小反刍兽疫病毒M基因截短表达及鉴定

目的将小反刍兽疫病毒M蛋白基因截短表达后用于特异性单抗制备及临床抗体水平检测。方法:用在线分析软件BepiPred分析小反刍兽疫病毒M蛋白潜在的B细胞线性表位,以本实验室构建的pCR2.1T-PPRV M质粒为模板,扩增三段截短的M基因,纯化后的PCR产物分别与克隆载体pCR2.1T连接,筛选出的阳性重组质粒经双酶切后,分别与表达载体pET-32a(+)及pGEX-6p-1连接,再将鉴定为阳性的重组质粒转化入E.coli BL21(DE3)菌株诱导表达,并用SDS-PAGE及Western blot验证。结果 PCR产物电泳,得到与预期大小相符的特异性片段。对连接克隆载体及表达载体的重组质粒双酶切后,均出现与预期一致的片段,DNA测序表明,插入片段序列与小反刍兽疫Nigeria75/1株M蛋白基因完全一致。重组蛋白经SDS-PAGE及Western blot鉴定,证明所构建的重组蛋白均获得高效表达,并均具有良好的反应原性。结论成功表达了小反刍兽疫截短M基因的蛋白,为制备特异性单抗及小反刍兽疫抗体检测奠定了一定基础。

Truncated Expression and Identification of the Peste des Petits Ruminants Virus M Gene

Objective Truncated expression of the M protein gene of peste des petits ruminants virus for specific monoclonal antibody preparation and the clinical antibody level detection. Methods The potential B-cell linear epitopes peste des petits ruminants virus M protein were analyzed by online analysis software BepiPred and were amplified by PCR with pCR 2.1T-PRRV M plasmid as model.Then the amplified products were insered into the cloning vector pCR 2.1 T. Positive recombinant plasmid were cutted by enzyme digestion and insered into the prokaryotic expression vector pET-32a （＋）andpGEX-6p-1. Positive recombinant plasmid were transformed into E.coli BL21（DE3 ）and were induced to express with IPTG and the truncated M protein expression was analysed with SDS-PAGE and Western blot.Results Electrophoresis of the PCR product showed specific fragment was acquired. By restriction enzyme digestion, the recombinant plasmid were digested into the fragments in line with the expectation and the recombinant proteins were in high-level expression .Conclusion The truncated M protein of peste des petits ruminants virus was successfully expressed, thus providing the foundation for the preparation of specific monoclonal antibodies and antibody detection of peste des petits ruminants.