Patent Abstract:
compositions comprising biologically pure pfs230 and nucleic acids which encode them are provided . the proteins can be used induce transmission blocking immune responses in susceptible hosts .

Detailed Description:
the present invention provides recombinantly produced pfs230 and fragments derived from the protein that are useful for inducing an immune response when injected into a human or other host animal . pfs230 and homologs in other plasmodium species can be used to block transmission of a number of parasites associated with malaria . four species of the genus plasmodium infect humans , p . vivax , p . ovale , p . malariae , and p . falciparum . in addition other plasmodium species infect other animals . for instance , p . gallinaceum is responsible for avian malaria . pfs230 is expressed by the parasite while it undergoes gametocytogenesis in the human host . this antigen has been identified on day 2 of gametocytogenesis and continues to be produced as the gametocyte is taken up by the mosquito in a blood meal and emerges from the erythrocyte in the mosquito midgut . once the parasite emerges from the erythrocyte , pfs230 is exposed on the surface of the parasite , and is thus in contact with the components of the bloodmeal including antibodies and complement . the 9 . 4 kb open reading frame of the nucleic acid encoding pfs230 predicts a protein with a molecular weight of 363 , 243 daltons . pfs230 exists in at least two forms , a 360 kda form that does not radiolabel with 125 i and a 125 i radiolabeled form isolated from surface labeled gametes . the labeled form when sized under reducing conditions migrates as a 310 , 000 molecular weight band . these results suggest that the full - length 360 kda protein is processed to a 310 kda protein that is expressed on the surface of the gamete . a prior art mab 1b3 has been reported to immunoprecipitate a 230 kda protein from radiolabeled surface proteins of newly formed gametes and zygotes . this monoclonal antibody was reported to recognize two proteins of 260 , 000 and 230 , 000 mr on western blots . quakyi , et al ., j . immunol . 139 : 4213 - 4217 ( 1987 ), which is incorporated herein by reference . evidence provided here shows that the protein encoded by the gene of the present invention is the same protein as that recognized by mab 1b3 . in particular , antisera raised against fusion proteins expressed from the nucleic acids of the invention recognized bands similar to those reported for pfs230 . the antisera also immunoprecipitates 125 i - labeled pfs230 and reacts with the surface of intact gametes as assayed by indirect immunofluorescence . seq . id . no . 2 is the deduced amino acid sequence of the 9 . 4 kb gene . the deduced amino acid sequence of pfs230 codes for a 363 kda polypeptide having five distinct characteristics : 1 ) consistent with pfs230 being a non - integral membrane protein ( kumar & amp ; wizel , mol . biochem . parasitol ., 53 : 113 - 120 ( 1992 )), there is a presumptive signal sequence at the amino - terminus , but no other predicted hydrophobic or transmembrane regions ; 2 ) starting at amino acid 280 , there are 25 contiguous e residues ; 3 ) beginning with amino acid 379 , a four amino acid ( e - e - v - g ) ( seq id no : 3 ) repeat is repeated tandemly 8 times followed by 4 copies of an eight amino acid ( e - e - v - g - e - e / g - e / v - g ) ( seq id no : 4 ) repeat ; 4 ) there are three regions of highly negative net charge , including amino acids 273 - 325 , which contain the 25 e residues , amino acids 1147 - 1205 , and amino acids 1604 - 1668 ; and 5 ) there are six copies of a seven cysteine motif with the consensus sequence . the pfs230 proteins of the invention may be recombinantly produced or may be purified from parasites isolated from infected host organisms . methods for purifying desired proteins are well known in the art and are not presented in detail here . for a review of standard techniques see , methods in enzymology , &# 34 ; guide to protein purification &# 34 ;, m . deutscher , ed . vol . 