiana)) was then added to the sporozoite solution. The sporozoites were incubated with the proteinase K solu¬ tion at 65°C for 1 hour 8.0 ml of phenol was added to the mix¬ ture. The mixture was gently rocked on an oscillating platform for 30 minutes at room temperature. The sample was then cen- trifuged in a Beckman JA20 rotor at 7, 000 rpm for 5 minutes at 15 °C. The aqueous phase was removed with a large bore plastic pipette. The phenol interface was re-extracted by the addition of 5.0 ml of TE buffer. This solution was mixed gently by rocking, and the phases separated by centrifugation as described above. The re-extraction process was repeated. The aqueous phases were then pooled, extracted with an equal volume of chloroform (CHC1_), and the pooled extractions were mixed by gently rocking. The CHC1, was separated from the aqueous phase by centrifugation and the aqueous phase was removed with a large-bore plastic pipette. The extracted aqueous phase was adjusted to 0.3 M sodium acetate by adding a 1/10 volume of a 3.0 M sodium acetate stock solution. The DNA was precipitated from this solution by adding 2.5 volumes of cold ethanol. Gentle mixing with the cold ethanol yielded a large, viscous mass of nucleic acid that was "spooled" out of solution using a small glass rod. This • material was then rinsed twice with 70% ethanol and allowed to air-dry. The dry nucleic acids were resuspended in 0.5 ml of TE buffer by rocking the solution for 16 hours at 4°C. In order to remove any contaminating RNA from the E. tenella DNA preparation, the nucleic acids were treated with RNase A. Five-tenths milliliter of the sporozoite nucleic acid preparation was treated with 100 ug/ml RNase A (Miles La¬ boratories, Elkhart, Indiana) at 37°C for 30 minutes. More RNase A was added to a final concentration of 200 ug/ml, and the solution incubated at 37 °C for an additional 60 minutes. An equal volume of phenol was added to the material and the mixture was gently rocked for 5 minutes. The phases were sepa¬ rated by centrifugation in a Brinkmann Microfuge for 1.0 minute at room temperature. The aqueous phase was removed with a large-bore pipette and extracted with an equal volume of CHC1,. The aqueous phase was separated from the chloroform by centrifugation and pipetting. The aqueous phase was adjusted to 0.3 M sodium acetate by adding a 1/10 volume of a 3.0 M stock solution. The DNA was precipated from this solution by the addition of 2.5 volumes of ethanol. The ethanol precipita¬ tion yielded a large viscous pellet that was spooled from the solution and rinsed with 70% ethanol. After air drying, the DNA was resuspended in 0.5 ml of TE buffer. Integrity of the E_^ tenella DNA was analyzed by electro- phoresis through an agarose gel and subsequent staining of the gel with ethidium bromide. These results indicated that the DNA was of high molecular weight and was relatively free of RNA. The concentration of the DNA was determined by optical " absorbance at 260 nm. This particular preparation yielded 400 ug of purified DNA. 3. Preparation of Small E^ tenella Genomic DNA Fragments " In order to align a given fragment from the E. tenella genome in the proper reading frame for transcription and trans¬ lation of the native polypeptide, it was necessary for the fragment to be adapated to the correct reading frame. The frame correctors must provide an EcoRI cohesive end for cloning into the EcoRI site of the beta-galactosidase gene by lambda gtll. The following frame correctors were used: NEB 1018 5'-GGAATTCC n NEB 1004 5 '-CGGAATTCCG n+1 NEB 1019 5 r -CCGGAATTCCGG n+2 The frame correctors were obtained from New England Biolabs (Beverly, MA) and phosphorylated with polynucleotide kinase (P.L. Biochemicals) using standard phosphorylation conditions. Because the frame correctors are blunt-ended, any restric¬ tion endonuclease that leaves a blunt end may be used to frag¬ ment the E. tenella genomic DNA. For the library described here, Rsal was selected, which recognizes the tetranucleotide sequence 5'-GTAC and cleaves between the T and A residues, generating a blunt end. Treatment of E. tenella genomic DNA with Rsal theorectically yields a cut in the DNA once every 256 base bairs on average residues, thereby generating an array of small fragments . One hundred micrograms of E. tenella genomic DNA were cleaved using a 10-fold excess of the restriction endonuclease Rsal (New England Biolabs) . The enzyme was then inactivated by heating the reaction mix to 70°C for 10 minutes. The resultant mixture was extracted with an equal volume of phenol, and re¬ sidual phenol was removed by ether extractions. The reaction was made 0.3 M with sodium acetate, then 2.5 volumes of ethanol were added to precipitate the DNA. The precipitated DNA was pelleted by centrifugation, and the pellet rinsed with 70% eth¬ anol. The Rsal fragmented DNA was resuspended in a small vol¬ ume of TE buf er. In order to protect any E_. tenella genomic DNA fragments that might have internal EcoRI sites, it was necessary to modi¬ fy the DNA. EcoRI methylase (New England Biolabs) was reacted with the fragmented- E. tenella DNA in the presence of S- .denosyl methionine using conditions recommended by the sup¬ plier. The methylase was inactivated by phenol extraction of the reaction mix, and residual phenol was removed by ether extractions. The mix was then adjusted to 0.3 M sodium ace¬ tate, and 2.5 volumes of ethanol were added. After 30 minutes at -70°C, the precipitated DNA was pelleted by centrifugation. This DNA was resuspended in TE buffer. The Rsal cleaved and EcoRI methylated E. tenella genomic DNA was divided into three aliquots. A different length frame corrector was added to each aliquot of small fragment E. tenella DNA. Addition of the frame corrector was such that the final concentration of corrector was 5-10 uM and in vast excess of the number of ends of fragmented genomic DNA present in each reaction. T4 DNA ligase (P.L. Biochemicals) was added to each of the three aliquots and incubated at 4°C for 16 hours. The ligation reaction was terminated by heat inactivating the mix¬ tures at 70°C for 10 minutes. Sodium chloride to 0.1 M and excess EcoRI endonuclease were ' then added to the mixtures, and the reactions were incubated at 37°C for several hours. The EcoRI was heat inactivated at 70°C for 10 minutes. Removal of excess frame correctors from the adapted insert DNA was accomplished by gel purification of the inserts. The adapted insert DNA was size-fractionated on a 2.5% agarose gel. The insert DNA was retarded by the gel matrix in such a way that adequate separation from monomer correctors was obtained after a few minutes of electrophoresis . The insert-containing region of the gel was then excised, and the DNA electroeluted from the gel . 4. Ligation of Fragmented and Frame-Corrected DNA to lambda gtll and JLn Vitro Packaging of the Recombinant DNA Phosphatized and EcoRI-cleaved lambda gtll DNA was mixed with frame-corrected small fragments of E. tenella. These DNAs were ligated using T4 DNA ligase (P.L. Biochemicals) overnight at 14°C. A small aliquot of each frame-corrector, ligation re¬ action mixture was analyzed by gel electrophoresis to monitor the ligation reaction. Each 1 of the three reaction mixture ali¬ quots was heated at 70°C for 5 minutes and then mixed with lambda in vitro packaging extracts. The packaging reaction was allowed to proceed for 60 minutes at room temperature at which time a drop of chloroform was added to prevent bacterial growth. 