牛病毒性腹泻病毒非结构蛋白p20的原核表达  

Prokaryotic Expression of Non-structural Protein p20 of Bovine Viral Diarrhea Virus

作　　者：王婉怡 李娅婕 王桂花[1] 韩晓东[1] WANG Wan-yi;LI Ya-jie;WANG Gui-hua;HAN Xiao-dong(School of Life Science,Inner Mongolia Agricultural University,Hohhot,Inner Mongolia 010018,China)

机构地区：[1]内蒙古农业大学生命科学学院,内蒙古呼和浩特市010018

出　　处：《动物医学进展》2024年第10期48-52,共5页Progress In Veterinary Medicine

基　　金：内蒙古自然科学基金项目(2022SHZR1769)。

摘　　要：牛病毒性腹泻是由黄病毒科、瘟病毒属中的牛病毒性腹泻病毒(BVDV)感染牛引起的一种传染病,是一种全球性牛传染病。为了在原核表达系统表达BVDV非结构蛋白p20,并分离纯化蛋白进行晶体学研究。首先对BVDV-1 p20蛋白基因进行密码子优化,合成后克隆到pET30表达载体上。测序鉴定成功的pET30-p20表达质粒转化大肠埃希氏菌表达菌株BL21(DE3),IPTG诱导获得的p20蛋白经过Ni柱、分子筛纯化。最终,经过多重缓冲溶液的筛选,p20蛋白可以稳定存在于含有5 mmol/Lβ-巯基乙醇、5%甘油、150 mmol/L氯化钠(pH8.0)的Tris-HCl缓冲液中。高纯度均一性的p20蛋白获得,为后续p20晶体学研究以及单克隆抗体的制备提供原核表达纯化方案。Bovine viral diarrhea is an infectious disease caused by bovine viral diarrhea virus(BVDV),which belongs to Flaviviridae and Pestivirus genus.BVDV is a global infectious disease of cattle.This study expressed and purified the BVDV nonstructural protein p20 in prokaryotic expression system for crystallographic study.The codon of BVDV p20 protein gene was optimized and cloned into pET30 expression vector.The pET30-p20 expression plasmid was successfully identified by sequencing,and transformed into E.coli expression strain BL21(DE3).The p20 protein was induced by IPTG and purified by Ni column and molecular sieve.By screening the multiple buffer solutions,p20 protein can stably existed in Tris-HCl buffer(pH8.0),containing 5 mmol/Lβ-mercaptoethanol,10%glycerol and 150 mmol/L sodium chloride.High-purity and homogeneous p20 protein was obtained,which provided a prokaryotic expression purification scheme for subsequent p20 crystallography research and the preparation of monoclonal antibodies.

关 键 词：牛病毒性腹泻病毒 原核表达系统 分子筛 p20蛋白质 

分 类 号：S852.653[农业科学—基础兽医学] S858.23[农业科学—兽医学]