狐源犬瘟热病毒CDV-FOX-TA株核蛋白基因的原核表达及鉴定

应用RT-PCR技术扩增出犬瘟热病毒(CDV) 核衣壳(N)蛋白基因抗原性好的高保守基因片段，将其TA克隆至pMD18-T载体中，再利用酶切、连接的方法将测序正确的N基因目的片段亚克隆到原核表达载体pET24b中6×His Tag编码基因的上游，并将该重组质粒转化大肠杆菌Rosetta 2 (DE3)株，经IPTG诱导，N基因融合蛋白获得了高效表达。SDS-PAGE 分析和Western blot 分析的结果显示，表达产物的分子质量为15 kD，与CDV标准阳性血清呈阳性反应，间接ELISA结果也表明重组表达产物具有良好的抗原性，能够有效区分CDV标准阳性与阴性血清，表明大肠杆菌表达的CDV N 蛋白在免疫原性上具有与天然N蛋白同样的特性，可作为检测CDV的间接ELISA包被抗原。

Prokaryotic expression and identification of nucleocapsid gene of Canine Distemper Virus CDV-FOX-TA strain from Fox

The high conservative sequence of Canine Distemper Virus (CDV) N gene was amplified with RT-PCR, cloned into pMD18-T vector , and then constructed into upstream site of His Tag coding sequence in prokaryotic expression vector pET24b. The N fusion protein was highly expressed when the recombinant plasmid was transformed E coli Rosetta 2(DE3) strain and induced with IPTG. SDS-PAGE and Western blot showed the 15kD expressed products reacted with standard positive serum against CDV. An indirect ELISA demonstrated recombinant expressed protein had good antigencity and can distinguish effectively anti-CDV positive serum and negative serum. The results showed expressed CDV N protein had similarity of antigencity with natural N protein, and can be used as diagnosis antigen. It provided evidence for establishment of an indirect ELISA method for CDV detection.