SARS-N蛋白真核表达质粒的构建及其免疫原性

目的　选取SARS CoVN蛋白编码基因为研究对象 ,构建真核表达质粒PVAX1/N ,并在体外转染COS 7细胞的基础上免疫Balb/c小鼠 ,测定血清总IgG水平及诱发的细胞免疫反应。方法　用RT PCR方法从感染SARS病毒的VeroE6细胞中扩增得到约 12 6 9bp的片段 ,构建重组真核表达质粒PVAX1/N ,转染COS 7细胞后 ,用细胞免疫化学方法鉴定目的蛋白的表达 ;免疫Balb/c小鼠后 ,用ELISA方法测血清总IgG水平 ,MTS/PES比色法测定脾T淋巴细胞增殖 ,ELISPOT法测定CD8+ T淋巴细胞的活性。结果　构建的重组质粒经酶切及测序鉴定 ,与GenBank中登录的序列完全一致 ;细胞免疫化学鉴定结果显示 :重组质粒可在COS 7细胞中表达有免疫原性的SARS CoVN蛋白 ;免疫小鼠血清中检测到较高滴度的抗体 ;T淋巴细胞增殖及CTL活性均明显优于对照组 (P <0 .0 1)。结论　成功构建真核表达质粒PVAX1/SARS CoVN ,并能诱导免疫小鼠产生特异性的抗SARS抗体及特异性的细胞免疫反应

Construction of PVAX1/SARS-CoV N and its immunogenicity

Objective To construct eukaryotic expression vector PVAX1/SARS-CoV N, to immunize Balb/c mice after infection of COS-7 in vitro, and to assay the total serum IgG levels and the cellular responses induced by the recombinant plasmid. Methods SARS-CoV N gene (1 269 bp) was amplified from SARS-CoV-infected Vero E6 cells by RT-PCR, then inserted into the constructed eukaryotic expression vector PVAX1/N. The expressions of N protein in infected COS-7 cells were detected by immunocytochemistry and SARS-specific antibody in serum of immunized Balb/c mice was detected by ELISA. T lymphocyte proliferation and CD8 + CTL were detected by MTS/PES and ELISPOT, respectively. Results The recombinant plasmid was confirmed using enzyme digestion and DNA sequencing. N protein with immunogenicity in COS-7 cells was fund and SARS-specific antibodies in serum of the mice were detected. T lymphocyte proliferation and activity of CTL were superior to that of the control group (P < 0.01). Conclusion The eukaryotic expression plasmid PVAX1-N has been constructed successfully, which could induce SARS-specific antibody and cellular immunological reactions.