H9N2亚型禽流感病毒非结构蛋白基因NS1的表达及其单克隆抗体的研制

Expression of the non-constructual protein-l(NS1)of avian influenza virus H9N2 and preparation of its monoclonal antibodies

利用RT-PCR技术扩增获得了H9N2亚型禽流感病毒的NS1基因,ORF长度为654bp。成功构建了重组表达载体pET-NS1,将其转化BL21,并诱导表达出了相对分子质量大约为28 000的NS1融合蛋白。NS1融合蛋白经His trap Hp Kit柱子纯化,采用缓慢稀释和透析相结合的方法复性H9N2-NS1蛋白,获得纯度较高的NS1蛋白,以纯化的NS1免疫BALB/c小鼠,间接接ELISA方法筛选阳性克隆,对获得的抗H9N2-NS1单克隆抗体(McAbs)进行特异性分析;建立了2株稳定分泌抗H9N2-NS1McAbs的杂交瘤细胞系6B9和6C2;腹水的特异性鉴定结果表明,2株McAbs能特异性的识别H9N2-NS1,而与H5N2亚型AIV、H9N2亚型AIV、NDV、IBV、IBDV、ILTV、EDS76均不发生反应。Western blotting鉴定表明,所获得的单抗只与NSl蛋白条带反应。亚类显示,6B9为IgG1,6C2为IgG2b。结果表明,2株抗H9N2-NS1McAbs的制备对H9N2亚型禽流感病毒检测试剂盒的研制以及该病的防治有重要意义。另外,NSl蛋白单抗的成功研制为进一步研究NSl蛋白的结构、功能与细胞的相互作用机制及AI遗传变异规律奠定了一定的基础。

Nonstructural gene 1（NS1） of H9N2 subtype avian influenza virus was amplified by RT-PCR,which nucleotide sequences is 654 bp in length.The fragment was cloned into the pET-28（a） vector to construct a recombinant expression plasmid.The recombinant expression plasmid was induced by 1mmol/L IPTG for 5 hours.The result showed that NS1 protein was highly expressed by inclusion body.Its molecular weight was 28 000 as expected.The expressed NS1 protein was denatured by SKL,then the denatured protein was refolded by slow diluting and dialyzing method.The result indicated that the high pure and active protein was obtained.This protein was used as antigen to immunize BALB/c mice intraperitoneally and an indirect ELISA assay coated with this purified H9N2-NS1 was used to screen hybridoma cells secreting specific monoclonal antibodies.In these ways,2 strains of celllines stably secreting H9N2-NS1-specific monocloanl antibodies were obtained,they were 6B9 and 6C2.As demonstrated by the examination of the mouse ascetic fluids,all the monoclonal antibodies produced by these 2 strains of cell lines could recognize H9N2-NS1specifically,yet without reactivity to avian influenza virus H5N2,H9N2,NDV,IBV,IBDV,ILTVand EDS76V.The subtype of 6B9 was proved to be IgG1 and 6C2 was proved to be IgG2b.These McAbs may be value for the development of diagnostic kit as well as for the prevention and treatment of avian influenza virus H9N2.Also,the successful preparation of its monoclonal antibodies laid the foundations for further studies on the structure,function of the nonstructural protein 1 and interaction with host cells and genetic diversity.