牛病毒性腹泻病毒NS3基因的序列分析、表达与抗原性鉴定

Sequence analysis, expression and antigenicity detection of bovine viral diarrhea virus NS3 gene

摘要:

本研究应用套式RT-PCR方法扩增出牛病毒性腹泻病毒VEDEVAC株编码NS3蛋白的基因，克隆至表达载体pET-30a(+)，并进行测序。对瘟病毒属病毒NS3基因进行氨基酸差异性分析，显示平均P-distance为0.07，系统进化树分析表明VEDEVAC株隶属于BVDV 1型。将构建成功的重组质粒转化大肠埃希氏菌Rosetta(DE3)，在IPTG诱导下表达NS3重组蛋白，经Ni-NTA亲和层析纯化和尿素梯度透析后进行反应原性鉴定。Western blotting结果显示表达的重组蛋白可以与牛病毒性腹泻病毒阳性血清反应，并与猪瘟阳性血清有交叉反应，ELISA结果显示该重组蛋白具有良好的反应原性。所获得的蛋白为建立针对NS3抗体的ELISA检测方法奠定了基础。

Abstract:

In this study, we cloned the NS3 gene from bovine viral diarrhea virus (BVDV) VEDEVAC strain. The result showed that the average P-distance of Pestivirus NS3 amino acid sequence was 0.07 and the VEDEVAC strain was classified to BVDV type 1. Using pET-30a(+) as vector and Escherichia coli Rosetta (DE3) as host, we obtained purified recombinant NS3 protein by Ni-NTA affinity chromatography. Western blotting analysis demonstrated that both BVDV positive serum and classical swine fever virus (CSFV) positive serum were able to recognize the recombinant NS3 protein. Indirect-ELISA assay indicated that the protein could be used as detection antigen.