182 ( 1990 ), which is incorporated herein by reference . for instance , pfs230 or its homologs in other species can be purified using affinity chromatography , sds - page , and the like . another aspect of the present invention relates to the cloning and recombinant expression of pfs230 and its homologs . the recombinantly expressed polypeptides can be used in a number of ways . for instance , they can be used as transmission - blocking vaccines , as described below . the recombinantly produced proteins can also be used for raising antibodies or for t cell and b cell epitope mapping . in addition , oligonucleotides from the cloned genes can be used as probes to identify homologous polypeptides in other species . the invention relies on routine techniques in the field of recombinant genetics , well known to those of ordinary skill in the art . a basic text disclosing the general methods of use in this invention is sambrook et al ., molecular cloning , a laboratory manual , cold spring harbor publish ., cold spring harbor , n . y . 2nd ed . ( 1989 ), which is incorporated herein by reference . pfs230 was immunoaffinity purified using mab 1b3 as described in detail below . the isolated protein was then digested with trypsin . the tryptic peptides were separated by reverse phase hplc and three well resolved peptides were microsequenced . from this amino acid sequence degenerate oligonucleotide probes were used to screen a p . falciparum sexual stage cdna library . other methods for isolating genes encoding pfs230 and its homologs can also be used . for instance , the amino acid sequence of the n - terminus can be determined and degenerate oligonucleotide probes , designed to hybridize to the desired gene , are synthesized . amino acid sequencing is performed and oligonucleotide probes are synthesized according to standard techniques as described , for instance , in sambrook et al ., supra . oligonucleotide probes useful for identification of desired genes can also be prepared from conserved regions of related genes in other species . for instance , probes derived from a gene encoding pfs230 may be used to screen libraries for homologous genes from other parasites of interest . other methods include the detection of restriction fragment length polymorphisms ( rflp ) between wild type and mutant strains lacking a pfs230 polypeptide . amplification techniques , such as the polymerase chain reaction ( pcr ) can be used to amplify the desired nucleotide sequence . u . s . pat . nos . 4 , 683 , 195 and 4 , 683 , 202 describe this method . sequences amplified by pcr can be purified from agarose gels and cloned into an appropriate vector according to standard techniques . genomic or cdna libraries are prepared according to standard techniques as described , for instance , in sambrook , supra . to construct genomic libraries , large segments of genomic dna are generated by random fragmentation or restriction enzyme degradation and are ligated with vector dna to form concatemers that can be packaged into the appropriate vector . two kinds of vectors are commonly used for this purpose , bacteriophage lambda vectors and plasmids . to prepare cdna , mrna from the parasite of interest is first isolated . eukaryotic mrna has at its 3 &# 39 ; end a string of adenine nucleotide residues known as the poly - a tail . short chains of oligo d - t nucleotides are then hybridized with the poly - a tails and serve as a primer for the enzyme , reverse transcriptase . this enzyme uses rna as a template to synthesize a complementary dna ( cdna ) strand . a second dna strand is then synthesized using the first cdna strand as a template . linkers are added to the double - stranded cdna for insertion into a plasmid or phage vector for propagation in e . coli . identification of clones in either genomic or cdna libraries harboring the desired nucleic acid segments is performed by either nucleic acid hybridization or immunological detection of the encoded protein , if an expression vector is used . the bacterial colonies are then replica plated on solid support , such as nitrocellulose filters . the cells are lysed and probed with either oligonucleotide probes described above or with antibodies to the desired protein . standard transfection methods are used to produce prokaryotic , mammalian , yeast or insect cell lines which express large quantities of the pfs230 polypeptide , which is then purified using standard techniques . see , e . g ., colley et al ., j . biol . chem . 