5. Amplification of the "Small Fragment" E. tenella Expression Library Packaged phage were diluted with lambda dil and adsorbed to E_. coli strain Y1088 as described by Young, R. and Davis, R. in PNAS j3£:1194 (1983). Amplification of the library on this strain ensured that the beta-galactosidase gene was not expressed? therefore, any phage containing coding sequences that might be deleterious to the host E. coli cell were not expressed and not lost from the library. Primary amplification gave libraries with the following complexity: corrector 8-mer 10-mer " 12-mer 1.5 x 10 6 5.1 x 10 5 * 1.1 x 10 6 The background level of nonrecombinants as indicated by a blue coloration on chromogenic substrate from each reaction was less than 10% of the total phage. Each of the three primary li- braπes had titers of from 5 x 10 9 to 2 x 1010 phage per ml of lysate. It was calculated, based on the number of phage in the genomic expression library, that a 50% probability of having every fragment in all three reading frames and all orientations was obtained. B. Sporozoite cDNA Expression Library The lambda gtll:sporozoite cDNA expression library was constructed in several steps which included purification of poly(A) mRNA from sporozoites, construction of an initial cDNA library in lambda gtlO, and transfer of the cDNA library from lambda gtlO to lambda gtll. 1. Isolation of Poly(A) mRNA Approximately 3 x 10 9 E^_ tenella sporozoites were obtained. The sporozoites were frozen in liquid nitrogen and stored at -70°C until used. Total cellular RNA was isolated by resuspending the sporozoites in 5 ml of RNA lysis buffer (4M guanidine isothiocyanate; 0.13 M sodium acetate, pH 5.2 ? 0.5% Sarkosyl? 1 M beta-mercaptoethanol; 1 mM EDTA), and trans¬ ferring the sporozites to a new tube. The original tube was rinsed with an additional 2 ml of RNA lysis buffer and the two solutions were combined. This mixture was vortexed 3 x 20 sec¬ onds to ensure lysis of the sporozoites. The mixture was layered over a 1.25 ml cushion of 5.7 M CsCl, 50 mM sodium acetate, pH 5.2 in a SW 50.1 rotor and cen- trifuged for 19 hours at 35,000 rpm. The supernatant was decanted and the bottom of the tube containing the RNA pellet was removed and transferred to a 15 ml corex tube. The RNA pellet was solubilized in 3 ml of RNA lysis buffer for 1 hour at 60 °C. After removal of the tube bottom, 6 ml of distilled water was added, followed by the addition of 18 ml of ethanol. The RNA was allowed to precipitate overnight at -20°C, and the precipitate was collected by centrifugation at 10,000 rpm for 10 minutes in a JA-10 rotor (Beckman Instruments) . The RNA was resuspended in 4 ml of 0.3 M sodium acetate, pH 5.2. The corex tube was washed with an additional 0.5 ml of 0.3 M sodium ace¬ tate pH 5.2. The RNA and wash solutions were combined and the RNA was precipitated by adding 10 ml of ethanol and letting the mixture remain at -20°C overnight. The RNA was pelleted by centrifugation as before and the pellet was washed with etha¬ nol, vacuum dried and resuspended in 400 microliters of dis¬ tilled water for a final volume of 500 microliters. The yield 9 of total cellular RNA from the 3 x 10 sporozoites was approxi¬ mately 800 micrograms . Poly(A) mRNA was isolated by oligo ( dT)-cellulose chroma- trography using standard techniques as described in Edmonds, M. et al . PNAS _68: 1336 (1971 ) . The total cellular RNA suspended in 1.2 ml distilled water was mixed with 300 microliters of 5X binding buffer (50 mM Tris-HCl, pH 7.5? 2.5 M NaCl) and passed through a 1 ml column of oligo ( dT)-cellulose (Type III, Collaborative Research, Lexington, MA) . The column was washed with 15 ml of IX binding buffer (10 mM Tris-CHl, pH 7.5; 0.25 M NaCl) and poly(A) mRNA was eluted by the addition of 3 ml of 10 mM Tris-HCl, pH 7.5. Fractions containing poly(A) mRNA were pooled, diluted with one-quarter volume of 5X binding buffer and passed through the rejuvenated oligo ( dT)-cellulose column. Fractions of the eluate containing poly (A) mRNA were pooled, mixed with one-tenth volume of 3 M sodium acetate, pH 5.2, and precipitated with ethanol overnight at -20°C. The RNA was collected by centrifugation in a SW 41 rotor for 30 minutes at 25,000 rpm. The RNA pellet was washed with ethanol, vacuum dried and resuspended in 40 microliters of distilled water. Approximately 13 micrograms of poly(A) mRNA were recovered. 2. Construction of the Lambda gtlO:Sporozoite cDNA Library Two micrograms of poly(A) mRNA were converted into cDNA using AMV reverse transcriptase (Life Sciences, St. Petersburg, FL) . The reaction was primed with oligo (dT.. ___.„) (Collaborative Research, Lexington, MA). A small molar amount of 32P-dATP was included m. the reacti.on to provi.de a tracer for subsequent steps. After 60 minutes at 42°C, the cDNA was separated from the unincorporated nucleotides and other small molecules by passage through a 1 ml column of Sephacryl S-300 using 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA. Fractions containing cDNA were identified by scintillation count, pooled, and recovered by ethanol precipitation. A string of dG resi¬ dues were added to the 3 ' ends of the cDNA using terminal deoxynucleotide transferase (PL Biochemicals) . At the comple¬ tion of this reaction, RNase A and oligo (dC, ___.„) (Collaborative Research) were added directly to the mixture which was then put in a boiling water bath for 45 seconds, cooled to room temperature and then put on ice. Second strand cDNA synthesis was performed by adding all four deoxynucleotides and E^_ coli DNA polymerase I to the reac¬ tion mixture and incubating the mixture at 15 °C for 18 hours. Double-stranded cDNA was then treated with EcoRI endonuclease. After phenol and ether extractions, the cDNA was passed through a. 1 ml Sephacryl S-300 column as described above. Fractions of eluate containing cDNA were pooled and the cDNA recovered by ethanol precipitation. The cDNA was ligated overnight to phosphorylated EcoRI linkers (New England Biolabs, Beverly, MA) and treated with several hundred units of EcoRI to digest excess linkers. The mixture was then passed through a 1 ml Sephacryl S-300 column and the fractions of eluate containing cDNA were pooled and precipitated with ethanol. The double-stranded cDNA was resuspended in a small volume of water and stored at -20°C. To clone the cDNA into the unique EcoRI site of lambda gtlO as described by Huyhn, Young, and Davis in DNA Cloning Techniques, A Practical Approach, Glover, D.M. [ed . ], IRL Press, Oxford, _irι press, lambda gtlO DNA was digested to com¬ pletion with EcoRI, ligated to cDNA overnight at 16°C and pack¬ aged _in vitro. Aliquots were plated on E^ coli C600 and C600 HflA to determine the number of total phage and the number of recombinant phage containing cDNA inserts. From serval liga- 7 tions and packagings, a total of 2 x 10 phage were recovered, 15-18% or approximately 3 x 10 of which contained cDNA in¬ serts . The phage in this library were amplified on E. coli C600 HflA to eliminate non-recombinant phage. The phage were plated 4 at a density of 5 x 10 per 150 mm plate, incubated at 37 °C until plaques appeared and overlaid with 12.5 ml of 10 mM Tris-HCl, pH 7.4; 10 mM MgS0 4 per plate. A total of 3.2 x 10 12 phage were recovered from the amplified library. Over 99% of the recovered phage were recombinants. 