两株H1N1亚型猪流感病毒HA蛋白中和性单克隆抗体的制备及抗病毒活性研究

Preparation and Antiviral Activity of Two Neutralizing Monoclonal Antibodies against HA Protein of H1N1 Swine Influenza Virus

为了制备抗欧亚类禽型(Eurasian avian-like,EA)H1N1猪流感病毒(SIV)血凝素(HA)蛋白的单克隆抗体(MAb)并对其生物学活性进行检测,利用SIV A/swine/Henan/11/2005(HN11)增殖、纯化的全病毒蛋白作为免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞与骨髓瘤细胞(SP2/0)融合,采用间接ELISA和HI检测方法进行筛选。结果获得了两株能稳定分泌抗HA蛋白MAb的杂交瘤细胞株,分别命名为2B6和4C7;两株MAbs的腹水ELISA和HI效价均分别高于1∶107和5log2;两株MAbs能够在IFA试验中与病毒HA蛋白发生反应,而在Western blot试验中不能检测到病毒HA蛋白;病毒中和试验结果显示这两株MAbs对EA H1N1SIV具有病毒中和活性;MAbs的抗病毒活性又进一步通过小鼠的感染试验进行了评估,结果表明接种了MAbs的小鼠获得了有效抵抗同源及异源H1N1SIV感染的保护效果。研究证实制备的两株MAbs具有较高的病毒中和活性和较强的抗病毒感染能力,可以将其进一步应用于抗EA H1N1SIV的感染及开展病毒抗原性变异的分子基础研究。

This study aimed to prepare monoclonal antibodies（MAbs）against HA protein of Eurasian avian-like H1N1（EA H1N1）virus and analyze their biological activities.After viral proliferation in chicken embryos,the purified whole-virus protein of A/swine/Henan/11/2005（HN11）was used to immunize BALB/c mice.The splenic cells of the mice were fused with the SP2/0 cells after the third immunization.After screening by indirect ELISA and hemagglutination inhibition test and three circles of sub-clone,two hybridoma cell strains secreting MAbs against HA protein of the HN11 were obtained,which was designated as 2 B6 and 4 C7,respectively.The ELISA titers of two MAbs were higher than 1：107,and HI titer were higher than 5 log2.These two MAbs could specifically recognize HA protein expressed by plasmid pCAGGS-H1 HA in 293 T cells by IFA,but not in Western-blot assay.The results of neutralization test indicated that two MAbs have specific neutralization activities for EA H1N1.The protective efficacies of two MAbswere further verified in BALB/c mice,which suggested that MAbs 2B6 and 4C7 can effectively provide protection against the infection with homologous and heterologous H1N1 influenza viruses.Two MAbs,with high viral neutralization activity and strong anti-viral infection ability,were prepared,could be used for further research on anti-viral infection and the molecular basis of antigenic variation of EA H1N1 SIVs.