SHUTTLE VECTOR

Abstract: Described is a shuttle vector containing a yeast derived DNA and a E. coli derived DNA and carrying the expression control region of acid phosphatase gene of yeast, which can be recombined with various genes under control of the phosphatase promoter, to give various recombinant plas­ mids. The shuttle vector is useful in genetic engineering industries.

Description: SHUTTLE VECTOR The present invention relates to a novel shuttle vector. Mbre particularly. it relates to a shuttle vector which contains a yeast derived DNA ard an Escherichia coli derived DNA and carries the expression control region of the repressible acid phosphatase (said region being hereinafter referred to as "acid phosphatase promoter or "acid phosphatase gene"), and can replicate both in E. coli and in yeast. With recent active studies on genetic engineering, various recombinant DNAs and transforittants therefrom have been developed. In the preparation of the recombinant DNAs, a vector for inserting a specific gene is used. Such a vector includes a vector which can replicate only in a certain microorganism, e.g. in Escherichia coli, and a so-called shuttle vector which can replicate in two or more kinds of microorganisms, e.g. both in E. coli and in a yeast, or both in a certain microorganism (e.g. E. coli) and in a certain animal cell. For instance, there is very recently reported a shuttle vector which can replicate both in E. coli and in eyeast: a vector utilizing the alcohol dehydrogenase (ADH1) expression control region to achieve downstream promotion of the HBsAg structural gene (cf. Nature, 298,347- 350 (22 July, 1982)) YasQver, the shuttle vector used in this rethod carries an ADHl promoter and, when a recombinant DNA inserted with HBs gene is prepared by utilizing the vector and then a transformant is prepared from the récoSbinant DNA, the transformant can produce the desired HBs proteins only in a small amount. The present inventors have extensively studied to develop an improved E. coli-yeast shuttle vector which can be recombined with various genes and can express them¯ As a result, it has been found that a specific shuttle vector containing both a yeast derived DNA and a E. coli derived Stand carrying the repressible acid phosphatase promoter of the yeast has desired characteristics and is useful for recombining various genes under the control of the phosphatase promoter to prepare recombinant DNAs which can give various transformed yeasts. An object of the present invention is to provide a novel shuttle vector which contains both a E. coli derived DRand a yeast derived D.^E and carries the repressible acid phosphatase pr=ster. Another object of the invention is to provide a shuttle vector which can be recombined with various genes under the control of the phosphatase promoter. These and other objects and advantages of the invention will be apparent to persons skilled in the art from the following description. The shuttle vector used in the present i=vention- is a plasmid vector which contains both a yeast derided DµE and a E. coli derived DNA and carries the repressible acid phosphaSse gene of the yeast. This plasmid vector can replicate both in E. coli and in yeast, and can be used for the preparation of recombinant DNAs which in turn are used for the preparation of transformed yeasts, wherein the recombinant plasmids are prepared by using E. coli and then a. yeast is transformed with the recombinant plasmid. The transformed yeasts thus prepared can produce the desired gene products in a large scale. In the step of the transformation of a yeast1 the vector does not need to contain the E. coli derived DNA. The yeast derived DNA contains usually a DNA sequence which is necessary for replication of a plasmid in the yeast as an episome, - for instance, a DNA sequence necessary for the autonomous replication of the yeast (which is designated "ars 1"), and a DNA sequence necessary for the replication of 2/m DNA (which is designated "2Llori"), and contains optionally a gene useful as a selective marker for the transformed yeast. The selective marker includes, for example, a leucine-producing gene, a histidine-producing gene, a tryptophane-producing gene, a uracil-producing gene, an adenine-producing gene, or the like, which may be used alone or in combination of two or more thereof. The E. coli derived DNA contains a DNA sequence necessary for the replication of the plasmid within E. coli, for example, a DNA sequence of a replication initiating region of the plasnid Col El, and preferably contains a gene useful as a selective marker for the transformed E. coli. The selective marker includes, for example, an ampicillinresistance gene, a kanamycin-resistancegene, a tetracycline-resistance gene, a ch1oraippbnicoiresistance gene, or the like, which may be used