3. Moving the Lambda gtlO: Sporozoite cDNA Library Into the Expression Phage Lambda gtll DNA was obtained from the amplified lambda gtlO:sporozoite cDNA library by growing plate lysates of the phage. Sixty-four 5 150 mm plates were seeded with 1 x 10 phage per plate and 0.7 ml of an overnight culture of E^_ coli C600 which had been resuspended in one-half volume of 10 mM MgSO.. After lysis, each plate was overlaid with 12.5 ml of 10 mM Tris-HCl pH 7.4, 10 mM MgSO. and stored overnight at 4°C. Phage were purified from the liquid overlay by conventional procedures using poly¬ ethylene glycol precipitation and CsCl banding in a step gradi¬ ent with a final banding in a CsCl equilibrium gradient. The DNA was obtained from the phage by extraction with .formamide as described by Davis, R.W. , et al . in Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, New York, 1980). A total of 1.4 mg of phage DNA was recovered. The cDNA inserts were obtained from this phage DNA by digestion with EcoRI endonuclease and size-fractionated on sucrose gradients . 200 micrograms of phage DNA were digested with 1,000 units of EcoRI, phenol extracted and ethanol precip¬ itated. Approxiamtely 5% of the DNA was labeled at the EcoRI sites with. 32P-dATP and the Klenow fragment of E^ coli DNA polymerase 1 to serve as tracer. The DNA was suspended in a 1.00 mM NaCl solution, heated to 42°C for 5 minutes to anneal the phage arms, and size-fractionated on a 10-40% sucrose gra¬ dient made up in 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, and 1 M NaCl. The sucrose gradient was centrifuged in an SW 41 rotor at 15°C for 24 hours at 30,000 rpm. Fractions containing cDNA were identified by scintillation count, pooled, dialyzed against 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and precipitated with ethanol. The resuspended cDNA was centrifuged through a second 10-40_% " sucrose gradient using identical procedures except that fractions of the gradient were analyzed by gel electrophoresis and. autoradiography to determine cDNA size. Fractions contain¬ ing cDNA larger than 500 bp were pooled, dialyzed against 10 • mM Tris-HCl, pH 8.0, 1 mM EDTA, ethanol precipitated and resuspended in a small volume of water. Purified cDNA was ligated to EcoRI-digested and phosphatized (using calf intestinal phosphatase) lambda gtll DNA, and packaged in vitro■ Aliquots of the packagings were plated on E^ coli Y1088 (using the method described by Young and Davis in Science 222: 778-782 (1983)) in the presence of X-GAL and IPTG in order to determine the total number of phage and the number of recombinant phage. Total phage recovered from several ligations and packagings were 1 x 10 , of which greater than 90% were recombinants. A pαrction of this library (7 x 10 phage) was amplified on plates using: E. coli Y1088. The phage were plated at a density of " 4- x 10 4- ' per 150 mm plate. The final amplified library had a titer of. " 2..2. x 10 phage per ml. The amplified lambda gtll:sporozoite cDNA library was used as a source of phage for screening with antibodies and other probes as described below. 4. Determination of cDNA Size DNA from positive phage was purified from plate lysates using similar procedures as described above for obtaining DNA from the lambda gtlO:sporozoite cDNA library except that E. coli_ Y1088 was used. as the host and the number of phage plated per plate was 3 x 10 . cDNA sizes were determined by digesting 0.5 micrograms of phage DNA with EcoRI and analyzing the products by agarose gel electrophoresis. Commercially avail¬ able lambda DNA digested with Hindlll and 0X 174 DNA digested with Haelll served as size markers. II. GENERAL METHODOLOGY A. Antibody Screening of the Lambda GT11:Tenella Expression Libraries The lambda gtll:tenella expression libraries ( cDNA or genomic) were plated at densities of 1 x 10 4 through 2 x 105 phage per 150 mm plate using E_ coli Y1090 as host as described by Young and Davis in Science 222: 778-782 (1983). Plates were incubated at 37 °C or 42 °C for 4 hour, then overlaid with nitrocellulose filters (BA-85, Schleicher and Schuell) that had been soaked in 10 mM IPTG and air-dried. After incubating overnight at 37 °C, filters were batch-washed 3 x 10 minutes in TBS (10 mM Tris-HCl, pH 8.0? 150 mM NaCl) . Non-specific pro¬ tein binding sites on the filters were blocked by incubating filters for 60 minutes in TBS + 2% bovine serum albumin (Frac¬ tion V, Miles Laboratories, Elkhart, IN). The filters were then incubated individually or in pairs for 2 hour with 10-20 ml of primary antibody (e.g., immune chicken serum., hyperimmune rabbit anti-sporozoite serum, mouse monoclonal anti-sporozoite antibodies) typically diluted 1:200 (1:500 for monoclonals) with TBS to which had been added 2% bovine serum albumin (BSA). The filters were washed 3 x 10 min with TBS containing 0.1% NP-40 (obtained from Sigma) , then incubated singly or in pairs for 60 min with 10-20 ml solution of a second antibody (e.g., peroxidase-conjugated goat anti-rabbit IgG, Cappel La¬ boratories) diluted 1:500 in TBS + 2% bovine serum albumin. Filters were batch-washed 3 x 10 min in TBS and stained in a solution comprising 200 ml TBS, 2.5 ml hydrogen peroxide and 40 ml of a 3 mg/ml solution of 4-chloro-l'-napthol in methanol . Staining was quenched by removing the filters to water. Posi¬ tive staining plaques were subjected to several rounds of rescreening with antibody as described above until pure. B. Elution Of Antibodies Bound To Proteins Immobilized On Nitrocellulose Membranes When proteins are immobilized on nitrocellulose membranes such as Western blot transfers of proteins separated on poly- acrylimide gels or replicas of phage plaques taken from agar plates, it is possible to bind antibodies which specifically recognize the immobilized proteins. Antibodies which do not specifically bind to the immobilized proteins remain in solu¬ tion and can be easily washed away, leaving behind only those specifically bound. The bound antibodies can be eluted by rinsing the filters in a low pH buffer (5 mM glycine, pH 2.3, 0.5 M NaCl, 0.5% Tween 20, 0.01% BSA), which dissociates the antibody—antigen complex. If the eluted antibodies are immediately neutral¬ ized,i.e., using a 50 mM Tris-HCl, final concentration, they retain full activity and can be used for a variety of analyti¬ cal purposes. Eluted antibodies have been used to screen the recombinant expression libraries and determine the sporozoite protein that corresponds to a _given insert clone using the methods that follow. 