H9亚型禽流感病毒HA1基因的克隆表达及其生物学活性的初步分析

Cloning,expression and preliminary studies on bioactivities of HA1 segment of H9 subtype avian influenza virus

根据GenBank已发表序列设计引物,通过RT-PCR成功获得了H9亚型禽流感病毒北京分离株A/Chicken/Beijing/1/96(H9N2)的HA1片段,经序列分析HA1片段与其他已发表序列同源性为95%～98%。将HA1片段克隆入pET-28a的多克隆位点构建重组质粒pET28-H9HA1并转化宿主菌BL21(DE3),用IPTG诱导表达,经SDS-PAGE电泳及Western blot分析证实,H9HA1片段得到表达,并且表达的蛋白带分为42Ku和23Ku两条。血凝实验和血凝抑制实验表明原核表达的HA1蛋白不具备血凝活性,其阳性血清不能引起血凝抑制。交叉反应实验证实原核表达的重组蛋白能与禽流感病毒H9亚型单特异性血清反应,而与禽流感病毒H5、H7亚型以及其他禽传染病病原微生物单特异性血清无交叉反应。说明表达的蛋白具有良好的反应原性和特异性,有开发成为H9亚型禽流感检测试剂的可能。

According to the sequence of HA that was published on GenBank, HA1 gene of an AIV isolate, A/Chicken/Beijing/1/96（H9N2）, was amplified. The sequence of HA1 gene showed 95%-98% homologous compared with other published HAl sequence. The fragment of HA1 was cloned into expression plasmid pET-28a. And the recombinant plasmid DNA was named as pET28-H9HA1. Then it was transformed in E.coli BL21 （DE3）. There were two pieces of recombinant fusion protein expressed in E.coli BL21 （DE3） induced by IPTG and the molecular weight were 42 Ku and 23 Ku, respectively, by SDS-PAGE and Western blot. The fusion protein did not cause hemagglutination by hemagglutination（HA） test. The positive serum did not cause hemagglutination inhibiting by hemagglutination inhibition （HI） test. The protein exhibited specific binding to antiviral antibodies of H9 subtype and has no cross-reaction with H5 or H7 subtype or other avian infectious diseases. The results above showed the expressed HA1 recombinant protein shared good reactinogenicity and specificity and could be developed as a new diagnostic reagent for H9 subtype AIV.