alone or in combination of two or more thereof. Commonly used E. coli derived DNA is the small plasmid-pBR322 which contains an ampicillinresistant gene and a tetracycline-resistant gene. The shuttle vector of the present invention is characteristic in that it carries the repressible acid phosphatase promoter of the yeast. This acid phosphatase promoter is usually a promoter controlling the eppression of a polypeptide of 60,000 daltons (p60) which constitutes the acid phosphatase. A representative example of the shuttle vector is a vector which is prepared by recombining a yeast derived DNA containing ars 1, 2 ori and a leucine producing gene (.leu 2) with the . E. coli plasmid pBR322. The shuttle vector is designated "pAT 77" (Fig. 1) and is prepared as follows. An EcoRI fragment of about 8,000 nucleotide base pairs (8kb) containing a gene coding for a polypeptide (p60) of 60,000 daltons which constitutes the repressibleacid phosphatase (cf. BUMS, 77, 6541-6545,-1980 and PNAS, 79, 2157-2161, 1982) is inserted into the EcoRI site of the E. coli plasmid pBR322 (cf. Sutcliffe, J.G.; Cold Spring Harbor Symposium, 43, 77-90, 1979) to give a plasmid, which is used as the starting material. Said EcoRI fragment (8 kb DNA fragment) contains a unique recognition site for the restriction enzyme Sal I at a position where it is divided into a fragment of about 2.8 kb and a fragment of about 5.2 kb. The starting plasmid is digested with the restriction enzyme Sal I and re-annealed with T4 DNA ligase to give a plasmid which has lost the 5.2 kb acid phosphatase gene fragment. This plasmid is designated "pAT 25". pAT 25 is a plasmid consisting of a 3.7 kb fragment extending from the EcoRI site to the Sal I site of pBR322 which contains the ampicillin-resistance gene and a fragment (about 2.8 kb) extending from the EcoRI site to the Sal I site of the yeast acid phosphatase gene. Into the EcoRI site of pAT 25 is inserted an EcoRI fragment (1.4 kb) containing a DNA sequence necessary for the autonomous replication of the ars 1 and trp 1 gene of- yeast (cf. PNAS, 76, 1035-1039, 1979) to give a plasmid (designated "pAT 26"). Said ars l-trp 1 fragment contains a unique recognition site for the restriction enzyme Hind III within the trp 1 gene. Into -this unique Hind III site of the reccenbinant plasmid pAT 26 its inserted a Hind III fragment containing the leu 2 gene of yeast and a DNA sequence necessary for the replication of 2rm DNA (2 ori) (cf. Tohe, A., Guerry, p., Wichener, R.B.; J. Bacteriol., 141, 413-416, 1980) to give the desired shuttle vector pAT 77. The pAT 77 and pAM 82 derived therefrom as described hereinafter have the structures as shown in Figure 1. In Figure 1, the dark line represents the EcoRI/Sal I - pBR322-fragment containing the ampicillin-resistance gene (Apr) linked to ars 1, 2 ss ori, and the leu 2 gene in this order and to the 2.8 kb EcoRI/Sal I - fragment of the acid phosphatase gene. The recombinant plasmid pAT 77 can replicate in E. coli because of the presence of the origin site of pBR322 and it can also replicate in yeast because of the presence of the ars 1 and 2 F ori genes. Moreover, this plasmid contains as markers in order to select for transformed E. coli the ampicillin-resistance gene (Apr) and for transformed yeast the leu 2 gene, and thereby fulfills the requirements to be used as a shuttle vector. The gene map around the acid phosphatase promoter of the shuttle vector pAT 77 is shown in Figure 2. The nucleotide sequence of the BstEII/Sal I region in this shuttle vector has been determined by the present inventors and is as shown in the accompanying Figure 3. The ATG codon (methionine) shown in Figures 2 and 3 is the start codon of the acid phosphatase structural gene. That is, the repressible about 2.8 kb acid phosphatase gene fragment of this vector contains the region of from about 2.7 kb upstream from the structural gene to-the 82nd nucleotide base pair (82 bp) of the structural gene. The shuttle vector pAT 77 is cleaved with the restriction enzyme Sal I, followed by treating with theexonuclease Bal 31, by which a part or the complete structural gene of the acid phosphatase as shown in Figures 2 and 3 and further optionally various regions upstream therefrom are depleted. This deletion is effected for appropriate regions before the acid phosphatase promoter region: TATATAA (Ibgness box), i.e. -100 bp. The regions to be' deleted can be controlled by the conditions for treating with the exonuclease Bal 31 and are usually in the range of from +1 to -100 bp, preferably from +1 to -50 bp. The loss of the Hogness-box by extensive deletion, i.e. over -100 bp implies the loss for the control region, resulting in a lower yield of the desired gene products in the cultivated transformed yeast cells. On the other hand, when the deletion is insufficiently