根据GenBank公开发表的禽流感病毒(AvianInfluenzaVirus,AIV)H9N2亚型血凝素基因(HA)序列设计引物,用PCR方法从重组质粒pUCHA中扩增H9N2亚型禽流感病毒去除信号肽的血凝素基因,将该片段定向插入到原核表达载体pET32a(+)中,构建原核表达载体pETHA。阳性质粒转化宿主菌BL21(DE3),经IPTG诱导,HA基因获得表达,经定位分析,目的蛋白以包涵体的形式存在于大肠杆菌中。通过改变IPTG的浓度和诱导时间,确定了表达HA基因的最佳诱导条件:IPTG终浓度为0.7mmol/L,诱导时间为3h。Westernblot分析表明,重组蛋白能与H9N2亚型AIV阳性血清发生特异性反应。以纯化后表达产物作为诊断抗原包被酶标板建立了检测H9亚型AIV抗体的间接ELISA方法。结果表明,抗原的最佳包被浓度为25μg/mL,血清的最佳稀释度为1∶80,阳性标准初步定为:OD待检血清0.5且OD标准阳性血清1.0;OD标准阴性血清0.1。

The complete HA gene of AIV H9N2 subtype was amplified with PCR method using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank. The PCR product was successively cloned into pMD18-T and pET-32a (+) to get a prokaryotic expression vector pET-HA. The target gene was successfully expressed in the E.coli BL21(DE3) host cell in inclusion body when induced with IPTG. The expression was optimized with proper inducing conditions of 0.7 mmol/L IPTG and 3 hours induction. The highest expression of the target protein was up to 30.8% of the total bacterial proteins. Western-blot analysis proved that the recombinant protein had good immunoreactivity against H9 subtype AIV antibody. The indirect ELISA method for the detection of H9 subtype AIV antibody in chicken serum was established after the optional working circumstance for the ELISA assay (antigen concentration:4μg/mL;serum dilution: 1:80 ) was tried out with chessboard titration . The positive criterion of this ELISA method