犬瘟热病毒核衣壳蛋白保守区的表达与鉴定

Expression of the conserved region of nucleocapsid protein of canine distemper virus

根据发表的犬瘟热病毒基因序列设计一对引物,利用PCR方法从重组质粒pcDNA-N中扩增到CDVNP保守区基因片段,并按预定的阅读框架插入表达质粒载体pGEX-6p-1中的谷胱苷肽转移酶(GST)基因的下游,获得重组质粒pNP-CDV并转化大肠杆菌BL21。序列测定表明,克隆到的基因片段大小为1065bp,与已发表的毒株核衣壳蛋白基因序列具有很高的同源性。通过对菌体裂解物的SDS-PAGE、Wester nblot鉴定以及IFA试验,证明携带重组质粒pNP-CDV的大肠杆菌的可以表达融合蛋白形式的核衣壳蛋白保守区域(命名为GST-NP,分子量约为64ku),该融合蛋白具有免疫原性和对CDV高免血清的反应活性。GST-NP的表达,将为临床检测CDV抗体提供诊断抗原。

A fragment of the NP-gene of canine distemper virus （CDV） was amplified from the recombined plasmid pcDNA-N using a pair of primers based on the conserved region of nucleocapsid protein gene. The PCR products were cloned into plasmid vector pGEX-6p-1 with the 5＇ terminus of the gene that code for the conserved region of nucleocapsid protein was genetically fused to the 3＇ terminus of the gene coding for the enzyme glutathione S-transferase （GST）. The resulting plasmids containing the conserved region of nucleocapsid protein gene of CDV was designed as pNP-CDV. Sequence analysis showed that the gene was 1065 bp length and shared 100 % identity with the published sequence. The pNP-CDV vector was transferred into E.coli BL21 and high level expression of a 64 ku fusion protein （GST-NP） was detected by SDS-PAGE analysis, Western blot and immunofluorescence assay. The expressed protein was able to react with polyclonal antibody against CDV, indicating that GST-NP could be used as a potential antigen in the detection of the antibody specific for CDV.