猪流感病毒H1亚型HA蛋白特异性单克隆抗体的研制

Preparation of monoclonal antibodies against HA protein of subtype H1 of swine influenza virus

目的:研制猪流感病毒抗H1亚型HA特异性单克隆抗体(mAb)。方法:构建H1亚型猪流感病毒A/Swine/Guangdong/LM/2004(H1N1)HA基因表达质粒,基因免疫6～8周龄雌性BALB/c小鼠,加强免疫后取其脾细胞与骨髓瘤细胞Sp2/0进行融合。通过ELISA、HI试验筛选阳性克隆。应用ELISA、HI试验和Western blot试验测定mAb的反应性和特异性。结果:共获得3株分泌抗H1亚型猪流感病毒HA蛋白的杂交瘤细胞株,分别命名为8C4、8C6、9D6。mAb腹水ELISA效价在1∶16000～1∶256000之间;HI效价为1∶512～1∶256000;对A/Swine/Guangdong/LM/2004的中和效价分别为:10-2.83、10-6.4、10-5.8。Westernblot分析结果显示,3株mAb只与H1亚型猪流感病毒HA蛋白反应。结论:HA蛋白特异性mAb的研制为H1抗原变异分析及H1亚型猪流感诊断方法的建立奠定了物质基础。

AIM：To prepare the monoclonal antibodies（mAb）against the HA protein of subtype H1 of swine influenza virus（SIV）.METHODS：To construct recombinant expression plasmid subtype H1 of SIV HA gene of A/Swine/Guangdong/LM/2004（H1N1）.BALB/c mice of 6-8 weeks old were immunized endemically with the recombinant plasmid.The splenocytes from the immunized mice were fused with Sp2/0 myeloma cells after the last immunization.Hybridoma cells were screened by ELISA and hemagglutination inhibition（HI）tests.The activity and specificity of mAbs were evaluated by HI test and Western blot assay.RESULTS：Three hybridoma cell lines secreting specific mAbs named 8C4,8C6,and 9D6 were developed.The ELISA titer of these mAbs was 1∶16 000-1∶256 000,the HI titer was 1∶512-1∶256 000;The neutralized titer of 8C4,8C6,and 9D6 to A/Swine/Guangdong/LM/2004（H1N1）was 10-2.83,10-6.4 and 10-5.8.Western blot analys confirmed that mAbs only reacted with HA protein of subtype H1 of SIV.CONCLUSION：These mAbs can be used as a useful tool to analyze the antigenic variation.They also provide the effective reagents for the rapid detection of subtype H1 of SIV.