1. Screening Recombinant Expression Libraries With Eluted Antibodies Antibodies eluted from specific bands on a Western blot were used to screen recombinant expression libraries in order to find recombinant clones expressing antigens corresponding to the protein band on the Western blot. This technique allowed isolation of a number of the insert clones. In order to obtain enough antibodies to screen the li¬ brary, a Western blot was prepared from SDS-polyacrylimide gel consisting of a single wide lane of site functional E. tenella sporozite proteins. After non-specific protein binding sites _ on the nitrocellulose were blocked using 2% BSA in TBS, 1 hour at room temperature, the blot was washed 3 x 10 minutes in TBS at room temperature. Polyclonal rabbit anti-sporozoite serum diluted 1:50 in TBS + 2% BSA was bound to the blot overnight at room temperature. Unbound antibodies were removed by washing in TBS + 0.2% Tween 20 (Sigma) . A thin strip was cut from each side of the blot for staining in order to indicate the positions of bound antibodies on the central part of the blot. Second antibody, peroxidase- conjugated goat antirabbit IgG (Cappell Laboratories)? 1:500 in TBS + 2% BSA was bound to the strips. The strips were washed 2 x 10 minutes in TBS + 0.2% Tween 20 followed by a 10 minute wash in TBS) and subsequently stained for 20 minutes at room temperature in 50 ml TBS + 10 ml 4-chloro-l-naphtol (3 mg/ml in methanol) + 0.6 ml 3% H- 2 °2 ' The stained strips were lined up with the main body of the blot, and strips were cut in the main blot which corresponded to the desired bands on the side strips. The main blot strips were placed in long polypropylene sealable test tubes. A small volume ( 0.5 ml) of elution buffer (5 mM glycine, pH 2.3? 0.5 M NaCl, 0.5% Tween 20? 0.01% BSA) was added to each tube and the tubes were rocked end to end to allow the buffer to wash along the filter strip. After 30 seconds, the buffer was removed and immediately neutralized with a 1/20 volume of 1 M Tris-HCl, pH 7.4. The elution was repeated two more times for 30 seconds each with fresh elution buffer. All three washes were pooled. The eluted antibodies were diluted in TBS + 0.2% Tween 20 to an appropriate volume for screening the recombinant libraries, as described in Section IIA, infra. 2. Determination of Sporozoite Protein Corresponding to Insert Clone Antibodies eluted from plaque replicas of a purified recombinant clone were used to determine which sporozite pro¬ tein corresponded to that clone. By eluting antibodies bound to plaque replicas of the recombinant clones and using those antibodies to probe Western blots of sporozite proteins, it was possible to determine which protein was encoded in the recombinant clone. Five thousand to ten thousand plaques from a single purified recombinant clone were plated on a 100 mm plate, and a plaque lift was made as described in Section IIA, supra. The lift filters were washed 3 x 10 minutes in TBS and non-specific protein binding was blocked by incubation in TBS + 10% BSA for 1 hour. The filters were washed 3 x 10 minutes in TBS. A strip 5 mm wide was cut from the center of the filter disc. Polyclonal anti-sporozite serum was bound to the strip, washed and eluted as described for the Western blot elutions in Sec¬ tion III.A.l above. The eluted antibodies were then used to probe a Western blot of sporozite proteins. III. CONSTRUCTION OF THE LAMBDA EMBL3::EIMERIA TENELLA GENOMIC LIBRARY The construction of a lambda EMBL3: :E. tenella genomic DNA library was accomplished in five steps. First, _in vitro bac- teriophage lambda packaging extracts were prepared for effi¬ cient cloning of the recombinant phage. Second, lambda EMBL3 vector DNA was prepared. Third, preparation of fragmented E. tenella genomic DNA was accomplished by partial restriction endonuclease cleavage and size selection. Fourth, the E. tenella DNA was ligated to the lambda EMBL3 cloning vector and packaged _in vitro. Finally, the _ln vitro packaged recombinant phage were amplified on a recombinant selective host strain. A. Lambda in vitro Packaging Reagents Bacteriophage lambda in vitro packaging extracts from E. coli lysogens BSB2690 (Dam prehead donor) and BHB 2688 (Earn packaging protein donor) were prepared and used as described by Sternberg et al. in Gene _1: 255 (1977). In some experiments, lambda in vitro packaging extracts were purchased from Boehringer Mannheim and used according to the manufacturer's directions. The strains were grown separately, induced by temperature shift, and concentrated by centrifugation. Cells were broken by sonication (preh.ead donor) and by lysozyme treatment fol¬ lowed by rapid freezing and thawing (packaging protein donor) . After centrifugation to remove cellular debris and DNA, the extracts were frozen in small aliquots. Packaging was then accomplished by adding lambda DNA to a mixture of both extracts followed by incubation at • room temperature. Using various com¬ binations of extracts, a packaging efficiency of 4 x 10 plaque-forming units per microgram of lambda DNA was obtained. This efficiency was found to be adequate for library construc¬ tion. B. Preparation of Lambda EMBL3 Vector DNA Lambda EMBL3 is a replacement vector with a nominal cloning capacity of 9 to 20 kb (77 to 105%) of wild-type lambda. A lysate of lambda EMBL3 was obtained from Dr. Matt Scott (MCDB, University of Colorado) . EMBL3 allows for a dou¬ ble selection against non-recombinant phages. First, there is a genetic selection against religated vector by amplifying the library on a P2 lysogenic host E. coli (Q359), as described by Karn et al . in Proc. Nat. Acad. Sci. 2Z : 5172 ~ 5176 (1980). E. coli Q359 is non-permissive for intact spi EMBL3 phage. Sec¬ ond, EMBL3 allows a biochemical selection against the ligation event reinserting the phage middle fragment into the phage by double cleavage with BamHI and EcoRI and removal to the small linker fragment by isopropanol precipitation. Lambda EMBL3 phage stock was prepared essentially as described by Davis, R.W. , et al ■ in Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, New York, 1980) using the plate lysis method. E. coli Q358 cells were grown overnight in L-broth + 0.2% maltose and harvested by centrifugation. The pelleted Q358 were resuspended in 10 mM MgCl„ and lambda EMBL3 phages were added to the cells at a multiplicity of infection of 1. The phage were allowed to adsorb to the cells for 30 minutes at room temperature, mixed with 7.5 ml of HT soft agarose and plated on a 150 mm HT plate. Ten plates were prepared in this manner and the plates were incubated at 37°C for 5 hours, at which time confluent lysis had occurred. The plates were placed at 4°C, and each was overlayed with 12.5 ml of cold lambda dil (10 mM MgCl ? ; 10 mM Tris-HCl, pH 7.4 ? 