Construction, Selection And Immunogenicity Of Recombinant Fowlpox Vaccine Co-expressing HIV-1Gag And Gp145

Objective: To construct and select expressing HIV-1gag and HIV gp145protein inrecombinant fowlpox virus(rFPV) vaccine for Chinese population.Methods: Primers were designed and synthesized. HIV-1gag and gp145were amplified byPCR and ligased with pMD18-T Simple Vectors respectively. The sequencing results of HIVgag and gp145were correct, and then they were inserted into pTKET which was made in ourlaboratory to construct the recombinant plasmid pTKET-HIV-1gag-HIV gp145. The plasmidpTKET-HIV-1gag-HIV gp145and282E4strain fowlpox virus were co-transfected80%confluent Chicken Embryo Fibroblast (CEF) cells to select the rFPV with EGFP as thereporter gene. The rFPV which could express HIV-1gag, gp145and EGFP genes was got byselecting, and then the reporter gene was knocked out by Cre/Loxp system and got the rFPVwithout EGFP. The rFPV was verified and identified by PCR, RT-PCR and Western blot, thenexogenous gene of the rFPV was analyzed for genetic stability. On the basis of above research,the immunogenicity and safety of rFPV was analyzed through measuring the level ofHIV-specific antibody, the interferon enzyme-linked immune spot test, and detecting residualvirus in BALB/c mice models.Results: The target gene was obtained successfully after PCR amplification and genesequencing were validated, the recombinant plasmid with HIV-1gag, HIV gp145and EGFPgenes was constructed successfully. The rFPV was gained by homologous recombination, thegenes have been integrated into rFPV genome by PCR after10times plaques screening, andthe results of RT-PCR and Western blot showed that HIV-1gag and gp145were successfullyexpressed in infected cells. The rFPV which was continuously passaged20times also showedexogenous genes integration, transcription and expression by PCR, RT-PCR and Western blot.FPV-TK and EGFP were not amplified by PCR and showed that the HIV-1gag and gp145genes have good genetic stability in rFPV which have been purified. The result of measuringthe level of HIV-specific antibody, the iInterferon enzyme-linked immune spot test andresidual virus showed that the rFPV vaccine has good immunogenicity and safety.Conclusion: The rFPV expressing HIV-1gag and gp145protein was successfully generated,which has good immunogenicity and safety in BALB/c mice models. This experimentprovides a foundation for the following clinical study about HIV rFPV vaccines.