Prokaryotic And Eukaryoyic Expression Of The Shortened Hepatitis B Surface Antigen And Their Antigenic Analysis

Objective: To construct prokaryotic and eukaryocytic expression plasmids of the shortened hepatitis B surface antigen, and express the target proteins by IPTG induced in Escherichia coli. as well as in eukaryocyte (HepG2 and COS-7), then detect their antigenity as a basis study and explore of the choice of immunogen for preventive and therapeutic vaccines of Hepatitis B.Methods: The gene fragments coding 152aa (SI) and 124aa (S2) of the carboxyl terminus of HBsAg were amplified by PCR from plasmid pEcob6 with a pair of primers containing different endonuclease sites and were cloned into multiple cloning sites of plasmid pBKS(+). After identification with both PCR and enzyme digestion methods, accuraterecombinants were sent to detect their sequences. Reported sequences show that the sequencec is the same as the gene we wanted.Prokaryotic expression vectors pET32a(+)-S1 . pET32a(+)-S2 ; PQE30-S1 pQE30-S2 (pET32a(+)-S pQE30-S constracted based on the pBKS-S) and eukaryotic expression vectors pCDNA3.1 -S1 pCDNA3.1 -S2 pCDNA3.1-S had been constructed. Then, expression of recombinant genes in prokaryotic bacteria and eukaryotic cells were performed. Detections with immunocytochemistry, SDS-PAGE and Western blot , ELISA were made.Results:1. The gene fragments SI and S2 were successfully amplified by PCR from plasmid pEcob6 and were also successfully cloned into plasmid pBKS (+). And the recombinant plasmids were selected through identification with PCR ,enzyme digestion methods, and detection of their sequences. Reported sequences show that the sequencec is the same as the gene we wanted.2.Prokaryotic expression vectors pET32a(+)-S 1 pET32a(+)-S2; and PQE30-S1 , pQE30-S2 pET32a(+)-S pQE30-S and eukaryotic expression vectors pCDNA3.1-S1 pCDNA3.1-S2 pCDNA3.1-S had been successfully recombinanted.3.The recombinant plasmids pET32a(+)-S1 pET32a(+)-S2; andPQE30-S1 pQE30-S2 pET32a(+)-S, pQE30-S were transformed into Escherichia coli. BL21 and SG13009 respectively. The target proteins were expressed in their corresponsive host bacteria under the induction of IPTG. Their expression amounts were up to 20% of total bacteria proteins, and their molecular weight was in correspondence with theoretic values. While eukaryotic expression vectors pCDNA3.1-S1 pCDNA3.1-S2 pCDNA3.1-S were expressed in HepG2 and COS-7. Details are as follows:1). The expressed protein of S1 gene can be detected in BL21 (pET32a (+)-SlX SG13009 (pQE30-Sl) and in HepG2 (pCDNA3.1-Sl X COS-7 (pCDNA3.1-S1) cells, which can be detected by HBsAg a monoclonal antibody;2). The expressed protein of S2 gene can be detected in BL21 (pET32a (+)-S2) SG13009 (pQE30-S2) and in HepG2 (pCDNA3.1-S2) COS-7 (pCDNA3.1-S2) , which also can be detected by HBsAg a monoclonal antibody;3). The expressed protein of S gene can not be detected in BL21 (pET32a(+)-S) SGI3009 (pQE30-S) while can be detected in HepG2(pCDNA3. 1 -S) COS-7 (pCDNA3.1 -S) and can be detected byHBsAg a monoclonal antibody.Conclusions: Prokaryotic and eukaryocytic expression plasmids ofthe shortened hepatitis B surface antigen were successfully constracted ,and the target proteins expressed by IPTG induced in Escherichia coli. as well as in eukaryocyte (HepG2 and COS-7), then their antigenity were detected. ALL these as a basis study and explore of the choice of immunogen for preventive and therapeutic vaccines of Hepatitis B.