1 mM EDTA) and incubated overnight. The overlay solutions were then pooled and centrifuged in a Beckman JA-20 rotor at 10,000 rpm for 10 minutes at 4°C to remove cel¬ lular debris. The phage were then pelleted from the cleared lysate by centrifugation at 20,000 rpm in a JA-20 rotor for 3 hours at 4°C. The phage pellet was resuspended in lambda dil and lay¬ ered atop a CsCl gradient prepared in an SW 41 (Beckman Instru¬ ments) centrifuge tube. The gradient was spun at 35,000 rpm for 60 minutes at 15°C. The phage band was removed from the gradient by extraction with a syringe and then mixed with an equal volume of 1.7 g/ml CsCl. This solution was dispensed un¬ derneath another CsCl gradient in an SW 41 tube and then cen¬ trifuged at 35,000 rpm for 60 minutes at 15°C. The resultant phage band was removed with a syringe and the contents placed into dialysis tubing. The phage were dialyzed against 50% formamide, 0.1 M Tris-HCl (pH 8.5) for 24 hours at room temper¬ ature to release the phage DNA from the intact bacteriophage. The released DNA was dialyzed versus 0.1 M NaCl, 50 mM Tris-HCl (pH 7.5), and 1 mM EDTA for 16 hours with four changes of buffer in order to remove the formamide. The purified DNA was then removed from the dialysis membrane and stored at 4°C in a test tube. The DNA concentration of this preparation was determined by absorbance at 260 nm and gave a yield of 120 mg/ml. Purified lambda EMBL3 DNA was cleaved with the restriction endonuclease BamHI. After digestion, the BamHI enzyme was inactivated by adding EDTA and heating the reaction mix at 70°C for 10 minutes. The DNA solution was adjusted to 0.3 M sodium acetate and 2.5 volumes of ethanol were added to precipitate the DNA. The DNA was recovered by centrifugation, and the pel¬ let was resuspended in TE buffer as described in Section I.A1 (page 37), supra. The BamHI-cleaved DNA was then cleaved with EcoRI at 37°C for 60 minutes . The EcoRI was inactivated with EDTA, and the resultant mixture was then extracted with an equal volume of phenol. Residual phenol was removed by ether extraction of the aqueous phase. The doubly-cleaved DNA was then separated from the small EcoRI-BamHI polylinker sequences by precipitation of the mixture with isopropanol. The resultant pellet was resuspended in buffer and reprecipitated with ethanol. The DNA was then resuspended in 1/10 TE buffer and used for library construction. C. Preparation of Fragmented E. tenella Sporozoite Genomic DNA The bacteriophage lambda EMBL3 is designed to accept recombinant DNA inserts with cohesive termini that are compati¬ ble with termini generated by cleavage with BamHI, i.e., termini with end structures of 5 '-GATC. Sau3A is a restriction endonuclease that recognizes the tetranucleotide sequence 5'-GATC in DNA and cleaves on the 5' side of the G residue, generating a cohesive end appropriate for the EMBL3 BamHI cloning site. E. tenella sporozite DNA prepared as described above was used in small-scale digests with Sau3A to empirically determine the reaction conditions that would generate approximately 20 kb partial digestion products. These conditions were then scaled up and 100 ug of sporozoite DNA was partially digested with Sau3A, and the enzyme was immediately inactivated by phenol extraction of the reaction mix. After the phases were sepa¬ rated by centrifugation, the aqueous phase was removed with a large bore pipetter and residual phenol removed by chloroform extraction. The sample was adjusted to 0.3 M Sodium Acetate (NaOAc) with a concentrated stock solution, and 2.5 volumes of ethanol were added to precipitate the DNA. The precipitated DNA was concentrated by centrifugation and the pellet resuspended in TE buffer. This DNA was layered on top of a 10-40% sucrose gradient preformed in an SW28 (Beckman) centri¬ fuge tube. The gradient was centrifuged at 26,000 rpm at 20°C for 24. hour . The gradient was fractionated by pumping liquid from the bottom of the tube through a glass capillary in 0.5 ml ali¬ quots. The fractions were analyzed by gel electrophoresis to determine which aliquots harbored the appropriately sized frag¬ ments (15-20 kb) . Those fractions were pooled, placed into a dialysis membrane and dialyzed against TE buffer. After dialysis, the sample was extracted 3 times with an equal volume of butanol to concentrate the sample.. The salt concentration was adjusted to 0.3 M with sodium acetate, and the DNA precipi¬ tated with 2.5 volumes of ethanol. The DNA was concentrated by centrifugation, rinsed with 70% ethanol, and resuspended in a small volume of TE buffer. This DNA was analyzed by gel electrophoresis to confirm that it was of appropriate size (15-20 kb) . The yield was 500 ng . D. Ligation of Fragmented E. tenella DNA to EMBL3 Vector and in vitro Packaging of the Recombinant DNA Lambda EMBL3 DNA cleaved with BamHI and EcoRI and purified as described above was mixed at a 2:1 mass ratio with sized E. tenella genomic DNA prepared by partial Sau3A digestion. These DNAs were ligated with T4 DNA ligase overnight at 14°C. A small aliquot was analyzed by gel electrophoresis to monitor the ligation reaction. After brief incubation at 70°C, the li¬ gation reaction was mixed with the in vitro packaging extracts and incubated for 60 minutes at room temperature. Chloroform was then added to prevent bacterial growth and the phages were ready for amplification. E. Amplification of the E. tenella Library Packaged phage were diluted with lambda dil and adsorbed to fresh E. coli Q359 plating cells. The cells were mixed with NZC soft agarose and plated on NZCYM plates (described by Ma¬ niatis, T. et al . , in Molecular Cloning, Cold Spring Harbor Laboratory, New York, (1982). Plates were incubated at 37°C for 10 hours. The complexity of the library was determined by counting plaques in representative small regions of the plates. The total yield of recombinants was 150,000. The recombinant phage were eluted from the primary amplification plate by overlayering with lambda dil. The titer of the amplified li- 7 brary was 3 x 10 plaque forming units (pfu) per ml. The back¬ ground of nonrecombinants in the library was determined by dif¬ ferential plating of the amplified library on E. coli Q359, which allowed only recombinant phages to form plaques on E. coli Q358 which allows both recombinant and nonrecombinant phages to form plaques, and on E. coli KRO which allows only nonrecombinant phages to form plaques. (Karn, _J.M. et al. 1980 PNAS ll Frischauf, A. et al . (1983), J. Mol. Biol. 170) E. coli Q358 and lambda EMBL3 from Dr. D. Hirsh, E. coli Q359 and KRO from Dr. M. Scott, both of MCDB University of Colorado. The background of nonrecombinant phages in the amplified li¬ brary was less than 0.01% of the plaques. Analysis of randomly selected recombinant phages indicated that the average size of the E. tenella DNA inserts was 17 kb. IV. PLASMID VECTORS FOR EXPRESSION OF FUSION PROTEINS A number of antigenically reactive E. tenella recombinant phage clones were identified in the sporozite cDNA expression library and the "small fragment" E. tenella genomic expression library. Since the lambda gtll lysogens appeared to make a limited quantity of fusion protein, a plasmid expression vector was constructed that would produce the fusion proteins in mil¬ ligram quantities. A. pSEV4 Plasmid pLG2 was obtained from Dr. L. Guarente (MIT) (Guarente, L. , in Cell, 20:543-553 (1980)). This vector is a pBR322 derivative which, like lambda g+11, has lac operator and promoter sequences in addition to a wild-type beta-galactosidase gene containing a single EcoRI site near the 3' end of the gene. In addition, pLG2 contains the lac re- pressor gene. Moving E. tenella DNA inserts from lambda gtll into the EcoRI site of this vector yields a fusion protein identical to that initially identified in the phage. Plasmid pLG2 was