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为了获得具有良好免疫活性的H9N2亚型禽流感病毒HA蛋白,本研究利用Bac-to-Bac昆虫-杆状病毒表达系统对H9N2亚型禽流感病毒HA蛋白进行表达。运用PCR对H9N2亚型禽流感病毒HA基因进行扩增,将HA基因连接于转移载体pFastBacDual的BamHⅠ和HindⅢ两个酶切位点之间,构成pFastBacDual-HA重组载体;将pFastBacDual-HA转化至大肠杆菌DH10Bac感受态细胞,利用蓝白斑筛选法和PCR方法对阳性菌落筛选重组杆粒Bacmid-HA;重组杆粒Bacmid-HA对Sf9昆虫细胞进行了转染,获得重组杆状病毒rBV-HA;运用SDS-PAGE电泳和Western blotting分析rBV-HA感染的细胞和细胞上清。结果表明,重组HA蛋白大小约为65 kD;Western blotting分析显示,HA蛋白与免疫H9N2禽流感病毒后获得的阳性血清具有良好的特异性反应,为进一步研制针对HA蛋白的H9N2亚型AIV亚单位疫苗和研发H9亚型ELISA抗体检测试剂盒奠定了基础。
In order to obtain the HA protein of H9N2 subtype avian influenza virus with good immunocompetence, we used the Bac-to-Bac insect-baculovirus expression system to express HA protein of H9N2 avian influenza virus. The HA gene of H9N2 subtype of avian influenza virus was amplified by PCR, and the HA gene was ligated between the two restriction sites of BamHⅠ and HindⅢ of the transfer vector pFastBacDual to construct pFastBacDual-HA recombinant vector; the pFastBacDual-HA was transformed into the large intestine Bacillus subtilis DH10Bac competent cells were screened by blue-white screening and PCR methods. The recombinant bacmid Bacmid-HA was screened by positive bacteria and recombinant baculovirus Bacmid-HA was transfected into Sf9 insect cells to obtain recombinant baculovirus rBV-HA. SDS-PAGE electrophoresis and Western blotting analysis of rBV-HA infected cells and cell supernatant. The results showed that the size of recombinant HA protein was about 65 kD. Western blotting analysis showed that the HA protein reacted well with the positive serum obtained after immunization with H9N2 avian influenza virus. To further develop the H9N2 subtype AIV subunit against HA protein Vaccine and research and development H9 subtype ELISA antibody test kit has laid the foundation.