Study Of Heat Shock Protein70 Recombinant Antigen Of Echinococcus Granulosus

Objective To clone the heat shock protein 70 (HSP70) gene of Echinococcus granulosus (Eg) , construct the recombinant plasmid , express prokaryotically , purify the recombinant protein , identify the immunogenicity of its recombinant protein and lay bases for screening candidate antigen of Eg. Methods①Total RNA was extracted from protoscoles of cysts.The HSP70 gene of Eg was amplified by RT-PCR and recombined into pGEM-T vector for sequencing and analyzing.②The sequence was analyzed and forcasted by DNAStar analytical program and SWISS-MODEL serve in internet.③The recombinant protein was obtained after the EgHSP70 gene had been separated from HSP70/ pGEM-T/JM109, inserted into expressive plasmid vector pGEX-6P-1 and expressed in BL21 under the induction of IPTG. Results of the induced expression level was detected by SDS-PAGE. The recombinant protein was purified by affinity chromatography and its biological activity was tested by Western blot analysis.④The purified proteins were injected into mouse . The immunogenicity of the specific antibody and its level was identified by Western blot and ELISA. Results①A cDNA sequence of HSP70 gene which open reading frame is 402bp, has been amplified successfully by RT-PCR. Compared with the published DNA sequence in the Genbank , the homologies are 96%,which deduced that the amino acid sequence of HSP70 of Echinococcus granulosus are 81% identities.②The result showed that the EgHSP70 consists of 133 amino acids sequence which molecular weight was 14.53KDa. Compared with the memmbers of heat shock protein70 ,the homology of amino acid was high conservative . As a result ,the epitope of EgHSP70 from the DNAStar accord with the homology of the peptide.③The recombinant plasmid HSP70/pGEX-6P-1/BL21 has been constructed successfully and confirmed by restriction endonuclease analysis and DNA sequencing analysis . A predominant protein band at～40KD is present in cell lysates obtained after IPTG induction. EgHSP70 which molecular weight is 14 KDa was purified by affinity chromatography.By Western blot analysis, its immune characteristics were identified preliminarily ,which revealed that the recombinant protein could be recognized by the sera from the rabbits immunized with Eg.④With Western blot and ELISA detected , the results showed that the purified protein and crude proteins including protoscoles and cystic fluid, could be recognized by its HSP70-immunized mouse.From the level of IgG analysis,It was concluded that immunizing the mice with recombinant EgHSP70 vaccine could elicit the humoral response. Conclusion①The HSP70 gene sequence of Eg was cloned successfully and its reading frame is integrity.②The recombinant plasmid HSP70/pGEX-6P-1/BL21 was successfully constructed and EgHSP70 gene could be expressed in E.coli BL21 with high efficiency. And the expressed proteins possess higher immunogenicity and may be a new vaccine candidate.③By the animal experiment,we ensured that the recombinant protein EgHSP70 had higher immunoenicity and may be a new vaccine candidate.④By literature reveiew, there is no report concerning the cloning and expresstion and characteristic indentification of EgHSP70 in China.