modifie ' d to remove an extra EcoRI site prior to its use for expression. Plasmid pLG2 was partially digested with EcoRI restriction endonuclease to linearize the plasmid. The plasmid DNA was then displayed on a preparative agarose gel and the linear-sized DNA band was eluted from the gel. The eluted DNA was precipitated by making the solution 0.3 M with sodium acetate and adding 2.5 volumes of ethanol. The DNA was pelleted by centrifugation and the pellet was resuspended in TE buffer. The Klenow fragment of E. coli DNA Polymerase I was mixed with the DNA in the presence of dATP and dTTP to fill in the EcoRI cohesive ends. After heat inactivation at 70°C for 10 minutes, T4 DNA ligase (P.L. Bio¬ chemicals) ' was added and the mixture was incubated at 4°C for 16 hours. The ligated DNA was then used to transform E. coli AMA1004. (Casadaban, M. , et al . , in Methods in Enzymology 100:293 (1983). Transformants were selected on ampicillin plates in the presence of a chromagenic substrate for beta-galactosidase activity (X-GAL) . Transformants with beta-galactosidase activ¬ ity were screened by cleaving the DNA with EcoRI. A plasmid, pSEV4, that had only a single EcoRI site near the carboxyl ter¬ minus of the beta-galactosidase gene was identified from the transformants and characterized. Plasmid pSEV4 has a unique EcoRI site near the carboxyl terminus of the beta-galactosidase gene. Plasmid pSEV4 con¬ tains the wild-type lac operator, promoter and repressor in addition to the beta-galactosidase gene. Upon induction with IPTG for 60 minutes, beta-galactosidase activity was increased by 300—fold. Uninduced cells containing pSEV4 produced approx¬ imately 1000 units/mg of total cellular protein, whereas IPTG- induced cells gave approximately 300,000 units/mg of total cell protein. Protein gel analysis of induced and uninduced cells also showed the overproduction of beta-galactosidase by induced cells. This new plasmid, pSEV4 (see Fig. 10), has been used to express a number of E. tenella antigens as fusion proteins. B. pSEV6 A successor to pSEV4 was constructed to allow polarized "cassette" subcloning of DNA inserts from lambda gtll directly into a plasmid expression vector. Because EcoRI inserts could be subcloned in either orientation in pSΞV4, each pSEV4 subclone for its antigenic reaction. Polarized subcloning using pSEV6 obviates the need for this extra analysis. Extensive mapping of pSEV4 located five restriction endo¬ nuclease sites in the lac operon 5 ' to the beta-galactosidase gene's unique EcoRI site. Three of these sites are unique and two were made unique by deletion of the superfluous DNA between the lacl gene and the pBR322-deπved amp gene. Only one use¬ ful restriction site was found 3' to the EcoRI site, the Ncol site, therefore, additional restriction enzyme sites were in¬ serted in this region using a chemically synthesized poly- 1inke . The construction of pSEV6 was done in two steps. First, pSEV4 was shortened by approximately 5,700 bp to eliminate su¬ perfluous DNA. Plasmid pSEV4 was cleaved with Sphl restriction endonuclease, and the enzyme was inactivated by heating at 70°C for 10 minutes. The DNA was then partially digested with Aatll and the resulting digest was displayed by electrophoresis on a preparative agarose gel. The 7,620 bp fragment was excised from the gel and electroluted. The electroluted DNA was precipitated from a 0.3 M sodium acetate solution by adding 2.5 volumes of ethanol and incubating at -70°C for 30 minutes. The DNA was concentrated by pelleting in a Brinkman micro-centrifuge for 15 minutes and the pellet was resuspended in TE buffer. T4 DNA polymerase (New England Biolabs) was added to blunt-end the cohesive ends generated by the Aat.II digest. After heat inactivation of the T4 DNA polymerase, T4 DNA ligase (P.L. Biochemicals) was added to ligate the blunt ends of the DNA fragment. The ligated DNA was used to transform competent AMA1004 E. coli cells. Lac transformants were screened for the 7,620 bp plasmid. One plasmid, pSEV5, was identified and characterized as having the appropriate structure. DNA from pSEV5 was purified by standard methods, and sub¬ sequently cleaved with the restriction endonuclease Ncol, which cleaved at a unique site 3' of the beta-galactosidase gene. An oligonucleotide adapter molecule that would regenerate the Ncol site and which also contained Bglll and Kpnl sites was chemi¬ cally synthesized. This oligonucleotide was ligated to the Ncol cleaved pSEV5 with T4 DNA ligase (P.L. Biochemicals) . The ligated DNA was used to transform competent E. coli AMA1004 cells. DNA from the resulting lac transformants was screened for the presence of the unique Kpnl and Ncol sites. A plasmid was identified from this screening with all of the designed sequences. This plasmid, pSEV6 (see Fig. 11), has been used for the expression of various of the antigenically reactive fusion proteins. V. PURIFICATION OF THE CLONED ANTIGENS FOR ANIMAL TESTING The beta-galactosidase: :E. tenella antigen fusion proteins have been purified either by use of a substrate analog affinity column for beta-galactosidase or by classical methods of pro¬ tein purification. A. Preparation of Extracts Two liters of Luria broth, pH 7.5, described by Ma¬ niatis, T. et al . , in Molecular Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982) containing 50 ug/ L of ampicillin were inoculated with 10-20 ml of an over¬ night culture of E. coli AMA1004 containing one of the recombinant plasmids. The cells were allowed to grow at 37°C to mid-log phase ( , 00 = 0.2). Isopropyl-thiogalactoside (IPTG) was added to a final concentration of 1 mM to induce formation of the fusion protein. The cells were allowed to grow out for 2 hours, and then harvested by centrifugation at 5000 x G for 15 minutes at 4°C. All subsequent operations were carried out at 4°C. The cells were resuspended in 20 mL of breaking buffer (50 mM Tris-HCl, pH 7.5? 250 mM NaCl? 10 mM MgCl 2 ? 5% glycerol? 1 mM phenymethyl sulfonyl fluoride (PMSF) ) at 4 β C and centrifuged again at 5000 x G for 15 minutes. The cells were again sus¬ pended in 20-40 mL of breaking buffer (See Section VA (page 67), supra. ) . The cells could be frozen at this point and stored at -20°C if desired. The unfrozen or thawed cells were broken with two passes through a French pressure cell (Aminco) at 20,000 psi. Cell debris was removed by centrifugation at 30,000 x G for 30 minutes . Further clarification of the extract could be obtained at this point by ultracentrifugation at 100,000 x G for 30 minutes. The fusion protein was then precipitated by the addition of ammonium sulfate to a final concentration of 20 to 40% saturation. The optimal concentration of ammonium sulfate required for precipitation of the fusion protein varies with the individual protein and must be determined experi¬ mentally using, for example, procedures set forth in Heppel, L. in Methods in Enzymology, 1:570-576 (1955). The precipitate solution was stirred for one hour and the precipitate was removed by centrifugation at 30,000 x G for 15 minutes. The pellet was redissolved in 10 to 15 ml of starting buffer (50 mM Tris-HCl, pH 7.5? 250 mM NaCl? 10 mM MgCl-; 1 mM dithiothreitol (DTT)? 