SARS病毒s1基因片段真核表达载体的构建及免疫效果

Construction of the Eukariotic Expression Vector for s1 Gene Fragment of SARS-CoV and Study of Its Immunological Effects

本研究根据GenBank登录的BJ01株SARS-CoV序列合成801bpS1基因片段,该片段被亚克隆至真核表达载体pcDNA3.1(+)得到重组质粒pcDNA3.1(+)/S1;转染Hela细胞,SDS-PAGE、Western-Blotting鉴定蛋白表达;肌注免疫BALB/c小鼠,利用ELISA法检测免疫后小鼠的抗SARS-CoVIgG及IFN-γ水平,MTT法检测T细胞增殖活性。结果显示,重组质粒pcDNA3.1(+)/S1可在Hela细胞内表达S1蛋白,免疫后小鼠的T细胞增殖活性增强,抗SARS-CoVIgG与IFN-γ水平升高。本实验说明pcDNA3.1(+)/S1可诱导小鼠产生一定的体液免疫和细胞免疫应答。

The 801 base pairs of S1 gene fragment were synthesized based on the Sever acute respiratory syndrome associated coronavirus（SARS-CoV）BJ01 strain registered in GenBank. The synthetical DNA was subcloned into the appropriate site of pcDNA3.1 （＋） eukariotic expression vector to construct a recombinant plasmid pcDNA3.1（＋）/S1;The recombinant plasmid pcDNA3.1（＋）/S1 was transfected into Hela cells and the expressed protein was identified By SDS-PAGE and Western-blotting;BALB/c mice were immunized with pcDNA3.1（＋）/S1 by i. m. The level of anti-SARS-CoV IgG and IFN-γ, in BALB/c mice after immunization were detected by ELISA and T cell proliferation activity was tested by MTT. It was found that the recombinant plasmid pcDNA3. 1 （＋）/S1 could express S1 protein in Hela cells, T cell proliferative activity, the level of anti-SARS-CoV IgG and IFN-γ increased after immunization. It revealed that pcDNA3.1 （＋）/S1 could induce moderate cellular and humora immunological reaction in BALB/c mice.