Study on the Knockout and the Soluble Prokaryotic Expression of VP5 Protein Transmembrane Region of IBDV  

传染性法氏囊病病毒VP5蛋白跨膜区的敲除及可溶性原核表达研究(英文)

作　　者：严孝金[1] 李锋[1] 秦立廷[2] 李倩倩[1] 韩翠晓[1] 冯舵[1] 王笑梅[2] 高伟[1] 

机构地区：[1]北京林业大学理学院,北京市海淀区100083 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001

出　　处：《Agricultural Science & Technology》2011年第4期621-624,共4页农业科学与技术（英文版）

基　　金：Supported by the National Natural Science Fundation Item of China(30970578,31070651);"Excellent Talent Support Plan in NewCentury"of Ministry of Education(NECT-08-0731)~~

摘　　要：[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.[目的]构建敲除传染性法氏囊病病毒(IBDV)VP5蛋白跨膜区序列的原核表达载体,并进行目的蛋白的表达、分离和纯化。[方法]利用PCR技术分别扩增IBDVVP5基因的胞外片段和胞内片段,然后将2个片段及pET-28b(+)同时相接,即载体-胞内片段-胞外片段-载体,构建了敲除跨膜区基因片段的VP5重组表达质粒pET-VP5-FC及改进后的pET-VP5-SC。将表达质粒转化BL21(DE3),IPTG诱导后经Ni亲和层析及凝胶过滤层析纯化重组蛋白。[结果]得到可溶性表达的IBDVVP5。[结论]为进一步研究VP5蛋白的结构与功能奠定了良好的基础,另外为本文的研究方法对其它跨膜蛋白的可溶性表达及进一步研究也有一定的借鉴意义。

关 键 词：IBDV VP5 Transmembrane region knockout Prokaryotic expression 

分 类 号：S852.65[农业科学—基础兽医学]