小反刍兽疫病毒Ｍ基因的截短表达及 多抗血清的制备

目的截短表达小反刍兽疫病毒M蛋白基因并将其用于多抗血清的制备。方法之前有研究未能表达完整的M蛋白,而若在抗原性较弱的区域将其一分为二,以截短的形式进行表达,却能达到较理想的水平。因此根据GenBank上公布的PPRV M基因的序列,设计1对特异性引物,扩增出480bp的目的基因,将其克隆至原核表达载体pET-32a(+)中,得到重组表达质粒pET-32a-PPRV-M1,后转化至Rosetta感受态细胞中,IPTG诱导表达后,通过SDS-PAGE和Western blot试验对重组蛋白进行鉴定,将纯化后的重组蛋白免疫6周龄BALB/c雌鼠,制备多克隆抗体血清。结果经SDS-PAGE及Western blot鉴定,证明截短的M基因的蛋白主要以包涵体形式高效表达并具有良好的反应原性。结论成功克隆表达了截短的小反刍兽疫病毒M基因的蛋白并制备了多抗血清,为建立血清学相关的检测方法及临床治疗奠定了基础。

Expression of truncated M gene of Peste des petits ruminants virus and preparation of the polyclonal antibody serum

Abstract: Objective To express truncated M gene protein of Peste des petits ruminants virus (PPRV) for the preparation of polyclonal antibody serum. Methods According to the sequence of PPRV M gene published in GenBank, a pair of specific primers was designed to amplify a 480 bp target gene which was then cloned into the prokaryotic expression vector pET-32a(+). The resulted recombinant plasmid pET-32a-PPRV-M1 was transformed into Rosetta competent cells. The expression of the recombinant protein was induced by using IPTG, and identified by using SDS-PAGE and Western blot. The purified recombinant protein was used to immunize six-week-old BALB/c female mice to prepare polyclonal antibody serum. Results The results of SDS-PAGE and Western blot showed that the truncated M gene protein was mainly expressed in the form of inclusion bodies with good reactivity. Conclusion The protein of truncated PPRV M gene was successfully cloned and polyclonal antibody serum was prepared, which laid the foundation for the establishment of serological related detection methods and clinical treatment.