264 : 17619 - 17622 , 1989 ; and guide to protein purification , supra . the nucleotide sequences used to transfect the host cells can be modified to yield the pfs230 polypeptide or fragments thereof , with a variety of desired properties . for example , the polypeptides can vary from the naturally - occuring sequence at the primary structure level by amino acid , insertions , substitutions , deletions , and the like . these modifications can be used in a number of combinations to produce the final modified protein chain . the amino acid sequence variants can be prepared with various objectives in mind , including facilitating purification and preparation of the recombinant polypeptide . the modified polypeptides are also useful for modifying plasma half life , improving therapeutic efficacy , and lessening the severity or occurrence of side effects during therapeutic use . the amino acid sequence variants are usually predetermined variants not found in nature but exhibit the same immunogenic activity as naturally occurring pfs230 . for instance , immunogenically active fragments comprising about 6 to about 300 amino acids are typically used . shorter fragments comprising bout 100 to about 200 amino acids , preferably about 130 to about 160 , may also be used . for use as vaccines , immunologically active fragments are typically preferred so long as at least one epitope capable of eliciting transmission blocking antibodies remains . preferred polypeptide fragments of the invention include those comprising one or more of the six copies of the seven - cysteine motif noted above . other modifications include the addition of a membrane anchoring sequence to the expressed protein . such modifications allow the protein to be expressed on cell surfaces and thereby improve immunogenicity . in general , modifications of the sequences encoding the homologous polypeptides may be readily accomplished by a variety of well - known techniques , such as site - directed mutagenesis ( see , gillman and smith , gene 8 : 81 - 97 , 1979 ) and roberts , s . et al ., nature 328 : 731 - 734 , 1987 ). one of ordinary skill will appreciate that the effect of many mutations is difficult to predict . thus , most modifications are evaluated by routine screening in a suitable assay for the desired characteristic . for instance , the effect of various modifications on the ability of the polypeptide to elicit transmission blocking can be easily determined using the mosquito feeding assays , described in quakyi et al ., supra . in addition , changes in the immunological character of the polypeptide can be detected by an appropriate competitive binding assay . modifications of other properties such as redox or thermal stability , hydrophobicity , susceptibility to proteolysis , or the tendency to aggregate are all assayed according to standard techniques . the particular procedure used to introduce the genetic material into the host cell for expression of the pfs230 polypeptide is not particularly critical . any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used . these include the use of calcium phosphate transfection , spheroplasts , electroporation , liposomes , microinjection , plasmid vectors , viral vectors and any of the other well known methods for introducing cloned genomic dna , cdna , synthetic dna or other foreign genetic material into a host cell ( see sambrook et al ., supra ). it is only necessary that the particular procedure utilized be capable of successfully introducing at least one gene into the host cell which is capable of expressing the gene . the particular vector used to transport the genetic information into the cell is also not particularly critical . any of the conventional vectors used for expression of recombinant proteins in prokaryotic and eukaryotic cells may be used . expression vectors for mammalian cells typically contain regulatory elements from eukaryotic viruses . sv40 vectors include psvt7 and pmt2 . vectors derived from bovine papilloma virus include pbv - 1mtha , and vectors derived from epstein bar virus include phebo , and p205 . other exemplary vectors include pre4 , pmsg , pav009 / a + , pmt010 / a + , pmamneo - 5 , bacculovirus pdsve , and any other vector allowing expression of proteins under the direction of the sv - 40 early promoter , sv - 40 later promoter , metallothionein promoter , murine mammary tumor virus promoter , rous sarcoma virus promoter , polyhedrin promoter , cytomegalovirus promoter , or other promoters shown effective for expression in eukaryotic cells . the expression vector typically contains a transcription unit or expression cassette that contains all the elements required for the expression of the pfs230 polypeptide dna in the host cells . a typical expression cassette contains a promoter operably linked to the dna sequence encoding a pfs230 polypeptide and signals required for efficient polyadenylation of the transcript . the term &# 34 ; operably linked &# 34 ; as used herein refers to linkage of a promoter upstream from a dna sequence such