牛传染性鼻气管炎病毒gB基因原核表达与鉴定

Prokaryotic Expression and Identification of gB Gene of Infectious Bovine Rhinotracheitis Virus

为给后续牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)单克隆抗体制备、酶联免疫吸附试验等研究提供基础,采用PCR技术扩增IBRV gB基因主要抗原区序列(312~529 aa位置),构建原核重组表达质粒pET-32a-gB,将其转化至BL21(DE3)感受态细胞进行诱导表达,通过对蛋白表达形式确认并对诱导剂(IPTG)浓度、诱导表达时间及诱导温度优化,表达并纯化获得gB重组蛋白,并对重组蛋白进行反应原性鉴定。结果显示:gB重组蛋白主要以包涵体形式表达,IPTG最佳诱导浓度为1 mmol/L,最佳诱导表达时间为6 h,最佳诱导温度为37℃;重组蛋白相对分子质量约60 ku,纯化后蛋白质量浓度为1.02 mg/mL;Western-blot鉴定发现,gB重组蛋白能被IBRV阳性血清特异性识别。结果表明,成功对IBRV gB基因进行体外原核表达与鉴定,为后期开展相应单克隆抗体制备等研究奠定了基础。

In order to support future researches on the preparation of monoclonal antibodies against bovine infectious rhinotracheitis virus(IBRV)and enzyme-linked immunosorbent assay(ELISA),the sequence in the main antigenic region(312-529 aa)of IBRV gB gene was amplified by PCR assay,a prokaryotic recombinant expression plasmid pET-32a-gB was constructed and transformed into the competent cell BL21(DE3)for inducible expression,the protein expression form was confirmed,and the concentration of IPTG,time of induction expression and induction temperature were optimized,gB recombinant protein was expressed and purified and its reactogenicity was identified.The results showed that the recombinant protein was mainly expressed in the form of inclusion body,the optimal induction concentration of IPTG was 1 mmol/L,time of induction expression was 6 h,and induction temperature was 37â;the relative molecular weight of the recombinant protein was about 60 ku,and the mass concentration of the purified protein was 1.02 mg/mL;Western blot analysis revealed that gB recombinant protein could be specifically recognized by IBRV positive serum.In conclusion,a foundation was laid by in vitro prokaryotic expression and identification of IBRV gB gene for subsequent researches on preparation of corresponding monoclonal antibodies.