effected so that a part of the acid phosphatase structural gene remains, a fusion protein of a foreign gene product and a phosphatase peptide may be formed which is not desirable. After deleting a part or the complete structural gene of the acid phosphatase and optionally some regions upstream therefrom, a synthetic or natural linker, for example Sal I linker or Xho I linker, is recombined thereto to give a circular plasmid, by which there is obtained a shuttle vector in which a foreign gene can be inserted the expression of which is controlled by the acid phosphatase promoter. This shuttle vector can readily be. cleaved at a site to be recombined by treating with a conventional restriction enzyme, such as Sal I or Xho I, and hence, is preferably used in order to recombine with the desired gene. The shuttle vector of the present invention can De used for the preparation of various recombinant plasmids by recombining it with various genes downstream of the acid phosphatase promoter and further for the preparation of various transformed yeasts by the recombinant plasmids, and hence, is very useful in the field of genetic engineering industries. For instance, the shuttle vector of the present invention is used for the preparation of a recombinant plasmid inserted with an HBs gene and further the preparation of a transformed yeast therefrom which can produce HBs antigen in a large scale. The HBsAg obtained behaves like the natural HBs antigen from human donors in terms of immunological properties and hence is useful for the preparation of Hepatitis B virus vaccine. The present invention is illustrated by the following Example and Preparation. Example Preparation of shuttle vectors pAM 81, 82, 83 and 84 An EcoRI fragment of about 8,000 nucleotide base pairs (8 kb) containing a polypeptide (p60) gene of 60,000 daltons which constitutes the repressible acid phosphatase (available from Yeast S288C gene bank, Clarke, L. and Carbon, J., Cell, 9, 91-99, 1976) is inserted into the EcoRI site of the E. coli plasmid pBR322 to give a plasmid, which is used as the starting material. The starting plasmid is digested with the restriction enzyme Sal I and re-annealed with T4 DNA ligase to give a plasmid pAT25 which is deficient from the Sal I site to the acid phosphatase gene fragment 5.2 kb ±said plasmid pAT 25 being a plasmid consisting of a fragment (about 3.7 kb) of from EcoRI site to Sal I site of pBR322 which contains the ampicillin-resistancegene and a fragment (about 2.8 kb) of from EcoRI site to Sal I site of the yeast acid phosphatase gene, wherein both fragments link at each corresponding terminal. thereof). Into the EcoRI site of the above pAT 25 is inserted an EcoRI fragment (1.4 kb)containingthears 1 and trp 1 gene which is prepared by treating a plasmid YRP 7 (cf. Struhl, K. et al, Proc. Natl. Acad. Sci. U.S.A., 76, 1035-1039, 1979) with EcoRI to give a plasmid pAT 26. Said ars l-trp 1 fragment has aunkFe recognition site of a restriction enzyme Hind III within the -trp 1 gene. Into the Hind III site of the above pAT 26 is inserted a Hind III fragment containing aleu 2 and 2fl ori which is prepared by treating the plasmid pSLE 1 (cf. Tohe, A. et al, J. Bacteriol., 141, 413-416, 1980) with Hind III to give the desired shuttle vector pAT 77.The pAT 77 harboured in Saccharomyces cerevisiae (i.e. Saccharomyces cerevisiae AH 22/pAT 77) has been deposited at the sermentation Research Institute, Agency of Industrial Science and Technology, Japan according to the Budapest Treaty as "vERM BP-324". The pAT 77 thus obtained (irk) is cleaved with Sal I and then is treated with an exonuclease Bal 31 (0.1 U) in a solution (50 l) of 20 mMTris-HCl (pH 8.2), 12 St CaCI2, 12 mSI MgC12, 0.2 M NaCl and 1 mM EDTA for 30 seconds to one minute. The reaction mixture is subjected to phenol extraction and ethanol precipitation in the same manner as described in Preparation (1) (ii) (A) hereinafter. The resulting precipitates are treated with Xho I linker (1 pmol) and T4 DNA ligase under the same conditions as described in Preparation (1) (ii) (A) hereinafter for 12 hours. E. coli X1776 is treated with the above reaction mixture by the procedure as described in R.Curtiss III et al, "Molecular cloning of recombinant DNA" eds. W. A. Scott and R. Werner, page 99, Academic Press (1977) so as to transform the E. coli 1776 to give an ampicillin-resistant transformant. From the resulting transformant colonies, plasmid DNAs are prepared by the procedure as described by K. Matsubara (j. Virol., 16, 479, 1975). According to Maxam-Gilbert method (cf. Maxam, A. & Gilbert, W.; Pro. NAS., 74, 560-564), the nucleotide sequence of the resulting DNAs is determined. Furthermore the region of the acid phosphatase gene deleted withBal 31 is determined. Among these DNAs, the desired plasmids PAM 81, pAM 82, pAM 