摘要
将鸡Mx基因克隆入pET-32a载体,筛选鉴定后将重组阳性质粒转化到大肠埃希菌株BL21进行诱导表达,表达产物用Western blot鉴定,并经镍亲和层析法纯化及透析复性,得到的蛋白用微量细胞病变抑制法检测其抗病毒活性。结果显示,构建的载体经PCR扩增得到长度为2 118 bp的特异片段,测序结果证实与鸡Mx基因同源性99.95%,Western blot结果显示在78 ku处检测到特异性蛋白条带,微量细胞病变抑制试验结果显示,表达产物经8倍和256倍稀释后可分别保护50%鸡胚成纤维细胞免受新城疫病毒(NDV)和水疱性口炎病毒(VSV)的感染。结果表明,成功表达了鸡Mx蛋白,且表达产物具有一定的抗DNV和VSV活性。
To investigate the antiviral activity of chick Mx protein,Mx gene was cloned into the prokaryotic expression vector pET-32a.The positive recombinant plasmid was transformed into E.coliBL21 strain,and then induced by IPTG.The product was identified by Western blot and purified via metal chelate affinity chromatography.The anti-viral activity of renaturated protein was detected by CPEI50.Results showed that a 2118bp PCR product,99.95% homology with chicken Mx gene,was obtained from pET-cMx.Western blot showed the expression product can bind to chicken Mx antibody specifically.CPEI50 showed 8 and 256 fold diluted renaturated protein could preserve 50% CEF from NDV and VSV infection,respectively.Results indicated that the chicken Mx protein was successfully expressed in E.coli BL21,and the product had antiviral activity against NDV and VSV.
出处
《动物医学进展》
CSCD
北大核心
2011年第4期6-9,共4页
Progress In Veterinary Medicine
基金
佛山市科技发展专项基金(31-2-1)
佛山市产学研基金(2006A031)
关键词
MX蛋白
原核表达
抗病毒活性
鸡
Mx protein
prokayrotic expression
antiviral activity
chick
