NS2A序列对日本脑炎病毒NS1基因在原核系统中的体外表达的影响  

Effect of NS2A on the Expression of Japanese Encephalitis Virus NS1 Gene in E. coli

作　　者：仇华吉[1] 金吉东[1,2] 田志军[1] 周艳君[1] 王云峰[1] 童光志[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室 [2]延边大学农学院动物医学系,龙井133400

出　　处：《中国农业科学》2004年第4期614-618,共5页Scientia Agricultura Sinica

摘　　要：用RT-PCR扩增并克隆了日本脑炎病毒弱毒株SA14-14-2的NS1、NS1'、NS1-2A的 cDNA片段,分别将其亚克隆到原核表达载体pET-30b(+),构建了重组原核表达质粒pET30b-NS1、pET30b-NS1'、pET30b-NS1-2A,转化大肠杆菌BL-21(DE3),用IPTG诱导培养。结果表明,仅有pET30b-NS1质粒转化的菌株获得表达,而另外2种重组质粒转化的菌株均无表达。表达的融合蛋白分子量约为46kD,约占菌体总蛋白的30.8%,Western blotting分析表明表达产物具有抗原活性。动物试验证实,重组NS1免疫小鼠蛋白可以抵抗强毒的攻击。笔者研究证实NS2A或部分NS2A的存在对于NS1的表达具有不利影响。Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and a zoonotic pathogen. NS1, a nonstructuralglycoprotein of JEV, is able to elicit protective immune response. The cDNA fragments encoding NS1, NS1'and NS1-2Awere amplified by RT-PCR from JEV genomic RNA, and then cloned into an expression vector pET-30b(+), resulting inrecombinant plasmids pET30b-NS1, pET30b-NS1', and pET30b-NS1-2A. Each of the recombinant plasmids was transformedinto E.coli strain BL-21(DE3). After induction by IPTG, the transformed bacterial cultures were subjected to SDS-PAGEanalysis. A recombinant His-tagged protein with an apparent molecular weight of about 46kD was obtained for pET30b-NS1, accounting for 30.8% of the total bacterial cellular lysates. Whereas, no expression was detected for the other tworecombinant plasmids. The recombinant protein showed reactivity with both monoclonal antibody against His-tag andspecific antisera against JEV in Western blotting. The mice immunized with the recombinant protein were protected fromlethal challenge. The results showed that NS2A has an adverse effect on the in vitro expression of NS1.

关 键 词：日本脑炎病毒 NS1基因 原核系统 体外表达 NS2A序列 RT-PCR 

分 类 号：S852.64[农业科学—基础兽医学]