注册 | 登录 邮箱

非洲猪瘟病毒结构蛋白p54的原核表达与反应原性鉴定

27789 2019-12-16 10:10:24 官丽娟,邵 钰,任伟杰,等 中国动物检疫
0

p54蛋白为非洲猪瘟病毒(ASFV)的主要结构蛋白之一,参与病毒对靶细胞的吸附与进入。为深入研究p54结构蛋白的抗原性,根据GenBank序列号(MK128995)对应的E183L基因序列,设计1对特异性引物扩增其整个CDS区,并克隆至pET-30a(+)载体,构建了表达p54蛋白的重组质粒pET-30a(+)-p54。用BL21(DE3)转化该质粒,经IPTG诱导表达后进行SDS-PAGE电泳,可见重组质粒表达出1条分子量约为20 kDa的特异性条带,且重组表达蛋白以融合表达蛋白形式存在于上清。进一步通过His亲和层析法纯化目的蛋白,用ASFV阳性血清进行蛋白质免疫印迹反应,发现表达的重组p54蛋白能与ASFV阳性血清产生特异性反应,表明p54蛋白表达成功。本研究为ASFV抗体ELISA检测方法的建立奠定了基础。

Prokaryotic Expression and Reactivity Identification

of Structural Protein p54 of African Swine Fever Virus

As one of the major structural proteins of African swine fever virus(ASFV),p54 protein helps the virus adsorb and enter into the target cells. In order to further study its antigenicity,one pair of specific primers were designed to amplify the whole CDS domain of E183L gene according to the serial number(MK128995)in GenBank. Then the amplification products were cloned into the vector pET-30a(+),and the recombinant plasmid pET-30a(+)-p54 was constructed,after transformation into BL21(DE3),induced expression by IPTG and SDS-PAGE electrophoresis. one specific band with the molecular weight of 20 kDa was obtained and the recombinant protein appeared in the supernatant in the form of fusion protein. Further,the recombinant protein was purified by His-tag affinity chromatography,and the positive serum of ASFV was used for Western blotting. it was found that the expressed recombinant p54 protein could specifically react with ASFV positive serum,indicating that p54 protein was successfully expressed. The study would lay a foundation for the development of ELISA kit for detection of ASFV.

全文下载链接:https://kns.cnki.net/KCMS/detail/37.1246.S.20191206.0907.028.html


最新评论