Molecular Cloning And Expression Of Combined Vaccine Candidate Protein (Cholera Toxin B Subunit And EG95) And Construction And Transformation Of The Chloroplast Expression Vector To Echinococcosis

Cystic hydatid disease (CHD) is a serious zoonosis caused by infection with the metacestode stage of the typeworm Echinococus granulosus in humans and many livestock such as sheep, goat, cattle, yak and horse et al.., the infection occurs endemically in many countries of the world and is particularly prevalent in the China,Russia, parts of Africa, Australia and the southern zone of South America.Vaccine is an important way to control CDS. Field of CHD vccine research involved dead vaccine, molecular vaccine, synthetic peptide vaccine, DNA vaccine and recombinant Salmonella typhimurium vaccine. Eg95 is an effective candidate vaccine antigen which expessed at the oncosphere stage and can induce a high level of immunologic protection. Eg95 genes are highly conservative among all strains of echinococcus granulosus.Cholera toxin is a heat-labile enterotoxin secreted by vibrio cholerae , the B subunit of it can binding to GM1-ganglioside, a ganglioside exist on surface of most mammalian cell. The B subunit of cholera toxin can elicit strong immune response and be used as adjuvant or carrier protein of antigen.By RT-PCR, we amplified the cDNA of EG95 from the oncospheres of Echinococcus granulosus and constructed pUC57/EG95 vector. As well as using specific primer amplified CTxB gene from vibrio cholerae (strain) DNA extraction. The fusion gene of CTxB and EG95 was constructed by recombinant DNA technique, then was subcloned into the Escherichia coli expression pET28a(+) plasmid and transformed to E. coli DH5a, After that, recombinant was obtained by antibiotic selection. DNA sequencing for the recombinant plasmid demonstrated the obtained fragment are completely identic with previous expected design.The CTxB part of the fousion gene was 375bp, terminal codon TGA was deliberately knocked out by designing primer in order to construct fusion gene. The results showed that the EG95 cDNA with a 471 bp of open reading frame, was 481 bp in length and the sequence of which was the same as that from the Australian strain of GenBank. The total CTxB-EG95 gene of recombinant was 846 bp, which was expected for expression of a 31 KDa CTxB-EG95 fusion protein . The recombinant transformants of E. coli BL21 were induced with IPTG. by SDS-PAGE analysis, the molecular weight of the 31 KDa expressed fusion protein in recombinant E. coli was verified, which were the same as the expected results. The Western-blotting test showed that the fusion protein could be recognized by the polyclonal antibodies of CT. All of above results demonstrated the expected fusion antigen protein CTxB-EG95 have been correct expressed.Restriction enzymes digested fusion gene CTxB-Eg95 was ligated with chloroplast expression vector pLCT-B which containing chloroplast gene rp12-trnH-psbA-trnK, promoter and terminator of chloroplast gene PpsbA, selective labeled gene aadA. Then a chloroplast transformation and expression vector pLCT-B/Eg95/ctxB was constructed and transformated by gene-gun method. into tobacco and medicago et al. transformated materials are selecting on induction nutrient medium which containing spectinomycin. The current result is: tobacco was selected by 500mg/L spectinomycin for 2~3 weeks, induced a mosaic of white and green leaves;Alfalfa was selected by 10mg/L spectinomycin for 5 weeks, in addition to the albinism leaves, the remaining had formed meat yellow callus;Cole was selected by lOmg/L spectinomycin for 3~4 weeks, induced albinism adventitious bud and green adventitious bud.