Construction And Preliminary Evaluation Of A Vibrio Cholerae Serogroup O139 Vaccine By Genetic Engineering

Background and obejectiveCholera is caused by Vibrio cholerae,resulting to a severe infectious diarrheal disease that may spread rapidly.The world's seventh pandemic,has not yet been effectively controlled,spreading to the world's five continents,more than 100 countries since 1961.There are approximately 200 serogroups recognized 'O' serogroups of V.cholerae,only serogroups O1,containing classical and El Tor biotype,as well as O139 leading to Cholera.The classical biotype,the sixth pandemic strain,was responsible for cholera epidemics until 1961,when the El Tor biotype displaced it and started the seventh pandemic,which is still continuing.In 1992,a newly recognized serogroup,O139,emerged,and resulted in massive cholera epidemics in the Indian subcontinent,and there is low cross-protection between V. cholerae serogroups O139 and O1 vaccine.Several lines of evidence have suggested that strain O139 arose from an El Tor biotype by acquiring the O139 biosynthesis gene cluster from a non-O1 strain.Thus,O139 combines the virulent properties of O1 strains and the presence of a capsule of a non-O1 strain.The main pathogenic factor of Vibrio cholerae are cholera toxin(CT),encoded by ctxAB gene on the CTX element,which is influenced and regulated by environmental cues.CTX element comes from bacteriophage CTXΦ,and is highly probable to be transferred among diverse V.cholerae strains in the environment. CTXΦis a single- stranded filamentous bacteriophage with a small genome of 6.9 kb, and is composed of two parts,phage regulatory genetic element RS2,of 2.4kb,and a core region of 4.5 kb.The RS2 element contains three genes 59-rstR-rstA-rstB-39, while the core region harbours six genes,including the ctxAB operon.CT composes of five surrounding B subunit of a toxic A subunit.When CT binds GM1(ganglio-side),it begins to affect intestina parva,enhancing the enzyme activity of adenylate cyclase and then inner intestinal cyclic adenosine monophosphate (cAMP) levels and secreting chloride and bicarbonate into the intestina parva, resulting in water coming out from blood vessels and extracellular space into enteric cavity quickly,named watery diarrhea.TLC(toxin-linked cryptic) factor and the RTX(repeat in toxin) gene cluster locate on the upstream and downstream of CTX unit.TLC locates at 842 bp upstream of RS1 region,and its function is related to acquirement and duplication of the CTXΦ. TLC is the form of pTLC,a 4.7 kb plasmid integrating into the chromosome.There are cri and orf2-3 factors associated with the acquirement and duplication of the CTXΦin pTLC.Because TLC function is related to acquirement and duplication of the CTXΦ,in the TCP-positive and CTX-negative strains,it is possible that obtaining TLC is an intermediat step to acquire CTXΦ.TLC component may make attRS1 insertting in Vibrio cholerae chromosome I so that Vibrio cholerae can acquire CTXΦphage.Because of that,Faruque SM infer that TLC components of Vibrio cholerae is a conditions of the seventh pandemic.Another large gene cluster of RTX (repeat in toxin) is located downstream of the CTX element 693 bp Agency,which related to cytotoxicity,and its activity is not dependent on CTX,belong to RTX toxin family.Because of TLC,CTX and RTX gene clusters are linked together,they can be deleted together through homologous recombination.Gene knock-out technology began to develop since the late 80's of 20th century and have a broad application prospects in the life sciences domain.Homologous recombination is a method of gene knock-out.And the suicide plasmid is effective carrier to build seamless Mutant through homologous recombination,precisely positioning the integration site by homologous fragment of the both ends of the target gene.Using the "upstream fragment - inserted gene - the downstream fragment" model can avoid polar effect of strain and inactivate target genes completely.With the development of the intestinal mucosal immunity,molecular biology of Vibrio cholerae as well as gene recombination technology,it is gradually tending to develop attenuated live V.cholerae vaccine candidates through genetic engineering methods to delete cholera toxin