BVDV NS5A蛋白在原核系统中的优化表达与纯化鉴定  

Optimal Expression and Purification of BVDV NS5A Protein in Prokaryotic System

作　　者：高明阳 杨宣叶 吴玉湖 王进千 胡欣妍 周建华 GAO Ming-yang;YANG Xuan-ye;WU Yu-hu;WANG Jin-qian;HU Xin-yan;ZHOU Jian-hua(Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730010)

机构地区：[1]西北民族大学生物医学研究中心/生物工程与技术国家民委重点实验室,兰州730030 [2]西北民族大学生命科学与工程学院,兰州730010

出　　处：《中国奶牛》2025年第1期35-41,共7页China Dairy Cattle

基　　金：国家自然科学基金(No.32360874);甘肃省自然科学基金(No.23JRRA715);西北民族大学引进人才科研项目(xbmuyjrc202225)。

摘　　要：本研究构建了牛病毒性腹泻病毒NS5A蛋白重组质粒并在原核表达系统中表达,但分析过程中发现其主要形式为包涵体,因此通过更换条件使BVDV NS5A蛋白可溶,同时可纯化出蛋白,为后面结构功能分析、抗体制备以及酶联免疫吸附实验奠定基础。以实验室保存的毒株为试验材料,采用逆转录PCR(reversetranscription PCR,RT-PCR)技术扩增NS5A基因,构建pET-28a-NS5A和pET-32a-NS5A表达质粒,转化于大肠杆菌BL21(DE3)和Rosetta gami 2(DE3)感受态细胞中,使用IPTG进行诱导表达,对表达产物进行SDS-PAGE和Western-blot分析,最后根据表达条件的摸索,纯化出可溶性的NS5A蛋白。成功克隆出了BVDV NS5A基因,序列全长为1 488bp,编码497个氨基酸,理论分子质量为55.8ku。成功构建了原核表达质粒pET-28a-NS5A和pET-32a-NS5A,其在大肠杆菌BL21(DE3)和Rosetta gami 2(DE3)可表达以包涵体形式为主的NS5A重组蛋白,分子质量分别约为61ku和75ku,经过体表达条件的摸索,pET-32a-NS5A质粒在大肠杆菌BL21(DE3)中,经过25℃低温诱导出现较多可溶性,并纯化出NS5A蛋白。本研究成功克隆出了1 488bp的NS5A基因,经不同条件的诱导表达后,得到可溶性NS5A重组蛋白。In this study,bovine viral diarrhea virus NS5A protein recombinant plasmid was constructed and expressed in the prokaryotic expression system,but during the analysis,it was found that its main form was inclusion body.Therefore,the BVDV NS5A protein could be soluble by changing the conditions,and the protein could be purified at the same time,which laid the foundation for the subsequent structural and functional analysis,antibody preparation and enzyme linked immunosorbent experiment.Strains preserved in our laboratory,using reverse transcription PCR(reversetranscriptionPCR,rt-pcr)technology amplification NS5A gene,then build a pET-28a-NS5Aa and pET-32a-NS5A prokaryotic expression plasmid.The protein was transformed into BL21(DE3)and Rosetta gami 2(DE3)Escoli receptor cells,induced by IPTG,and analyzed by SDS-PAGE and Western-blot.Finally,soluble NS5A protein was purified according to the expression conditions.The BVDV NS5A gene was successfully cloned with a total sequence of 1488bp,encoding 497 amino acids and a theoretical molecular weight of 55.8ku.Prokaryotic expression plasmid pET-28a-NS5A and pET-32a-NS5A were successfully constructed,which could express NS5A recombinant protein mainly in the form of inclusion bodies in Escoli BL21(DE3)and Rosetta gami 2(DE3),with molecular weights of about 61ku and 75ku,respectively.After exploring the expression conditions,pET-32a-NS5A plasmid was soluble in Escherichia coli BL21(DE3)after low temperature induction,and NS5A protein was purified.The NS5A gene 1488bp was cloned successfully,and soluble NS5A recombinant protein was obtained by inducing expression under different conditions.

关 键 词：牛病毒性腹泻病毒 NS5A蛋白 原核表达 鉴定 可溶性 

分 类 号：S858.23[农业科学—临床兽医学]