0.1% Triton X-100), and then dialyzed overnight against 500 ml of starting buffer. 2. Affinity Purification Procedure The use of a beta-galactosidase affinity column is based on the methods described by Steers and Cuatrecasas in Methods in Enzymology, 34:350-358 (1974). Affinity resin (p-amino- phenyl-beta-D-Thiogalactopyranoside-agarose, obtained from Sigma) was packed into a 1.5 cm diameter by 15 cm column. The column was washed with 10 column volumes of starting buffer of 50 M Tris-HCl (pH 7.5), 250 mM NaCl, 10 mM MgCl_, and 1.0 mM dithiothreitol (DTT) and 0.1% Triton-XlOO before use. The col¬ umn can be regenerated after use by washing extensively with elution buffer 0.1 M sodium borate (pH 10), 250 mM NaCl, 1 mM DTT or by washing with 6M guanidine hydrochloride in 50 mM Tris-HCl, pH 7.5. After washing, the column is reequilibrated with 10 column volumes of starting buffer. For affinity chromatography, dialyzed material was applied to the pre-equilibrated affinity column at a flow rate of about 0.2 ml/min. After the sample was applied to the column, the column was washed with 15 ml of starting buffer at the same flow rate, then with 30 ml of starting buffer at about 0.5 ml/min followed with 180 ml starting buffer at about 1 ml/min. Finally, the column was washed with 120 ml of starting buffer without Triton X-100 at the same flow rate. The absorbed protein was eluted with 0.1 M sodium borate, pH 10.0? 250 mM NaCl? and 1 mM DTT using 120 ml at a flow rate of about 1 ml/min. The peak-protein containing fractions are pooled and could be concentrated if desired to about 10 ml using an Amicon ultrafiltration device (Model 8050) containing an YM-30 membrane. 3. Ultracentrifuge Purification An alternative purification useful for some of the fusion proteins (e.g., pSEV4: :CH2-13 ) is accomplished by obtaining dialyzed protein as set forth above. The dialyzed material is subjected to ultracentrifugation at 100,000 x G for 30 minutes. The pellet containing the bulk of the fusion protein is redissolved in a small volume of dialysis buffer 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 10 mM MgCl,, and 1.0 mM DTT and 0.1% Triton-XlOO. This method yield's material that is not as pure as that generated by the affinity column when judged by SDS- polyacrylamide electrophoresis. 4. Analysis The purified materials obtained by these- methods were ana¬ lyzed for protein by the Bio-Rad (Richmond, CA 94804) protein method described by the manufacturers. They were also sub¬ jected to analysis by SDS-polyacrylamide electrophoresis. These gels are visualized either by protein staining or by Western blot analysis. Protein staining was typically done with either the silver stain method as described by Wray, W.P. et al . in Anal. Biochem. 118:197-203 (1981) or the Bio-Rad pro¬ tein stain method. Western blot analysis is carried out as described by Renart, J. et al. , in PNAS (USA) 76:3116 (1979). The Western blot analysis involves electrophoretic trans¬ fer of the resolved protein bands to nitrocellulose, blocking the nitrocellulose paper with BSA, probing with a specific antibody (either anti-betagalactosidase or anti-sporozite sera) . After washing, the blots are probed with the appropri¬ ate peroxidase conjugated second antibody, followed by color development using the peroxidase catalyzed reaction. VI. PROCEDURES FOR OBTAINING THE ENTIRE E. tenella GENE ENCODED BY A cDNA OR SMALL GENOMIC FRAGMENT Many of the recombinant E. tenella clones that are re¬ active with serum from immune animals probably contain only a portion of the entire coding region for a particular polypep¬ tide. The small fragment genomic expression clones were frag¬ mented intentionally so that the antigenic polypeptide is on the order of 100 or so amino acids in length. Most of the "small fragment" clones encode polypeptides larger than 100 amino acids. The E_. tenella sporozoite cDNA clones may encode only a portion of the gene because the mRNA used to obtain the clone was truncated. The cDNA may have been shortened because the mRNA that was isolated was not full length, or the full-length cDNA contained a naturally occurring EcoRl restriction endonuclease site. In the latter situation, the lambda gtll cDNA expression library was obtained by moving the cDNA from lambda gtlO into gtll by cleaving the cloned cDNA with EcoRl. In some cases, it may be important to clone the entire coding sequence of a given antigenic E_. tenella polypep¬ tide in order to maximuze the immune response, or modulate the response. Consequently, large E. tenella genomic lambda EMBL3 recombinant phages have been isolated that encode the entire gene. Briefly, the antigenic E_. tenella cDNA or "small fragment" E. tenella genomic fragment is subcloned into the EcoRl site of the E . coli plasmid pBR325, described by Prentki,P. , et al.[1981] in Gene _14, 289, specifically incorporated hereain by reference. Plasmid DNA is then prepared from the chloramphenicol resistant transformant. The DNA is then 'nick translated' using E. coli DNA polymerase to incorporate 32 P-labeled deoxyn — ucleotides into the DNA. This labeled DNA is then used as a radioactive probe to select an homologus _E. tenella sequence from the large genomic recombinant lambda EMBL3 library. Phage selected from the recombinant library by this method are plaque purified, DNA is prepared, and the E. tenella specific insert is mapped with various restriction endonucleases . The resulting map is compared with a similar map derived from the initial clone to confirm the identity of the large genomic phage. Because of the presence of introns in some of the genes that have been characterized, it is not certain that the entire coding region is contained within the large genomic phage. However, given the deposited phage, the restriction map, and the E . tenella large genomic library in lambda EMBL3, it is possible for anyone skilled in the art to obtain the entire gene. This is done by "walking" along the E. tenella genome by isolating phage that contain flanking E. tenella genomic DNA using the method described by Bender, W. et al . , in JMB 168: 17-33 (1983), specifically incorporated herein by refer¬ ence. VII. PROCEDURES FOR ISOLATING LAMBDA EML3 E. tenella GENOMIC PHAGE THAT CONTAIN THE REPEATING TRINUCLEOTIDE CAG E_. tenella genomic and cDNA lambda gtll expression phage that encode polypeptides F, G and M contain repetitions of the trinucleotide 'CAG, and consequently these phage all cross- hybridize with each other at the DNA level even though they encode different polypeptides. Because of this repeating structure, probing the lambda EMBL3 E_. tenella library with the insert subcloned into pBR32'5 will yield many unrelated phage that happen to contain the repeating structure. In order to obtain the entire gene for polypeptides containing the repeat, a different selection is required that makes use of synthetic oligonucleotides . This method has been used to obtain lambda ET: :CHAD—19 which encodes polypeptide G. The method described below may also be used to isolate the entire gene for any of the polypeptides that contain the repeat. Once it has been determined that a given lambda gtll E. tenella expression clone contains the repeating CAG trinucleotide (by DNA cross-hybridization or by hybridization with a synthetic CAG containing oligonucleotide) the phage DNA is cleaved with EcoRl and the E . tenella insert is subcloned into the M13 mp8 or mp9 bacteriophage, described by Messing et al.in Gene _19_: 269-276 (1982), specifically incorporated herein by reference, and single stranded DNA is prepared from E. coli strain JM103 that has been transfected with the recombinant phage. This DNA serves as a template for the dideoxy DNA sequencing method developed by Sanger, F. et al. as described in PNAS(USA) 74:5463-5467 (1977), specifically incorporated herein by reference. Using standard procedures the DNA se¬ quence for the E . tenella insert was obtained for CHAD-19. The sequence consists of the 255 nucleotides shown below. Of the 85 residues encoded by the clone, there are 30 glutamine residues, and the CAG codon is used for 24 of the residues. 