水貂肠炎病毒VP2基因的原核表达及多克隆抗体的制备

Prokaryotic expression of VP2 gene of mink enteritis virus and preparation of anti-serum

利用原核表达系统表达水貂肠炎细小病毒(MEV)VP2衣壳蛋白,以制备抗VP2超免疫血清。根据VP2的基因序列,设计1对特异性引物,以MEV全基因组重组质粒p B-MEV-L为模板,利用PCR技术扩增出MEV VP2片段,并定向克隆到pET-32a载体中,经酶切与序列测定,证明成功构建了VP2基因原核表达质粒pET-MEV-VP2。将其转化大肠杆菌BL21(DE3),经IPTG诱导后实现了高效表达。免疫印迹试验表明,获得的重组蛋白具有较好的反应原性。以该蛋白为免疫原免疫新西兰兔,制备抗血清,四免后用ELISA方法检测其效价达到1∶1 024。免疫荧光试验证明,获得的抗体具有良好的结合活性。为MEV的病原生物学研究奠定基础。

The study aims to express the capsid protein VP2 of mink parvovirus in Escherichia coli（E.coli） and prepare its anti-serum. At first,according to the sequence of VP2 gene,two specific primers were designed. The recombinant plasmid pB-MEV-L with full length MEV genome was used as template in PCR amplification. After insertion of VP2 gene into prokaryotic expression vector pET-32a,the recombinant plasmid pET-MEV-VP2 was constructed successfully. Then transformation of pET-MEV-VP2 into E.coli BL21（DE3） and high expression was induced by IPTG. After 4 times immunization with the purified VP2 protein,the antiserum titer for ELISA was 1 ： i 024. The result of immunofluorescence assay showed that the rabbit antiserum had a good combined activity. All the results laid a solid foundation for further study of the biological characteristics of MEV.