that the promoter mediates transcription of the dna sequence . the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting . as is known in the art , however , some variation in this distance can be accommodated without loss of promoter function . the dna sequence encoding the pfs230 polypeptide will typically be linked to a cleavable signal peptide sequence to promote secretion of the encoded protein by the transformed cell . additional elements of the cassette may include selectable markers , enhancers and , if genomic dna is used as the structural gene , introns with functional splice donor and acceptor sites . enhancer elements can stimulate transcription up to 1 , 000 fold from linked homologous or heterologous promoters . enhancers are active when placed downstream from the transcription initiation site . many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues . for example , the sv40 early gene enhancer is suitable for many cell types . other enhancer / promoter combinations that are suitable for the present invention include those derived from polyoma virus , human or murine cytomegalovirus , the long term repeat from various retroviruses such as murine leukemia virus , murine or rous sarcoma virus and hiv . see , enhancers and eukaryotic expression , cold spring harbor pres , cold spring harbor , n . y . 1983 , which is incorporated herein by reference . in addition to a promoter sequence , the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination . the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes . if the mrna encoded by the structural gene is to be efficiently translated , polyadenylation sequences are also commonly added to the vector construct . two distinct sequence elements are required for accurate and efficient polyadenylation : gu or u rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides , aauaaa , located 11 - 30 nucleotides upstream . termination and polyadenylation signals that are suitable for the present invention include those derived from sv40 , or a partial genomic copy of a gene already resident on the expression vector . efficient expression and secretion in yeast is conveniently obtained using expression vectors based on those disclosed in barr et al ., j . biol . chem . 263 : 16471 - 16478 , 1988 , or u . s . pat . no . 4 , 546 , 082 , which are incorporated herein by reference . in these vectors the desired sequences are linked to sequences encoding the yeast α - factor pheromone secretory signal / leader sequence . suitable promoters to use include the adh2 / gapdh hybrid promoter as described in cousens et al ., gene 61 : 265 - 275 ( 1987 ), which is incorporated herein by reference . yeast cell lines suitable for the present invention include bj 2168 ( berkeley yeast stock center ) as well as other commonly available lines . any of a number of other well known cells and cell lines can be used to express the polypeptides of the invention . for instance , prokaryotic cells such as e . coli can be used . eukaryotic cells include , chinese hamster ovary ( cho ) cells , cos cells , mouse l cells , mouse a9 cells , baby hamster kidney cells , c127 cells , pc8 cells , and insect cells . following the growth of the recombinant cells and expression of the pfs230 polypeptide , the culture medium is harvested for purification of the secreted protein . the media are typically clarified by centrifugation or filtration to remove cells and cell debris and the proteins are concentrated by adsorption to any suitable resin or by use of ammonium sulfate fractionation , polyethylene glycol precipitation , or by ultrafiltration . other routine means known in the art may be equally suitable . further purification of the pfs230 polypeptide can be accomplished by standard techniques , for example , affinity chromatography , ion exchange chromatography , sizing chromatography , his 6 tagging and ni - agarose chromatography ( as described in dobeli et al . mol . and biochem . parasit . 