摘要
克隆了猪瘟病毒(CSFV)石门株E2和Npro基因,并分别插入pGEX-6p-1表达载体中,构建了原核重组表达质粒。经IPTG诱导后,应用SDS-PAGE电泳检测结果表明,重组质粒均得以成功表达。使用超声法破碎菌体获得蛋白,将带有GST标签的融合蛋白进行亲和层析,其中E2和Npro的表达形式分别为包涵体表达和可溶性蛋白表达。将E2变性后重折叠,应用亲和层析法纯化蛋白,Western blot检测发现CSFV蛋白E2能够被CSFV阳性血清识别,具有良好的反应原性,而Npro不能与CSFV阳性血清反应。
Two genes encoding proteins E2 and Npro of classical swine fever virus were cloned in this study,respectively, which were inserted in pGEX-6p-1 expression vector. Two prokaryotic recombinant expression plasmids were con- structed. The SDS-PAGE showed that all the two recombinant proteins were successfully expressed. The Escherichia coli were crused by ultrasonic method for acquiring proteins. Fusion proteins with GST-tag were processed by affini- ty chromatography. The protein E2 were expressed in precipitate as inclusion body,and Np~~ expressed in liquid su- pernatant as soluble protein. The inclusion body of E2 was denaturated and refolded to get soluble protein. Fusion proteins with GST-tag were purified by affinity chromatography. The Western blot was used to examine reactogenic- ity of purified proteins. It was found that prokaryotic recombinant protein Npro did not react to CSFV positive serum, but E2 can react with CSFV positive serum.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2012年第8期1101-1104,共4页
Chinese Journal of Veterinary Science
基金
国家高技术研究发展计划("863"计划)资助项目(2004AA213101)
