两株不同年代H9N2亚型禽流感病毒HA蛋白免疫原性研究

Study on the immunogenicity of HA proteins of two strains of H9N2 subtype avian Influenza A virus from different ages

为探究两株不同年代H9N2亚型禽流感病毒HA蛋白的免疫原性,试验采用RT-PCR方法分别扩增出A/Chicken/Henan/01/2006(HN106株)和A/Chicken/Henan/13/2010(X-13株)的HA基因,将其定向克隆到原核表达载体pET-32a(+),并转化至大肠杆菌BL21(DE3)感受态细胞,获得重组表达质粒pET-06-HA和pET-13-HA。将鉴定正确的阳性菌株用IPTG诱导表达后,对表达产物进行纯化和Western-blot分析。将纯化的pET-06-HA和pET-13-HA重组蛋白分别制备成亚单位疫苗免疫SPF鸡,用血凝抑制(HI)和间接ELISA试验分别检测免疫后0,7,14,21天06-HA蛋白免疫组、13-HA蛋白免疫组、对照组的血清抗体水平。结果表明:诱导表达出的重组蛋白pET-06-HA分子质量为80.3 ku,以包涵体的形式存在;重组蛋白pET-13-HA分子质量为80.8 ku,以可溶的形式存在。重组蛋白均能与相应的阳性血清发生特异性免疫反应,而不发生交叉反应。以HN106株和X-13株为4单位抗原分别检测血清抗体效价,各免疫组免疫7,14,21 d后与对照组相比平均抗体效价显著上升(P<0.01)。两种重组蛋白均能引起免疫鸡只较高的体液免疫水平;以06-HA蛋白为包被抗原测定06-HA蛋白免疫组和13-HA蛋白免疫组免疫14,21 d后血清抗体,两组之间抗体水平差异显著(P<0.01)。说明两株不同年代H9N2亚型禽流感病毒HA蛋白免疫原性良好且抗原性具有一定差异,可用于H9N2亚型禽流感病毒HA蛋白功能研究以及亚单位疫苗的研制。

In order to explore the immunogenicity of the HA protein of two strains of H9 N2 subtype avian Influenza A virus from different ages, the experiment used RT-PCR to amplify HA genes of A/Chicken/Henan/01/2006(HN106 strain) and A/Chicken/Henan/13/2010(X-13 strain),which were cloned into the prokaryotic expression vector pET-32 a(+),and transformed into E.coli BL21(DE3) competent cells, the recombinant expression plasmid pET-06-HA and pET-13-HA were obtained. After the identified positive strains were induced to express with IPTG,the expressed products were purified and analyzed by Western-blot. The purified recombinant proteins were prepared into subunit vaccines to immunize SPF chickens;hemagglutination inhibition(HI) and indirect ELISA tests were used to detect serum antibody levels in 06-HA protein immunization group, 13-HA protein immunization group and control group on 0,7,14,21 days after immunization. The results showed that the successfully induced expressed the recombinant protein pET-06-HA had a molecular mass of 80.3 ku and existed in the form of inclusion bodies;the recombinant protein pET-13-HA had a molecular mass of 80.8 ku and existed in a soluble form. The recombinant protein can react specifically with the corresponding positive serum, and no cross-reaction was observed. The serum antibody titer was detected with 4 units of the antigens of HN106 strain and X-13 strain respectively, and the average antibody titer of each immunization group increased significantly compared with the control group on 7,14,21 days after immunization(P<0.01). The both strains of recombinant proteins can cause higher levels of humoral immunity in immunized chickens. The 06-HA protein was used as the coating antigen for determination, the significant difference in serum antibody levels was found between the 06-HA protein immunization group and the 13-HA protein immunization group on 14 and 21 days after immunization(P<0.01). The results indicated that the HA proteins of the two strains of H9 N2 subtype avian Influenza A virus from different ages had good immunogenicity and certain difference in antigenicity, which can be used for research on the function of HA protein of H9 N2 subtype avian Influenza A virus and the development of subunit vaccine.