41 : 259 - 268 ( 1990 )), or other protein purification techniques to obtain homogeneity . the purified proteins are then used to produce pharmaceutical compositions , as described below . a further aspect of the invention includes antibodies against pfs230 or its homologous polypeptides . the antibodies are useful for blocking transmission of parasites . thus , antibodies can be used as therapeutic agents to block transmission . the multitude of techniques available to those skilled in the art for production and manipulation of various immunoglobulin molecules can be readily applied to block transmission . as used herein , the term &# 34 ; immunoglobulin &# 34 ; refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes . the recognized immunoglobulin genes include the kappa , lambda , alpha , gamma , delta , epsilon and mu constant region genes , as well as the myriad immunoglobulin variable region genes . immunoglobulins may exist in a variety of forms besides antibodies , including for example , fv , fab , and f ( ab ) 2 , as well as in single chains . for a general review of immunoglobulin structure and function see , fundamental immunology , 2d ed ., w . e . paul ed ., ravens press , new york , ( 1989 ) which is incorporated herein by reference . monoclonal antibodies which bind pfs230 can be produced by a variety of means . the production of non - human monoclonal antibodies , e . g ., murine , lagomorpha , equine , etc ., is well known and may be accomplished by , for example , immunizing the animal with a preparation containing pfs230 . antibody - producing cells obtained from the immunized animals are immortalized and screened , or screened first for the production antibodies which bind pfs230 and then immortalized . for a discussion of general procedures of monoclonal antibody production see harlow and lane , antibodies , a laboratory manual cold spring harbor publications , new york ( 1988 ), which is incorporated herein by reference . it may be desirable to transfer the antigen binding regions of the non - human antibodies , e . g ., the f ( ab &# 39 ;) 2 or hypervariable regions , to human constant regions ( fc ) or framework regions by recombinant dna techniques to produce substantially human molecules . such methods are generally known in the art and are described in , for example , u . s . pat . no . 4 , 816 , 397 , ep publications 173 , 494 and 239 , 400 , which are incorporated herein by reference . alternatively , one may isolate dna sequences which encode a human monoclonal antibody or portions thereof that specifically bind to pfs230 by screening a dna library from human b cells according to the general protocol outlined by huse et al ., science 246 : 1275 - 1281 ( 1989 ), incorporated herein by reference , and then cloning and amplifying the sequences which encode the antibody ( or binding fragment ) of the desired specificity . the pfs230 polypeptides of the present invention are also useful as prophylactics , or vaccines , for blocking transmission of malaria or other diseases caused by parasites . compositions containing the polypeptides are administered to a subject , giving rise to an anti - pfs230 polypeptide immune response . the pfs230 polypeptide - specific antibodies then block transmission of the parasite from the subject to the arthropod vector , preventing the parasite from completing its life cycle . an amount of prophylactic composition sufficient to result in blocking of transmission is defined to be an &# 34 ; immunologically effective dose .&# 34 ; the isolated nucleic acid sequence coding for pfs230 or its homologous polypeptides can also be used to transform viruses which transfect host cells in the susceptible organism . live attenuated viruses , such as vaccinia or adenovirus , are convenient alternatives for vaccines because they are inexpensive to produce and are easily transported and administered . vaccinia vectors and methods useful in immunization protocols are described in , e . g ., u . s . pat . no . 4 , 722 , 848 , incorporated herein by reference . suitable viruses for use in the present invention include , but are not limited to , pox viruses , such as , canarypox and cowpox viruses , and vaccinia viruses , alpha viruses , adenoviruses , and other animal viruses . the recombinant viruses can be produced by methods well known in the art : for example , using homologous recombination or ligating two plasmids together . a recombinant canarypox or cowpox virus can be made , for example , by inserting the gene encoding the pfs230 , immunologically active segment of pfs230 or other homologous polypeptide into a plasmid so that it is flanked with viral sequences on both sides . the gene is then inserted into the virus genome through homologous recombination . the recombinant virus of the present invention can be used to induce anti - pfs230 polypeptide antibodies in mammals , such as mice or humans . in addition , the recombinant virus can be used to produce the pfs230 polypeptides by infecting host cells which in turn express the polypeptide . the present invention also relates to host cells