30 60 GAA TTC CCG ATG CCG CAA AAG CAG CAG CAG CAG CAG ACG AAG CCG ACG CAG CAA AGA ATG Glu Phe Pro Me Pro Gin Lys Gin Gin Gin Gin Gin Thr Lys Pro Thr Gin Gin Arg Met 90 120 CAG CAG CAG AGG CAG CAA AAG ATG AAG CTG CTG CGG GGA AAG CTG ATG CAG CAA AGG ATG Gin Gin Gin Arg Gin Gin Lys Met Lys Leu Leu Arg Gly Lys Leu Met Gin Gin Arg Met 150 180 CAG CAG CAG AAG CAG AAG ACG CTG CAG CAG CAG CAC CAA CAA AGO CGG AGG CCT CGG GCA Gin Gin Gin Lys Gin Lys Thr Leu Gin Gin Gin His Gin Gin Ser Arg Arg Pro Arg Ala -_J I 210 240 GCC CGG ATG ACC CGT CTG CAG CAG CAG CGG CAG CAG CAG TCT GAC GCA GAT GAG AAG CAA Ala Arg Met T Arg Leu Gin Gin Gin Arg Gin Gin Gin Ser Asp Ala Asp Glu Lys Gin AAG TTC GCG GAA TTC Lys Phe Ala Glu Phe Fro the DNA sequence of the E_. tenella insert two regions were selected that flank the repeating sequence and appear to be unique. For CHAD-19, nucleotides 163-195 [CH-1] and nucleotides 220-249 [CH-3] were selected. These regions were then checked against the published nucleotide squence of bacteriophage lambda to make sure that no significant regions of homology existed between the selected regions and the lambda EMBL3 cloning vector. Single-stranded DNA sequences corre¬ sponding to each of the two regions, CH—1 (33 nucleotides) and CH-3 (30 nucleotides), were then chemically synthesized using the solid phase system of Applied Biosystems Inc. The oligonucleotides were purified by polyacrylamide gel electro- phoresis, and end-labeled with 32P-ATP using polynucleotide kinase (P.L. Biochemicals). The lambda EMBL3 E_. tenella genomic library was then plated out on the Q359 E_. coli strain at a density such that 4 x 10 phage plaques are present on 20 large petri plates. When the plaques were about 2 mm in diameter, they were lifted onto nitrocellulose filters using the method of Benton and Davis, in Science 196:180 (1977). Two replicas of each plate were made by sequential nicrocellulose lifts. The phage DNA that was transferred to the filters was denatured, neutralized, and. baked. Each pair of the processed filters was then sepa- rately probed with each of the 32P-labeled synthetic oligonucleotides corresponding to the unique regions of the clone (CH-1 and CH-3) using stringent hybridization and wash conditions. The dry filters were exposed to X-ray film which was developed after a 24-hour exposure. Correct lamba EMBL3 E. tenella clones were identified by hybridization of both probes to the same plaque. These genomic phage were then purified, processed and mapped as described above. It is desired in these types of experiments that at least two probes be used in order to eliminated false-positives . By screening 4 x 10 EMBL3::E. tenella, a genomic lambda ET::CHAD-19 clone that cor¬ responds to the CHAD-19 cDNA expression clone was obtained. A map of the lambda ET::CHAD-19 phage is contained in Fig. 4. It will be apparent to those skilled in the art that vari¬ ous modifications and variations can be made in the processes SUBSTITUTE SHEET of the present invention. Thus, it is intended that the pres¬ ent invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents . "UTESHEET

Claims: What is claimed is: 1. Protein A, a 120 kd protein of Eimeria tenella, pro¬ duced by recombinant-DNA methods. 2. Protein B, an 100 kd protein of Eimeria tenella, pro¬ duced by recombinant-DNA methods. 3. Protein C, a 200kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 4. Protein D, an 80 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 5. Protein E, an 80 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 6. Protein F, a 45 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 7. Protein G, a 30 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 8. Protein H, a 25 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 9. Protein I, a 14 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 10. Protein J, a 10 kd protein of Eimeria tenella, pro¬ duced by recombinant DNA methods. 11. Protein K of Eimeria tenella, produced by recombinant DNA methods. 12. Protein L of Eimeria tenella, produced by recombinant DNA methods. 13. Protein M of Eimeria tenella, produced by recombinant DNA methods. 14. Protein N of Eimeria tenella, produced by recombinant DNA methods. 15. The protein of claim 7 wherein said protein comprises at least 5 glutamine residues . 16. The protein of claim 15 wherein said protein com¬ prises at least 21 glutamine residues . 17. The protein of claim 16 where said protein comprises a at least 45 glutamine residues. 18. The phage lambda ET::Rbl-17. 19. The phage lambda ET::Rbl-8. 20. The phage lambda ET::CH2-13. 21. The phage lambda ET::Rbl-19. 22. The phage lambda ET::Rbl-24. 23. The phage lambda ET : :CH2-15. 24. The phage lambda ET : : GRS-74. 25. The phage lambda ET : :CHAD-19. 26. The phage lambda ET::Cal-2a. 27. The phage lambda ET::Cal-3gn. 28. The phage lambda ET::Cal-4cn. 29. The phage lambda ET::CH2-3. 30. The phage lambda ET::Rbl-145. 31. The phage lambda ET::X. 32. A recombinant DNA method for the manufacture of poly¬ peptides analogous to Eimeria polypeptides which are capable of inducing an antigenic response comprising: (a) preparation of a DNA sequence capable of directing a host microorganism to produce a polypeptide pos¬ sessing antigenic properties analogous to these pos¬ sessed by a polypeptide produced by Eimeria organ¬ isms? (b) cloning the DNA sequence into a vector capable of being transferred into and replicating in a host mi¬ croorganism, such vector containing operational ele¬ ments for the DNA sequence; (c) transferring the vector containing the DNA sequence and operational elements into a host microorganism capable of expressing the antigenic polypeptides? (d) culturing the host microorganism under conditions appropriate for amplification of the vector and expression of the polypeptide? and (e) in either order: (i) harvesting the polypeptide? and (ii) causing the polypeptide to assume a structure whereby it possesses antigenic properties analo¬ gous to properties possessed by polypeptides produced by Eimeria organisms .

Inventors: COX GEORGE N, MCDONELL MICHAEL, COX, GEORGE, N., MCDONELL, MICHAEL
Applicants: SYNERGEN INC, SYNERGEN INCORPORATED
Application year: 1988
Main classification: C12P21
IPC codes: A61K39, C07K1, C07K14, C07K19, C12N15, C12P21, C12R1, G01N33