and other virulence-associated genes.There are CVD103- HgR,Peru-15 and V.cholerae 638 belonging to attenuated live Vibrio cholerae serogroup O1 vaccine candidate and Bengal-15 and CVD 112 belonging to attenuated live Vibrio cholerae serogroup O139 vaccine candidate.These vaccines evoke an immune response similar to that of natural cholera infection,but exhibit some side reactions in humans.Besides,an attenuated vaccine strain could reacquire cholera toxin genes and become virulent after a subsequent phage infection.In this study,through gene recombination technology,the major virulence gene cluster TLC,CTX and RTX on chromosome of ZJ199693 was knockouted,and the genes ctxB and rstR were inserted instead.Further more,the gene of green fluorescent protein(GFPuv) will be used to substitute hlyA,which codes hemolysin.Thus,we will construct an attenuated live Vibrio cholerae serogroup O139 vaccine candidate.Our aim is to delete major virulence genes of Vibrio cholerae serogroups O139,minimize the side reactions and toxicity reversion possibility of vaccine candidates,and easy to distinguish between vaccine candidate strains and wild strains in environment through the integration of green fluorescent protein.The function of TLC is related to acquirement and reproduction of CTXΦ,while CTX gene cluster carries an important virulent gene named ctxAB that encodes cholera toxin(CT) and attB site for the CTXΦintegration.In addition,the RTX toxin can cause cell-rouding pathogenic effects by cross-linking actin in mammalian cells, so it can make hep-2 cell toxic.Besides,both RTX toxin and hemolysin can cause intestinal infection in mice,and are primary factors that cause the side effect and reactionogenicity of Vibrio Cholerae live attenuated vaccine.After deleting TLC, CTX,RTX gene clusters and hlyA gene,toxicit and its possibility to reversion of Vibrio cholerae will be mostly reduced.Moreover,the product that rstR gene encodes can inhibit the transfection to Vibrio cholerae of the same type of phage,reducing the possibility for the strain to regain toxicity so that the vaccine will be enhanced its bio-safety,and the cholera toxin B subunit which ctxB gene encodes may give human body antitoxic immunity to the candidate vaccine strain.The fluorescent green protein(GFP) that extracted from Aequorea victoria shows a maximum excitation under 395-498 nm ultraviolet light(UV).The recombinant fluorescent green protein(GFPuv)expressed by transformed (pGFP,Clontech) cells of Escherichia coli,was developed by introducing point mutations in an wild GFP DNA,replacing three amino acids(Phe99 for Ser;Met153 for Thr;and Val163 for Ala,based on the amino acid numbering of wild GFP).The GFPuv is expressed faster in E.coli strains and shows brighter fluorescence than the native GFP,the maximum peaks for fluorophore excitation being at 360-400nm.The recombinant GFPuv is a globular protein,made up of 27 kDa monomers(with 238 amino acids).The protein fluorescence is stable between pH 5.5 and 11.5,with the optimum at pH 8.0.When the encoding gene of green fluorescent protein is inserted into the chromosome of vaccine candidate,the strain emit green fluorescent at 360-400nm UV excitation,which benefic to detect vaccine candidate strain in the environment.Methods1.Primers were designed according to the Genbank of N16961,El Tor biotype of Vibrio cholerae,and pGFPuv nucleotide sequence.2.The upstream fragm- ent of TLC gene cluster(TLCup),downstream fragment of RTX gene cluster(RTXdown) and the chloromycetin resistance(cat) gene were PCR amplified and ligated with the plasmid pU18 to create recombinant plasmid pUC18-TLCup-cat-RTXdown.Then the fragment containing TLCup- cat -RTXdown fusion was excised and cloned into the suicide plasmid pDS132 to create the recombinant suicide plasmid pDS132-TLCup- cat -RTXdown.3.The recombinant suicide plasmid pDS132-TLCup- cat -RTXdown was conjugal transferred into the O139 Vibrio cholerae ZJ199693,and the deletion mutant grows in a modified LB medium containing 20%sucrose and chloromycetin,tested by PCR and sequencing.4.The upstream fragment of TLC gene cluster(TLCup),downstream fragment of RTX gene cluster(RTXdown) and rstR gene as well as ctxB gene were PCR amplified and ligated with the