Construction Of PcDNA3.1(+)/S1 Eukariotic Expression Vector And Detection Of Its Immunological Activity

Objectives:801 base pairs(22382bp~23182bp) of SARS-CoV S1 gene fragment was synthesized aaccording to sequence of SARS-CoV BJO1 putative S gene (AY2784884, Genbank). A gene S1 of SARS associated coronavirus expressing eukariotic vector was constructed by molecular biological methods. BALB/c mice were immunized with pcDNA3. 1(+)/S1 by i.m. The levels of humoral and cellular immunity pcDNA3.1(+)/S1 induced in BALB/c mice were detected. These results provided foundation for further studies on biological activities of SARS-CoV and for development of DNA vaccine of SARS.Methods:A pair of primers was synthesized after being designed by PRIMER5.0 software aaccording to sequence of SARS-CoV BJOl putative S gene. The S1 gene fragment was amplified by PCR. The PCR products were purified and cloned into pUCm-T vector. After cleavage and sequencing, the S1 gene fragment was connected to pcDNA3.1(+) in the correct orientation. Recombinant plasmids was prepared and transinfected into Hela cells. The expressed protein was identified by immunocytochemistry and Western-Blotting. 6 weeks old BALB/c mice were immunized with pcDNA3. 1 (+)/S1; After 6 weeks, expression of the S1 gene on BALB/c mice was indentified by PCR and immunohistochemistry. The proliferation response of spleen cells was detected by MTT. Enzyme-linked immunosorbent assay(ELISA) was used for the quantitative detection of the cytokines IFN-γ in murine spleen lymphocyte culture medium after stimulating and anit SARS-CoV IgG in Sera.