甲型H1N1流感病毒NA蛋白的原核表达及其单克隆抗体制备

Prokaryotic Expression of Neuramidinase in Influenza A Virus H1N1 and its Monoclonal Antibody Preparation

目的:利用原核系统表达甲型H1N1流感病毒NA蛋白,制备NA特异性单克隆抗体。方法:采用RT-PCR技术扩增A/Shenzhen/71/09病毒株NA基因,通过原核表达系统表达含6×His标签NA重组蛋白,利用镍离子亲和柱进行纯化。使用NA蛋白以100g/只剂量免疫BALB/C小鼠,3次免疫后,利用杂交瘤技术筛选分泌NA特异性单克隆抗体的细胞株,采用Western-blot方法对单抗进一步验证。结果:成功克隆A/Shenzhen/71/09NA基因,长度约1400bp。将NA基因克隆入原核表达载体pET-21b(+),经IPTG诱导后在大肠杆菌包涵体中检测到目的蛋白,分子量约50kd。采用尿素变性处理后借助6×His标签对蛋白进行纯化。用纯化蛋白免疫小鼠,将脾细胞与SP2/0细胞融合,使用ELISA检测细胞上清NA抗体,共筛选1株持续分泌NA抗体的杂交瘤细胞株7C11。Western-blot验证该单克隆抗体可以特异性结合NA抗原。结论:成功表达甲型H1N1流感NA蛋白,获得1株分泌NA抗体的单克隆细胞株,从而为后续甲流诊断及疫苗研制奠定基础。

Objective To express the Neuramidinase （NA） protein of A/H1N1 influenza virus in prokaryotic expression system E.coli andprepare the specific monoclonal antibodies against NA. Methods NA gene was amplified using RT-PCR from A/Shenzhen/71/09 viral strain, and recombination protein NA with 6 × His tag wasexpressed in prokaryotic expression system E.coli, then the recombinant protein was purified with Nickel ion affinity column （Ni-column）. BALB/C mice were immunized with purified NA protein, and hybridoma cells which secreted anti-NA monoclonal antibodies were screened after 3 times immunization. Finally the monoclonal antibody （mAb） was identified with western blot method. Results NA gene fragment was cloned successfully with about 1400bp and inserted into pET-21b（＋） vector. Recombination protein NA was detected in the inclusion bodies after IPTG induction, whose molecular weight was about 50kDa. NA protein was purified with Ni-column after denaturation with Urea. Mice were immunized with purified NA protein, then, the spleen cells were fused with myeloma SP2/0 cells. A hybridoma cell line called 7C11 which secreted anti-NA mAb continuously was screened by detecting anti-NA mAb level in cell supernatant with ELISA. It was proved that mAb could bind with NA protein with Westemblot assay. Conclusion We expressed recombinant NA protein of A/H1N1 influenza virus successfully and got one hybridoma cell line which could secret anti-NA mAb. This result would benefit the diagnosis and vaccine research of A/H1N1 influenza in the future.