plasmid pUC18 to create recombinant plasmid pUC18-TLCup- rstR- ctxB- RTXdown.Then the fragment containing TLCup- rstR-ctxB -RTXdown fusion was excised and cloned into the suicide plasmid pCVD442 to create the recombinant suicide plasmid pCVD442-TLCup- rstR-ctxB-RTXdown.5.The recombinant suicide plasmid pCVD442-TLCup- rstR-ctxB-RTXdown was conjugal transferred into the former deletion mutant and the latter deletion mutant will grow in a modified LB medium containing 20%sucrose and chloromycetin,tested by PCR and sequencing.6.The toxin test of small intestine segment of rabbit by ligation Cholera toxin which is also called CT,encoded by ctxAB gene,is the most important factor to cause cholera.Vibrio cholerae was injected into the small intestine segment of rabbit by ligation,and then whether there is any congestion and effusion in small intestine segment would be observed at the second day,to determine if the Vibrio cholerae is toxin- producing.7.Hep-2 cytotoxicity test RTX toxin can cause cell-rouding pathogenic effects by cross-linking actin in mammalian cells,so the toxic gene deletion strain is examined by hep-2 cytotoxicity test.8.GM1-ELISA The CTB protein,cholera toxin B subunit,has the ability to bind GM 1(ganglioside),so CTB expression of recombinating strain that reacquire ctxB is detected by GM1-ELISA.9.the upstream fragment of hlyA gene(hlyAup),downstream fragment of hlyA gene cluster(hlyAdown) and lacz-GFPuv gene were PCR amplified and ligated with the pUC18 to create recombinant plasmid pUC18-hlyAup-laczGFPuv-hlyAdown. Then the hlyAup-laczGFPuv-hlyAdown fragment was PCR amplified and excised and cloned into the suicide plasmid pCVD442 to create the recombinant suicide plasmid pCVD442- hlyAup-laczGFPuv-hlyAdown.Results1.The recombinant suicide plasmid pDS132-TLCup-cat-RTXdown was verified by PCR and enzyme digestion.The deletion of TLC,CTX and RTX gene clusters of O139 serogroup Vibrio cholerae ZJ199693 was identificated by PCR and sequencing,and deletion mutant 9693-1 was gained.2.The recombinant suicide plasmid pCVD442-TLCup- rstR-ctxB-RTXdown was verified by PCR and enzyme digestion.The deletion of TLC,CTX and RTX gene clusters and insertion of rstR and ctxB genes on chromosome were identificated by PCR and sequencing,and vaccine candidate O139-45 was gained.3.The toxin test of small intestine segment of rabbit by ligation showed that deletion mutant 9693-1 and vaccine candidate O139-45 were deleted of the chief toxin gene ctxAB.4.The toxic effect of TLC,CTX and RTX gene deletion mutant 9693-1 on Hep-2 cells was negative.5.GM1-ELISA showed that it was well for ctxB to express in vaccine candidate O139-45.6.The recombinant suicide plasmid pCVD442- hlyAup-lacz-GFPuv-hlyAdown was verified by PCR and enzyme digestion.E.coli carrying plasmid pCVD442- hlyA up-lacz- GFPuv-hlyAdown emits green fluorescent by the long-wave UV excitation, which indicates that it is well to express green fluorescent protein.Conclusion1.Construction of a Vibrio cholerae Serogroup O139 Vaccine by Genetic Engineering Vaccine candidate O139-45 was constructed that TLC,CTX and RTX gene clusters was deleted while ctxB and rstR was integrated in the same site on chromosome DNA,and it can be a vaccine candidate strain for further evaluation of immune effects. 2.Preliminary Evaluation of a Vibrio cholerae Serogroup O139 Vaccine by Genetic Engineering The toxin test of small intestine segment of rabbit by ligation showed that deletion mutant 9693-1 and vaccine candidate O139-45 were deleted of the chief toxin gene ctxAB.The toxic effect of TLC,CTX and RTX gene deletion mutant 9693-1 onHep-2 cells was negative.GM1-ELISA showed that it was well for ctxB to express in vaccine candidate O139-45.3.Construction of suicide plasmid contained Upstream and downstream gene fragements of hemolysin and GFPuv The recombinant suicide plasmid pCVD442-hlyAup-lacz-GFPuv-hlyAdown was constructed,which can express green fluorescent protein well.It can be used to further recombination of the Candidate vaccine strain O139-45,knockouting hlyA gene and inserting GFPuv gene,so that candidate vaccine can be observed green fluorescence through the long-wave Ultraviolet irradiation,which benefit to detect vaccine candidate strain in the environment.