infected with the recombinant virus of the present invention . the host cells of the present invention are preferably eukaryotic , such as yeast cells , or mammalian , such as bsc - 1 cells . host cells infected with the recombinant virus express the pfs230 polypeptides on their cell surfaces . in addition , membrane extracts of the infected cells induce transmission blocking antibodies when used to inoculate or boost previously inoculated mammals . in the case of vaccinia virus ( for example , strain wr ), the sequence encoding the pfs230 polypeptides can be inserted into the viral genome by a number of methods including homologous recombination using a transfer vector , ptkgpt - ofis as described in kaslow et al ., science 252 : 1310 - 1313 , 1991 , which is incorporated herein by reference . the pfs230 polypeptides , or recombinant viruses of the present invention can be used in pharmaceutical and vaccine compositions that are useful for administration to mammals , particularly humans , to block transmission of a variety of infectious diseases . the compositions are suitable for single administrations or a series of administrations . when given as a series , inoculations subsequent to the initial administration are given to boost the immune response and are typically referred to as booster inoculations . suitable formulations are found in remington &# 39 ; s pharmaceutical sciences , mack publishing company , philadelphia , pa ., 17th ed . ( 1985 ), which is incorporated herein by reference . the pharmaceutical compositions of the invention are intended for parenteral or oral administration . preferably , the pharmaceutical compositions are administered parenterally , e . g ., subcutaneously , intradermally , or intramuscularly . thus , the invention provides compositions for parenteral administration that comprise a solution of the agents described above dissolved or suspended in an acceptable carrier , preferably an aqueous carrier . a variety of aqueous carriers may be used , e . g ., water , buffered water , 0 . 4 % saline , 0 . 3 % glycine , hyaluronic acid and the like . these compositions may be sterilized by conventional , well known sterilization techniques , or may be sterile filtered . the resulting aqueous solutions may be packaged for use as is , or lyophilized , the lyophilized preparation being combined with a sterile solution prior to administration . the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions , such as ph adjusting and buffering agents , tonicity adjusting agents , wetting agents and the like , for example , sodium acetate , sodium lactate , sodium chloride , potassium chloride , calcium chloride , sorbitan monolaurate , triethanolamine oleate , etc . for solid compositions , conventional nontoxic solid carriers may be used which include , for example , pharmaceutical grades of mannitol , lactose , starch , magnesium stearate , sodium saccharin , talcum , cellulose , glucose , sucrose , magnesium carbonate , and the like . for oral administration , a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients , such as those carriers previously listed , and generally 10 - 95 % of active ingredient and more preferably at a concentration of 25 %- 75 %. in therapeutic applications , pfs230 polypeptides or viruses of the invention are administered to a patient in an amount sufficient to prevent parasite development in the arthropod and thus block transmission of the disease . an amount adequate to accomplish this is defined as a &# 34 ; therapeutically effective dose .&# 34 ; amounts effective for this use will depend on , e . g ., the particular polypeptide or virus , the manner of administration , the weight and general state of health of the patient , and the judgment of the prescribing physician . the vaccines of the invention contain as an active ingredient an immunogenically effective amount of the pfs230 polypeptides or recombinant virus as described herein . useful carriers are well known in the art , and include , e . g ., thyroglobulin , albumins such as human serum albumin , tetanus toxoid , polyamino acids such as poly ( d - lysine : d - glutamic acid ), influenza , hepatitis b virus core protein , hepatitis b virus recombinant vaccine and the like . the vaccines can also contain a physiologically tolerable ( acceptable ) diluent such as water , phosphate buffered saline , or saline , and further typically include an adjuvant . adjuvants such as incomplete freund &# 39 ; s adjuvant , aluminum phosphate , aluminum hydroxide , or alum are materials well known in the art . in addition , the compositions can be administered in slow release particles as described in langer , science 249 : 1527 - 1533 ( 1990 ). vaccine compositions containing the polypeptides or viruses of the invention are administered to a patient to elicit a transmission - blocking immune response against the antigen and thus prevent spread of the disease through the arthropod vector . such an amount is defined as an &# 34 ; immunogenically effective dose .