H3N2亚型猪流感病毒HA1、HA2基因的原核表达及抗原性分析  

Prokaryotic Expression and Antigenic Analysis of H3N2 Swine Influenza Virus HA1 and HA2 Genes

作　　者：宋玉慧[1] 王翔宇[1] 王洁琼[1] 潘梦梦 张剑[1] 刘琳[1] 李新生[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《中国畜牧兽医》2016年第10期2762-2767,共6页China Animal Husbandry & Veterinary Medicine

基　　金：河南省科技创新人才计划(144100510009);郑州市前沿技术研究开发项目(141PQYJS566);河南省产学研合作项目(15210700096)

摘　　要：血凝素(hemagglutinin,HA)蛋白是猪流感病毒(swine influenza virus,SIV)的一个重要蛋白,在疾病预防和治疗中具有重要作用。本试验旨在克隆和表达H3N2亚型猪流感病毒A/swine/Henan/1/2010(H3N2)的HA1和HA2基因。用含有H3N2亚型SIV的鸡胚尿囊液提取RNA,RT-PCR扩增后将目的基因定向克隆到pET-28a(+)原核表达载体上,并将其转入宿主菌BL21(DE3)pLyS进行表达,IPTG诱导后经SDS-PAGE检测并用该病毒重组HA蛋白制备的特异性单克隆抗体1C10作为一抗对两种蛋白进行Western blotting分析。SDS-PAGE结果显示,得到HA1和HA2大小分别为34.8、23.2ku的重组蛋白,Western blotting结果表明,HA1蛋白与1C10单克隆抗体具有良好的反应原性,且1C10单克隆抗体表位在HA1蛋白上。本试验结果为进一步研究血凝素蛋白的结构和功能,以及建立快速诊断方法和基因工程疫苗提供材料。Hemagglutinin protein plays an important role in disease prevention and treatment of the swine influenza virus（SIV）.In order to clone and express subtype H3N2 SIV A/swine/Henan/1/2010（H3N2）HA1and HA2 genes with chicken embryo allantoic fluid containing the H3N2 SIV after extraction of RNA.The HA1 gene and HA2 gene were amplified from the total RNA using RT-PCR and they were inserted into prokaryotic expression vector pET-28a（＋）to construct recombinant expression vector.Then the vector was transformed and expressed in E.coli BL21（DE3）pLyS.Then the bacteria were induced by IPTG and their lysates were analyzed by SDS-PAGE and Western blotting,respectively.The monoclonal antibody 1C10,which was specific to HA protein,was used as primary antibody in Western blotting analysis.It was found that the expressed recombinant HA1 protein was 34.8ku and recombinant HA2 protein was 23.2ku analyzed by SDS-PAGE.As demonstrated by Western blotting,this HA1 expressed products showed the capacity of reacting with monoclonal antibody 1C10.All the results suggested that the expressed HA1 and HA2proteins of H3N2 influenza virus would be very helpful in the development of rapid diagnosis method and vaccine development,which would facilitate further study on the function of HA at the same time.

关 键 词：H3N2亚型猪流感病毒 HA1基因 HA2基因 原核表达 

分 类 号：S852.659.5[农业科学—基础兽医学]