83 and pAM 84 which are deficient in te complete structural gene of phosphatase are selected and isolated. Designating "A" in the codon ATG encoding the first amino acid (methionine) of the product n60 of the phosphatase structural gene as "+1", the following regions are deleted in these shuttle vectors, pAM 81: till +2, pAM 82: till -33, pAM 83: till -50, and pAM 84: till -51. The pAM 81, pAM 82, pAM 83 and pAM 84harboured in Saccharomyces cerevisiae (i.e. Saccharomyces cerevisiae AH 22/pAM 81, AH 22/pAM 82, AH 22/pAM 83 and AH 22/pAM 84, respectively) have been d¯mDsited at theFermentation Research Institute, Agency of Industrial Science and Technology, Japan according to the Budsst Treaty as "FERM BP-325 ", "FERM BP-313", "FERM BP-327 ", and "FERM BP-326 ", respectively. An embodiment or preparing recombinant plasmid, a transformed yeast and HBsAg by recombination of HBV DNA using the shuttle vector pAM 82 is illustrated in the following Preparation. Preparation (1) Preparation of HBV DNA (i) Preparation of virus DNA Pooled blood plasma (700 ml) obtained from ten patients who are positive in HBsAg (subtype adr) and HBcAg is centrifuged at 5,000 r.p.m. for 20 minutes to remove dissolved substances. The resulting solution is centrifuged at 40C, 18,000 r.p.m. for 8 hours, and the resultant precipitatej is re-dissolvd in 10 ml of a buffer (pH 7.5) of 10mM Tris-HCl, 0.1 M NaCl and ImM EDTA. The solution is added to the top of a centrifuge tube containing 30 % sucrose, which is centrifuged at 40C, 39,000 r.p.m. for 4 hours. The resultant precipitate is redissolved in the same buffer as above. In order to allow the following operations the buffer solution is subjected to the reaction with HBV DNA polymerise by treating it in a mixture (500 l) of 67 mbI Tris-HCl (pH 7.5), 80 mM NH4C1, 25 mM MgC12, 0.5 % NP40 {Tergitol#, manufactured by Sigma Co.), 0.1 %. 2-mercaptoethanol, 330 M dCTP (deoxycytidine triphosphate), dGTP - (deoxyguanosine triphosphate), and dATP (deoxyadenosine triphosphate), 0.5 fiN 0.5 M &alpha;-[32P]dTTP (deoxythymidine triphosphate) at 370C for 3 hours To the reaction mixture is added the same volume of 100 mM EDTA solution. By the above DNA polymerase reaction, the single-stranded region of the DNA is repaired to a completely double-stranded structure which is [32p] labeled. This DNA is added to the top of a -centrige tube wherein 30 %, 20 % and 10 % aqueous solutions of sucrose are packed in layers in this order, and it is centrifuged at 40C, 39,000 r.p.m. for 4.5 hours. In order to digest the proteins strongly bonded to DNA, the precipitates obtained above are treated in a mixture (200 %l) of 1 mg/ml of Pronase E (manufactured by Waken Kagaku K.K.) and 0.2 % aqueous sodium lauryl sulfate solution at 370C for 2 hours. The resulting mixture is extracted with phenol (200 l) twice, and the resulting DNA-containing extract is washed with ether to remove the phenol and to give a solution of HBV DNA. The DNA thus obtained has a specific radioactivity of 2.5 x 106 cpm/ g and can be used for digestion with restriction enzymes. (ii) Cloning of HBV DNA The double-stranded circular HBV DNA obtained above is cloned by using -phage Charon 16A DNA as a vector and then is sub clone by using the known plasmid pACYCli7 as a vector as follows. (A) . Cloning in the system of Aphage Charon 16A host-vector: HBV DNA (20 ng) is treated with endonuclease Xho I in a mixture (209Ul) of 10 mM. Tris-HCl (pH 7.4), 7 mM MgC12, 100 mM NaCl and 7 mM 2-mercaptethanol at 370C for 2 hours. The resulting mixture is extracted with phenol (20 1) and further with ether, and to the aqueous layer is added a double volume of cooled ethanol to precipitate DNA. The mixture is kept at -700C for one hour and then centrifuged at 10,000 r.p.m. for 5 minutes, and the precipitated DNA is recovered. The precipitate thus separated is dissolved in a mixture (5 l) of 10 nDl Tris-HCl (pH 7.4) and 1 mM EDTA. The HBV DNA and an equimolar amount of A-phage Charon 16 A DNA (having a unique .5;ho I recognition site) obtained by cleavage with endonucleaseXho I in the same mariner as described above are reacted with T4 DNA ligase [a mixture of 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 10 mM dithiothreitol, 100 g/ml calf serum albumin, 0.5 mM ATP and 0.5 l enzyme preparation (T4 ligase, manufactured by Takara Biomedicals, 1-5x103 unit/ml)i at 40C for 18 hours. The reaction mixture is extracted with phenol and ether and then subjected to precipitation with ethanol in the same manner as described above. The precipitates thus obtained are dissolved in amixture (10)11) of 10 mM Tris-HCl (pH 7.4) and 1 mS1 EDTA. The thus annealed DNA is subjected to in vitro packaging operation to form A-phage in the same manner as described in "Methods in Enzymology", 68, 299-309 and further plaques (104) are formed