抗猪甲型H1N1流感病毒血凝素蛋白单克隆抗体的制备与特性分析  

Preparation and Characterization of Monoclonal Antibody Against Hemagglutinin Protein of 2009 Strain of H1N1 Swine Influenza Virus

作　　者：张健[1] 陈艳[1] 乔传玲[1] 杨焕良[1] 唐续[1] 辛晓光[1] 陈化兰[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室农业部动物流感重点开放实验室,哈尔滨150001

出　　处：《畜牧兽医学报》2011年第5期711-716,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：哈尔滨市科技攻关计划项目(2009AA6BN078)

摘　　要：本研究旨在制备猪甲型H1N1流感病毒血凝素(HA)蛋白的特异性单克隆抗体。采用RT-PCR方法扩增猪甲型H1N1流感病毒的HA基因,将其克隆至真核表达载体pCAGGS上,获得重组质粒pCAGGS-HA,转染293T细胞,通过间接免疫荧光(IFA)检测表明HA蛋白在293T细胞中得到表达。将pCAGGS-HA以100μg.只-1剂量免疫5周龄BALB/c小鼠,获得4株稳定分泌抗HA蛋白单克隆抗体(MAb)的杂交瘤细胞株,分别命名为11G7、3C10、3G3和2B11。其中11G7诱导小鼠产生的腹水HI效价为14log2,中和效价为1∶8 192,HI试验结果进一步表明11G7只与甲型H1N1流感病毒发生反应,而不与其他H1N1、H1N2、H3N2、H5N1及H9N2亚型流感病毒反应。该MAb的制备将为建立猪甲型H1N1流感病毒与传统亚型SIV的鉴别诊断方法奠定基础。This experiment was conducted to prepare monoclonal antibodies specific to the hemagglutinin protein of H1N1 swine influenza virus（SIV）,A/Swine/Heilongjiang/44/2009.Hemagglutinin（HA） gene of the SIV was amplified by RT-PCR,and then the HA gene was subcloned into the eukaryotic express vector pCAGGS.The recombinant plasmid was named as pCAGGS-HA.Expression of HA protein in 293T cell was confirmed by indirect immunofluorescence assay（IFA）.Five weeks old BALB/c mice were immunized with 100 μg pCAGGS-HA plasmid each time,and 4 hybridoma cell lines secreting MAbs against the HA protein were obtained,which were designated as 11G7,3C10,3G3 and 2B11,respectively.The mice ascite of 11G7 has a HI titer of 14log2,and a neutralization titer of 1：8 192.Furthermore,11G7 could only react with the 2009 H1N1 SIV,and not with other H1N1,H1N2,H3N2,H5N1 and H9N2 subtype SIVs,which provide an useful tool for differential diagnosis between 2009 H1N1 and other traditional subtype SIVs.

关 键 词：甲型H1N1猪流感病毒 HA MAB 

分 类 号：S852.659.5[农业科学—基础兽医学]