&# 34 ; in this use , the precise amounts again depend on the patient &# 39 ; s state of health and weight , the mode of administration , and the nature of the formulation , but generally range for the initial immunization ( that is for therapeutic or prophylactic administration ) from about 1 . 0 μg to about 1 mg of peptide for a 70 kg patient , followed by boosting dosages of from about 1 . 0 μg to about 100 μg of peptide pursuant to a boosting regimen over weeks to months . the following examples are offered by way of illustration , not by way of limitation . pfs230 was immunoaffinity purified using monoclonal 1b3 ( mab 1b3 ) ( quakyi , et al ., j . immunol ., 139 : 4213 - 4217 ( 1987 )). it was electroeluted from mab 1b3 - resin prepared as described in williamson , et al ., anal . biochem ., 206 : 359 - 362 ( 1992 ), reduced and alkylated , run in one lane of a 4 % gel and then transferred electrophoretically to nitrocellulose . the band corresponding to pfs230 was excised then digested in situ with trypsin . the tryptic peptides were separated by reverse phase hplc and three well resolved peptides were microsequenced . from this amino acid sequence degenerate oligonucleotide probes were designed utilizing p . falciparum codon bias and used to screen a p . falciparum sexual stage cdna library prepared according to standard techniques . fig1 shows the results of the samples from each of the purification steps which were size - fractionated on a 4 - 20 % sds - polyacrylamide gel and stained with coomassie blue . the lanes in gel are as follows : gamete / zygote extract before 1b3 - sepharose resin ( lane 1 ), proteins that did not bind to the 1b3 - sepharose resin ( lane 2 ), molecular weight standards ( amersham ) ( lane 3 ) and protein electroeluted from 1b3 - sepharose resin ( lane 4 ). the molecular weight ( mr × 10 3 ) is indicated on the left and the position of pfs230 is indicated on the right . oligonucleotide probes from each of the three tryptic peptides hybridized to a 4 . 4 kb insert of an isolated clone . sequencing revealed open reading frames at both the 5 &# 39 ; and 3 &# 39 ; ends of the 4 . 4 kb clone , therefore synthetic oligonucleotides probes corresponding to the ends were used to rescreen the library and obtain overlapping clones that extend the sequence . this process was continued until cdna clones covering the entire 9 . 4 kb open reading frame were isolated . the deduced amino acid sequence of the 9 . 4 kb gene ( seq . id . no . 2 ) contains all 3 tryptic peptides that were microsequenced . the pfs230 rna transcript is 12 . 5 kb and sexual stage - specific as shown in the northern analysis of p . falciparum rna in fig2 . equal amounts of rna were run in each lane ( 1 ) asexual , ( 2 ) gametocytes ( stage 2 & amp ; 3 ), and ( 3 ) zygotes / gametes ( 5 hours post emergence ). the blot was probed with the random - prime labeled 4 . 4 kb insert described above . the message is most abundant in gametocytes . with a long exposure of the northern a faint band can be seen in rna from 5 hour zygotes but there is no band with asexual rna . oligonucleotide probes from the extreme 5 &# 39 ; and 3 &# 39 ; ends of the orf hybridize to what appears to be the same transcript . the 9 . 4 kb open reading frame predicts a protein with a molecular weight of 363 , 243 kda , this is larger than the 260 , 000 and 230 , 000 mr reported for the proteins mab 1b3 recognizes by western blot . only the 230 , 000 band was shown to be radiolabeled when live gametes were surface - labeled with 125 i . since mab 1b3 does not react with reduced pfs230 it has been difficult to obtain an accurate molecular weight of the protein . quakyi , et al ., supra . prior art estimates of the size of the protein have been made with molecular weight standards having molecular weights less than 200 kda . to more accurately determine the molecular weight , radiolabeled pfs230 from surface labeled gametes was carefully sized under reducing conditions using molecular weight markers ranging from 100 , 000 to 500 , 000 . reduced 125 i labeled pfs230 migrated as a 310 , 000 molecular weight band ( fig3 ). to confirm that the cloned gene was indeed pfs230 , antibodies to a 2 . 