therefrom on an L-agar plate (23 cm x 23 cm) by using E. coli DP50 SupF (cf. Blattner, F.R. et al, Science 196, 161, 1977) as an indicator. These plaques are subjected to plaque hybridization using 32P-labeled HBV DNA prepared above as a probe (cf. Science, 196, 180, 1977) in order to select plaques formed from the phage having HBV DNA, by which a plurality of the desired pages are separated. (B) Sub cloning by using plasmid pACYC177 as a vector: From HBV DNA cloned in Charon 16 A as obtained in ste (A) a phage DNA is prepared by using E. coli DP5O-SupF as a bacteria to be infected in the same manner as described in "Methods in Enzymology", 68, 245-378, 1979. The DNA thus obtained is digested with Xho I under the same conditions as described above for 2 hours, and the resulting reaction mixture is subjected to an electrophoresis with 0.75 % agarose gel to isolate HBV DNA (3.2 kb). The HBV DNA is absorbed onto DEAE (diethylaminoethyl cellulose) paper (manufactured by Toyo Roshi, Japan) in order to separate it from the vector DNA and then eluted with I M NaCI aqueous solution to give an HBV DNA having Xho I terminals at both ends. Separately, plasmid pAC?C177 (cf. Chang, A.C.Y., Cohen, S. N.; J. Bacteriol., 134, 1141-1156, 1978) having a unique Xho I cleavage site within its kanamycin-resistance gene is digested with Xho I, and the product is purified by phenol extraction, ether treatment and ethanol precipitation in the same manner as described above. The thus obtained pACYC177 cleaved with Xho I is mixed with XhoI-terminal HBV DNA obtained above in a molar ratio of 1 : 5, and the mixture is annealed with T4 DNA ligase for 18 hours as described above. The annealed DNA preparation (10 l) obtained above is added to a liquid of E. coli (0.1 ml) which is prepared by treating a culture broth of E. coli X1776 [cf. R. Curtiss III et al, "Molecular cloning of recombinant DNA" eds. W.A. Scott and R. Werner, page 99, Academie Press (f977) by the procedure as described in MV. Norgard, Gene, 3, 279 (1918), and the mixture is mixed well and allowed to stand at 0 C for 25 minutes. The mixture is applied onto an L-agar plate containing ampicillin (20 g/ml), i-biotine (1ys g/ml), diaminopimelic acid (100 g/ml) and thymine (20/ gimp) and is incubated at 37 C overnight. The resulting colonies are applied onto both an agar plate containing kanamycin (20 g/ml) and an agar plate containing ampicillin (20 g/ml). . The colonies which grow only on the agar plate containing ampicillin are selected.pACvCl77 contains an ampicillin-resistancegene and a kanamycin-resistance gene, but when EBV D2T.A is inserted into the Xho I site of the kanamycin-resistance gene1 the kanamycin-resistance is lost. Accordingly, the selected colonies contain a recombinant DNA of PACYCJ77-HBV DNA. wron the colonies thus selected, a plasmid is prepared by the procedure as described by K. Matsubara (J. Virol., 16, 479, 1975). The plasmid thus obtained, i.e. the recombinant DNA of pACYC177-HBV DNA (which is designated tpHBV"), is treated with Xho I under the same conditions as described above to give total HBV DNA fragment (3.2 kb). Besides, when it is treated with Xho I and BamHI, there is obtained a fragment (about 1.3 kb) containing the HBsAg gene. (2) Preparation of HBsAg gene-expresssion plasmids (i) Preparation of plasmids inserted with the complete HBV DNA HBV'DNA obtained by treating a plasmid pHBV (pACYC 177-HBV DNA) with Xho I is recombined with Xho I cleaved shuttle vector, pAM 81, pAM 82, pAM 83 and pAM 84 ina molar ratio of 5 : 1 by annealing with T4 DNA ligase under the same conditions as described above. E. coli 1776 is transformed with the reaction mixture and a plasmid DNA is prepared from the resulting ampicillin-resistant transformant in the same manner as described hereinbefore. The DNAs thus prepared are analyzed with various restriction enzymes, such as Xho I, Xba I and Hind III, and thereby, insertion of HBV DNA into the vectors and its orientation are determined. The thus obtained HBsAg gene-expression plasmids contain te Is gene and ESc gene in this order downstream of the phosphatase promoter, and the plasmids is designated pAH 203. (ii) Preparation of plasmid inserted with HBsAg gene fragment An HBsAg gene fragment (3 g) prepared by cleaving plasmid pHBV with BamHI is treated with T4 DNA polymerase (0.2 U) in a solution (100/ 1) of 67 mM Tris-HCl (pH 8.6), 6.7 raM MgC12, 10 mM 2-mercaptoethanol, 6.7 M EDTA and 16.7 mI (NH4)2SO4 which contains 200 M o(ATP, CTP, &alpha;TTP and gGTP for 30 minutes in order to fill in the BamHI cleavage end. The reaction mixture is subjected to phenol extraction and ethanol precipitation as described above. The resulting precipitates are subjected to linking reaction with Xho I linker in a molar ratio of 1 : 10 with T4 DNA ligase under the same conditions