2.1亚型猪瘟病毒流行株E2蛋白单克隆抗体的制备及其抗原表位的鉴定  

Preparation and antigenic epitope mapping of a specific monoclonal antibody against glycoprotein E2 of classical swine fever subgenotype 2.1 field strain

作　　者：张艺潇 吴梦[3] 米士江 刘钟迪 涂长春 龚文杰 ZHANG Yi-xiao;WU Meng;MI Shi-jiang;LIU Zhong-di;TU Chang-chun;GONG Wen-jie(College of Veterinary Medicine,Jilin University,Changchun 130062,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Changchun 130012,China;Academy of Animal Sciences of Chongqing city,Chongqing 408599,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]中国农业科学院长春兽医研究所,吉林长春130012 [3]重庆市畜牧科学院,重庆408599

出　　处：《中国预防兽医学报》2024年第5期511-521,共11页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(32072843)。

摘　　要：为获得仅与我国基因2.1亚型猪瘟病毒(CSFV)流行株反应的单克隆抗体(MAb),并揭示疫苗株与流行株E2蛋白抗原氨基酸的差异,本研究利用基因2.1亚型重组质粒p FastBac1-JL23 E2通过杆状病毒表达重组E2蛋白(rE2),并采用Ni柱纯化后经SDS-PAGE检测r E2的表达及纯化效果;采用western blot鉴定r E2的反应原性。SDS-PAGE及western blot结果表明经杆状病毒表达了基因2.1亚型CSFV E2蛋白,且其纯化效果和反应原性均较好。采用杂交瘤技术制备2.1基因亚型CSFV E2蛋白的单克隆抗体(MAb),采用亲和层析法纯化该MAb,采用BCA法测定其浓度及亚类。将79株1.1、2.1、2.2和2.3基因亚型的CSFV及1.1基因亚型的HCLV株和SM株感染PK-15细胞,72 h后采用IFA检测MAb与不同基因型CSFV的反应性;采用western blot鉴定MAb与10个基因亚型共18种CSFV代表株E2蛋白的反应性。采用IFA鉴定MAb与不同基因型CSFV的中和活性。结果显示,经IFA筛选后共获得16株分泌E2蛋白MAb的杂交瘤细胞株。其中仅一株MAb TCH061与所有2.1基因亚型(2.1a、2.1b、2.1c、2.1g、2.1h、2.1i、2.1j、2.1k、2.1l、2.1m、2.1n)CSFV感染的细胞反应后出现绿色荧光,与基因1.1亚型HCLV疫苗株和SM株以及其他基因型CSFV感染的细胞反应后均无绿色荧光;western blot结果显示,该MAb能够识别2.1基因亚型CSFV E2蛋白(2.1a、2.1b、2.1c、2.1g、2.1h、2.1i、2.1j),在90 ku处出现特异性条带,而与其他基因型CSFV E2蛋白均不反应。表明TCH061为2.1基因亚型CSFV E2蛋白特异性的MAb。MAb的纯化结果显示,在50 ku和25 ku处有明显条带,其浓度为1.70μg/μL且该MAb TCH061重链为Ig G2a,轻链为κ链。中和活性结果显示,TCH061对JL^(23)株(2.1b)、GDLF1株(2.1c)有一定的中和活性,但不能中和C株。分别以GD53株E^(24)个不同区域的截短蛋白为抗原通过western blot初步确定MAb识别抗原表位的区域。利用CLC Sequence Viewer比对与MAb反应的CSFV E2蛋白氨基酸序列的差�In order to develop the differential monoclonal antibody(MAbs)that specifically reacts with the dominant subgenotype 2.1 field strains of classical swine fever virus(CSFV)in China and to reveal the antigenic difference of E2 protein between the vaccine C-strain and the field strains,this study used subgenotype 2.1 recombinant plasmid pFastBac1-2.1 JL 23 E2 by baculovirus expression recombinant E2 protein(rE2).The efficiency of expression and purification were assessed by SDS-PAGE following Ni column purification,and its reactivity was further evaluated using western blot analysis.The findings from both methods confirmed the expression of the subgenotype 2.1 CSFV E2 protein with commendable purification efficiency and reactivity.Monoclonal antibody(MAb)specific to the E2 protein of 2.1b subgenotype CSFV was prepared by hybridoma technique.79 strains of CSFV subgenotypes 1.1,2.1,2.2 and 2.3 were inoculated with PK-15 cells,and the reaction patterns of MAb to different genotypes of CSFV tested by IFA after 72 hours.The reactivity between MAb and 18 E2 proteins derived from CSFV belonging to 10 subgenotypes were verified by western blot.The MAb was subsequently purified through affinity chromatlography,and its concentration was precisely determined using BCA method and detection subclasses of MAb.The immunofluorescence assay(IFA)was used to identify the neutralizing activity of MAb against the diverse genotypes of CSFV.Following IFA identification,the positive nderwent additional subcloning,leading to the acquisition of 16 hybridoma cells that secreted the E2 MAb,The IFA results singled out one particular MAb,TCH061,which exhibited a unique reactivity profile,binding exclusively to cells infected with all variants of subgenotype CSFV 2.1(2.1a,2.1b,2.1c,2.1g,2.1h,2.1i,2.1j,2.1k,2.1l,2.1m,2.1n),while showing no reaction to the vaccine strain or other subgenotype CSFV.Western blot results showed that the TCH061 could specifically recognize the E2 protein subgenotype of CSFV 2.1(2.1a,2.1b,2.1c,2.1g,2.1h,2.1i,2.1j),with a

关 键 词：猪瘟病毒 单克隆抗体 病毒反应性 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]