0 - 2 . 2 kb section of the gene expressed in e . coli as fusions with maltose - binding protein ( rpfs230 / mbp - a e - b , described below ) were used to assay a western blot of triton x - 100 extracted p . falciparum gametes / zygotes . fig4 a and 4b show western blots of triton x - 100 extracted 125 i - surface - labeled gametes reacted with a 1 : 5 , 000 dilution of mpb antisera ( lane 1 ), rpfs230 / mbp - a antisera ( lane 2 ), and rpfs230 / mbp - b antisera ( lane 3 ). also shown is an autoradiograph of rpfs230 / mbp - b ( lane 4 ). when the extract was size - fractionated under nonreducing conditions the rpfs230 / mbp - a and - b antisera recognized bands of 325 , 000 kda and 275 , 000 kda , and under reducing conditions bands of 360 , 000 kda and 310 , 000 kda ( fig4 a and 4b , respectively . neither preimmune sera nor antisera to mbp alone reacted with any specific bands . the lower bands , under both reducing and nonreducing conditions comigrated with 125 i labeled pfs230 ( fig4 a and 4b ). this suggests that only the lower band was exposed on the surface of the gamete . possibly , the 360 , 000 protein is processed to a 310 , 000 form as it is moved to the surface of the gamete . to determine whether the rpfs230 / mbp antisera recognized the native ( nondenatured ) surface form of pfs230 , the antisera was used to immunoprecipitate radiolabeled pfs230 from a triton x - 100 extract of surface - labeled p . falciparum gametes / zygotes ( fig5 ). proteins immunoprecipitated by the following antibodies or antisera were loaded on the gel : mab 1b3 ( lane 1 ), rpfs230 / mbp a antisera ( lane 2 ), rpfs230 / mbp - b ( lane 3 ) and mbp antisera ( lane 4 ). the antibodies and antisera were incubated with a triton x - 100 extract of 125 - i surface labeled gametes and precipitated with protein a - sepharose as described above . the precipitated material was run out on a 4 - 20 % acrylamide gel . the radiolabeled bands were visualized by autoradiography . fig5 shows that 125 i - labeled pfs230 was precipitated by rpfs230 / mbp 1b antisera and monoclonal 1b3 but not mbp antisera . finally , an indirect immunofluorescence assay of intact gametes / zygotes was used to show that rpfs230 / mbp - a and b antisera recognized the surface of live gametes / zygotes ( fig6 a and 7a ). fig6 b and 7b , respectively , are the corresponding bright field image . fig8 a shows the results of the same experiment with mbp antisera . fig8 b is the corresponding bright field image . pfs230 open reading frame was pcr - amplified using a sense primer with a 5 &# 39 ; sma i site encoding amino acids 439 - 444 for rpfs230 / mbp - a or amino acids 2398 - 2405 for rpfs230 / mbp - b , and an antisense primer with a 3 &# 39 ; stop codon followed by a sal i site encoding amino acid 1127 - 1135 for rpfs230 / mbp - a or nucleotides 9607 - 9624 in the 3 &# 39 ; untranslated region for rpfs230 / mbp - b . gel - purified pcr products were ligated into stu i / sma i cut pih - 902 expression vector ( gift of paul riggs , new england biolabs ). iptg - induced rpfs230 - maltose binding protein fusion was purified from an extract of e . coli ( dh10b strain , brl ) on amylose resin and use to immumize nih outbred mice according to the method of rawlings , et al ., j . biol . chem ., 267 : 3976 - 3982 ( 1992 ). although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding , it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 4 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 9636 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : dna ( genomic )( ix ) feature :( a ) name / key : cds ( b ) location : 149 .. 9556 ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 4 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( xi ) sequence description : seq id no : 3 : glugluvalgly ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 8 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( ix ) feature :( a ) name / key : modified - site ( b ) location : 6 ( d ) other information : / product =&# 34 ; other &# 34 ;/ note =&# 34 ; xaa = glu or gly &# 34 ;( ix ) feature :( a ) name / key : modified - site ( b ) location : 7 ( d ) other information : / product =&# 34 ; other &# 34 ;/ note =&# 34 ; xaa = glu or val &# 34 ;( xi ) sequence description : seq id no : 4 : glugluvalglygluxaaxaagly15__________________________________________________________________________