as described hereinbefore. After phenol extraction and ethanol precipitation, the resulting plasmid is treated with Xho I to give an HBsAg gene fragment (about 1.3 kb) having the Xho I cleavage terminal at both ends. The fragment thus obtained is annealed with the shuttle vector pAM 82 which is cleaved with Xho I in a molar ratio of 5 : 1 by using T4 DNA ligase, and E. coli x1776 is transformed with the reaction mixture obtained above in the same manner as described in the step 3 (i) to give a plasmid DNA. Into the plasmid thus obtained is inserted the HBsAg gene in the correct orientation downstream of the phosphatase promoter of the vector pAM 82, which plasmid is designated pAS 101. (3) Preparation of transformed yeast The starting yeast is Saccharomyces cerevisiae AH22 (a, leu2, his4, canl (Cir+)], which has been deposited at the Fermentation Research Institute; Agency of Industrial Science and Technology, Japan according to the Budapest Treaty as "FEl! BP-3121'. The starting yeast is inoculated in YPD medium (100 ml) consisting of 2 % polypeptone, 1 % yeast extract and 2 % glucose, and the mixture is incubated at 300C overnight, and thereafter, the cells are collected by centrifugation. The cells thus collected are washed with sterilized water (20 ml), suspended in a solution (5 ml) of 1.2 M sorbitol and 100 Tg/ml Zymolyase@60,000 (manufactured by Seikagaku Kogyo K.K., Japan), and the suspension is allowed to stand at 300C for 30 minutes to give spheroplasts. The spheroplaststhus prepared are washed with 1.2 M sorbitol solution three times, and then suspended in a solution (0.6 ml) ot 2 M sorbitol, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5) The suspension thus prepared is divided into small test -.ubes in a volume of 60 1. To the suspension is added the solution of the recombinant plasmid pAH 203 (3Ofil) prepared in the above (3). After mixing well, 0.1 M CaCl2 (3 U1) is added thereto in a final concentration of 10 mM CaC12, and the mixture is allowed to stand at room temperature for 5 to 10 minutes. To the resulting mixture is added each 1 ml of a solution of 20 % polyethylene glycol 4,000, 10 mbl CaC12 and 10 mM Tris-HCl (pH 7.5), and the mixture is allowed to stand at room temperature for about 20 minutes. The resulting mixture (each 0.2 ml) is added to a medium (10 ml) consisting of 22 % sorbitol, 2 t glucose, 0.7 % yeast nitrogen base amino acid, 2 % YPD, 20/tg/ml histidine and 3 % agar, which is kept at a constant temperature of 450C. After gentle mixing, the mixture is added in a layer onto a plate of minimal medium containing 1.2 M sorbitol which is previously prepared and consists of 0.7 % yeast nitrogen base amino acid, 2 % glucose, 20 tg/ml histidine and 2 % agar and is set thereon. The plate is incubated at 300C to give colonies of a leucine-non-requiring yeast. The colonies areincubated in a BurkHolder minimal medium supplemented with histidine (20 ss g/ml) [cf. Tohe, A, et al; J. Bachterol., 113, 727-738, 1973] to give the desired transformed yeast: Saccharomyces cerevisiae pAH 203. In the same manner as described above, except that the recombinant plasmid PAS 101 is used instead of the recombinant pAH 203, Saccharomyces cerevisiae pAS 101 is prepared. (4) Production of HBsAg with the transformed yeast Each colony of the transformed yeasts obtained in the above {4) is applied onto an agar plate of BurkHolder minimal medium supplemented with histldine (20 Sg/ml) and incubated at 300C to form a colony (in order to confirm the transformant requiring no leucine). The resulting cells are separated from the colony inoculated into BurkHolder minimal medium supplemented with histidine (20/(g/ml) and incubated at 300C. After about 24 hours, the cells in exponential growth phase are collected by centifugation, suspended in a minimal medium (10 ml) containing no phosphoric acid (which is prepared by replacing KH2PO4 in BurkHolder minimal medium with KC1, followed by supplementing with 20 flg/ml histidine) in a cell concentration of about 4 x 106 cells/ml. After incubating at 300C for about 24 hours, the culture broth is centrifuged at 4,000 r.p.m. for 10 minutes to collect the cells. The cells thus separated are suspended in a solution (3 ml) of 1.2 M sorbitol, 50 mM phosphate buffer (pH 7.2), 14 mtl 2 R mercaptoethanol and 100 ssg/ml Zymolyase-60,000 (manufactured by Seikagaku Kogyo K.K., Japan), and the mixture is gently shaken at300C for 30 minutes to give spheroplasts, The spheroplass arecollected by centrifugation and are well suspended in a solution (1 ml) of 0.1 % TritoneRX-100 and 50 mM phosphate buffer (pH 7.2), stirred vigorously and then centrifuged at 7,000 r.p.m. for 10 minutes, and the resulting supernatant is taken as the yeast-lysed solution. The lysed solution (Z0 1) obtained above is tested with a ESs antigen RIA kit (manufactured by Abbott, U.S.A.) for the H3sAg activity. The results are shown in Table 1. Table 1 EMI21.1 <tb> Clone <SEP> No. <SEP> Host <SEP> Plasmid <SEP> HBsAg <SEP> activity <tb> <SEP> (cpm) <tb> <SEP> 1 <SEP> S. <SEP> Cerevisiae <SEP> pAH <SEP> 203 <SEP> 13,008 <tb> <SEP> AH22 <SEP> (FERN <SEP> BP-312) <tb> <SEP> 2 <SEP> .. <SEP> pAS <SEP> 101 <SEP> 11,200 <tb> Reference <SEP> . <SEP> <SEP> pAM <SEP> 82* <SEP> 320 <tb> *) This vector has no HBV or HBs gene and is used as a negative reference. (The negative control of RIA kit has an activity of 310 cpm, and the positive control thereof has that of 17,500 cpm) (5) Preparation of HBcAg gene expression plasmid An HBcAg gene fragment is prepared by the following procedure. Plasmid pHBV (3rig) is digested with the restriction endonuclease Rsa I in a usual manner. The reaction mixture is subjected to phenol extraction and ethanol precipitation as described above. The resulting precipitates are subjected to linking reaction with Xho I linker in a molar ratio of 1 : 10 with T4 DNA. ligase. After phenol extraction and ethanol precipitation, the resulting precipitates are subjected to Xho I digestion to give an HBc gene fragment (about 0.7 kb)- having Xho I cleavage termina3sat both ends. The fragment thus obtained is annealed with the shuttle vector pRi 82 which is cleaved with Xho I in a molar ratio of 5- : 1 by using T4 DNA ligase. The raction mixture is used to transform E. coli X1776 in the same manner as described in (1) (ii) (B) to give a plasmid DNA. Into the plasmid thus obtained is inserted the HBcAg gene in the correct orientation downstream of the phosphatase promoter of the vector -pAM 82, which plasmid is designated pHC 301. (6) Preparation of transformed yeast with HBcAg gene expression plasmid pHC 301 The starting yeast is Saccharomyces cerevisiae AH22 [a leu2 his4 cani (Cir +) 1, which has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan according to the Budapest Treaty as "FERM BP-312, The starting yeast is inoculated in YPD medium (100 ml) consisting of 2 % polypeptone, 1 % yeast extract and 2 % glucose, and the mixture is incubated at 30 C overnight, and thereafter, the cells are collected by centrifugation. The cells thus collected are washed with sterilized water (20 ml), suspended in a solution (5 ml) of R 1,2 M sorbitol and and g/ml 1ymolyase-60,000 (manufactured by Sebkagaku Kogyo K.K., Japan), and the suspension is allotted to stand at 300C for 30 minutes to give spheroplasts. The spheroplaststhus prepared are washed with 1.2 M sorbitol solution three times, and then suspended in a solution (0.6 ml) of 2 M sorbitol, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5). The suspension thus prepared is divided into small test tubesin a volume of 60 l. To the suspension is added the solution of the recombinant plasmid pAC 301 (30 l) prepared in the above (5). After mixing well, 0.1 M CaC12 is (3 l) is added thereto in a final concentration of 10 mmI CaC12, and the mixture is allowed to stand at room temperature for 5 to 10 minutes. To the resulting mixture is added each 1 ml of a solution of 20. % polyethylene glycol 4,000, 10 mM CaCl2 and 10 mM Tris-HCl (pH 7.5), and the mixture is allowed to stand at room temperature for about 20 minutes. The resulting mixture (each 0.2 ml) is added to a medium (10 ml) consisting of 22 % sorbitol, 2 % glucose, 0.7 % yeast nitrogen base amino acid, 2 % YPD, 20 g/ml histidine and 3 % agar, which is kept at a constant temperature of 45 C. After gentle mixing, the mixture is added in a layer onto a plate of minimal medium containing 1.2 M sorbitol which is previously prepared and consists of 0.7 % yeast nitrogen base amino acid, 2 % glucose, 20rg/ml histidine and 2 % agar and is set thereon. The mixture is incubated at 300C to give colonies of a leucine-non-requiring yeast. The. colonies are incubated in a BurkHolder minimal medium supplemented with histidine (20j < g/ml) [cf. Tohe, A, et al; J. Bachterol., 113, 727-738, 1973] to give the desired transformed yeast: Saccharomyces cerevisiae pHC 3C1. (7) Production of HBcAg with the transformed yeast Each colony of the transformed yeasts obtained in the above (6) is applied onto an agar plate of BurkHolder minimal medium supplemented with histidine (20g/ml) and incubated at 300C to form a colony (in order to confirm the transformant requiring no leucine). The resulting cells are separated from the colony, inoculated into BurkHolder minimal medium supplemented with histidine (20j±g/ml) and incubated at 300C. After about 24 hours, the cells in exponential growth phase are collected by centifugation, suspended in a minimal medium (10 ml) containing no phosphoric acid (which is prepared by replacing KH2PO4 in BurkHolder minimal medium with KC1, followed by supplementing with 20 Xg/ml histidine) in a cell concentration