H3N2亚型猪流感病毒血凝素基因的原核表达及间接ELISA诊断方法的建立  

Prokaryotic expression of swine influenza virus (H3N2) HA gene and establishment of indirect ELISA

作　　者：刘天强[1] 于海[1] 张强[1] 李国新[1] 王斌[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,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2008年第10期795-799,823,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点基础研究计划(2005CB523200)

摘　　要：采用RT-PCR扩增H3N2亚型猪流感病毒HA1基因。将该片段插入到原核表达载体pET-30a(+)中,构建原核表达载体pET-HA1。阳性质粒转化宿主菌BL21,经IPTG诱导,HA1基因获得表达,其重组蛋白以包涵体的形式存在。通过改变IPTG的浓度和诱导时间,确定表达HA1基因的最佳诱导条件。Western blot分析表明表达产物具有良好的抗原活性。用纯化的表达产物作为包被抗原建立了检测H3亚型猪流感病毒抗体的间接ELISA方法,确定抗原最佳包被浓度为4μg/mL,血清最佳稀释度为1∶100,阳性标准初步判定为:待检血清OD值>0.4,而且待检血清OD值/阴性血清OD值>2。应用此方法对309份临床血清样品进行了检测,并与HI方法进行了比较,结果表明基于重组HA蛋白的间接ELISA方法可用于H3N2亚型猪流感病毒感染的初步诊断。The HA1 gene of H3N2 subtype swine influenza virus （SIV） was amplified by RT-PCR using specific primers designed according to the nucleotide sequence of A/Swine/Heilongjiang/1/05 （H3N2）, cloned into pET-32a（＋）, and expressed in the host cell BL21 under IPTG induction. Western blot analysis showed that the recombinant protein reacted specifically with H3N2 subtype SIV positive serum. Indirect ELISA was developed for antibody detection of H3 subtype SIV using the recombinant protein as coating antigen. A total of 309 clinical serum samples were tested using this ELISA assay and the result was compared with that of HI test. It was suggested that the recombinant HA1 protein could be useful for primary diagnoses of pig infection with H3 subtype SIV.

关 键 词：H3N2亚型猪流感病毒 血凝素基因 原核表达 

分 类 号：S852.659[农业科学—基础兽医学]