H9N2亚型禽流感病毒HA-M2融合基因的真核表达及免疫原性研究

Eukaryotic Expression of HA-M2 Fusion Gene of H9N2 Subtype Avian Influenza Virus and Its Immunogenicity Analysis

选择H9N2亚型禽流感病毒(AIV)血凝素(HA)的两个保守T细胞表位基因和M2基因胞外保守序列(M2e),经人工合成并通过PCR扩增获得HA-M2融合表位基因,与载体p Fast Bac HTA连接,构建重组真核表达质粒p Fast HTA-HA-M2。阳性重组质粒转化DH10Bac感受态细胞,转染Sf9昆虫细胞获得含HA-M2基因的重组杆状病毒,重组杆状病毒感染Sf9细胞72 h后,进行SDS-PAGE、间接免疫荧光试验和Western blot鉴定。利用目的蛋白免疫3周龄SPF鸡,采用ELISA测定抗体滴度,MTT试验检测T淋巴细胞增殖。结果显示,目的蛋白HA-M2在昆虫细胞中有特异性表达,能被H9亚型AIV免疫阳性血清所识别,可诱导高滴度的AIV特异性抗体。试验结果为新型禽流感通用疫苗的研制奠定了基础。

In this study,two conserved T cell epitope genes of hamagglutinin （NA）of H9N2 subtype avian influenza virus （AIV） and M2 gene of extracellular domain were synthetized and joined as HA-M2 multiple-epitopes fusion gene by PCR. HA-M2 segment was amplified cloned into pFastBacHTA plasmid, and then the recombinant plasmid was transferred into DH10Bac competent cells to get the positive recombinant bacmid. After the transfection of Sf9 cell with recombinant baculovirus lasted for 72 h, the expression of interest protein was identified by indirect immunofluorescence assay,SDS-PAGE,and Western blot. The serum antibody titer and lymphocyte proliferation of immunized 3-week-age SPF chickens was determined by ELISA and MTY assay,respectively. The results showed that HA-M2 gene had expressed in Sf9 cells infected with recombinant baculovirus. The expressed protein could be specifically rec6gnized by positive serum against H9 subtype AIV,and induce high-titer specific antibody against AIV. It provided a basis for the study of new universal avian influenza vaccine.