of about 4 x 106 cells/ml. After incubating at 300C for about 24 hours, the culture broth is centrifuged at 4,000 r-.p.m. for 10 minutes to collect the cells. The cells thus separated are suspended in a solution (3 ml) of 1.2 M sorbitol, 50 mh phosphate buffer (pH 7.2), 14 SA 2 mercaptoethanol and 100 g/ml Zymolyase#60,000 (manufactured by Seikaga-Ku Kogyo K.K., Japan), and the mixture is gently shaken at 300C for 30 minutes to give spheroplasts. The spheroplasts are collected by centrifugation and are well suspended in a solution (1 ml) of 0.1 % TritoneRX-100 and 50 mM phosphate buffer (pH 7.2), stirred vigorously and then centrifuged at 7,000 r.p.m. for 10 minutes, and the resulting supernatant is taken as the yeast-lysed solution. The lysed solution (2Ofil) obtained above is tested with a HBc antigen RIA kitmanufactured by Abbott, U.S.A,) for the HBcAg activity. The results are shown in Table 2. Table 2 EMI25.1 <SEP> <tb> <SEP> Host <SEP> Plasmid <SEP> HBcAg <SEP> activity <tb> <SEP> (cpm) <tb> <SEP> S. <SEP> Cerevisiae <SEP> pHC <SEP> 301 <SEP> 4,989 <SEP> <tb> <SEP> AH22 <SEP> (FERM <SEP> BP-312) <tb> Reference <SEP> ti <SEP> pAM <SEP> 82 <SEP> 16,913 <tb> *) This vector has no HBV or HBs gene and is used as a negative reference. (The negative control of RIA kit has an activity of 17,740 cpm, and the positive control thereof has that of 446 cpm)

Claims: What is claimed is: 1, A shuttle vector, which comprises yeast gene anda Escherichia -coli derived DNA and carries the expression control region of the repressible acid phosphatase gene of yeast. 2. The shuttle vector according to claim 1, wherein said expression control region of the repressible acid phosphatase gene of yeast is a gene coding for a polypeptide of 60,000 daltons (p60) which constitutes the acid phosphatase, a part or the complete structural gene of the acid phosphatase and optionally a certain region upstream the phosphatase structural gene being deleted. 3. The shuttle vector according to claim 2, wherein the region to be deleted upstream the phosphatase structural gene in addition to the complete phosphatase structural gene is in the range of from +1 (ATG) to -100 bp. 4. The shuttle vector according to claim 3, wherein the region to be deleted is in the range of from +1 to -50 bp. 5. The shuttle vector according to claim 1, wherein said yeast derived DNA includes ars 1 and 2 F ori. 6. The shuttle vector according to claim 1, wherein said yeast derived DNA includes ars 1, 2 ss ori and a selective marker gene for a transformed yeast. 7. The shuttle vector according to claim 6, wherein said selective marker gene is a member selected from the group consisting of a leucine-producing gene, a histidine-proaucing gene, a tryptophane-producing gene, a uracil-producing gene, and an adenine-producing gene, and a combination of two or more thereof. 8. The shuttle vector according to claim 1, wherein said E. coliderived DNA includes a DNA sequence which is necessary for replication of.. ¯the D':IA in E. Coli. 9. The shuttle vector according to claim 1, wherein said E. coli derived DNA includes a D > JA sequence which is necessary for replication of the DNA in E. coli. and a selective marker gene for a transformed E. coli. 10. The shuttle vector according o claim 9, wherein the selective marker gene is a member selected from the group consisting of an ampicillin-resistance gene, a kanamycin-resistancegene, a tetracycline-resistance gene, and a chloramphenicol-resistancegene, and a combination of two or more thereof. 11. The shuttle vector according to claim 9, wherein said E. coli derived DNA is the plasmid pBR322 containing an ampici11in-resistance gene and a tetra cycline-resistance gene. 12. The shuttle vector according to claim 1, which consists of a yeast plasmid and a: E. coli plasmid, said yeast plasmid containing ars 1, 2 T ori, l.eu 2 and the expression control region of the repressible acid phosphatase gene in this order wherein a part or the complete the acid phosphatase structural gene and a certain region upstream the phosphatase structural gene are deleted, and said E. coli plasmid being the plasmid pBR322 which contains an ampicillin-resistance gene and a tetracycline-resistance gene. 13. The shuttle vector according to claim 12, wherein said yeast plasmid is deficient of the complete acid phosphaiase structural gene and of. a region upstream the phosphatase structural gene in the range of +1 to -100 bp. 14. The shuttle vector according to claim 13, wherein the deficient region upstream the phosphatase structural gene is in the range of from +1 to -50 bp,

Inventors: MATSUBARA, KENICHI, MIYANOHARA, ATSUSHI, TOH-E, AKIO
Applicants: JURIDICAL FOUNDATION THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE
Application year: 1983
Main classification: C12N15
IPC codes: C07K14, C12N15