H3N2亚型猪流感病毒血凝素单克隆抗体的制备及其抗原表位的鉴定  

Preparation and epitope identification of monoclonal antibody against hemagglutinin protein of H3N2 swine influenza virus

作　　者：陈艳[1,2] 王飞飞 杨焕良[2] 乔传玲[2] 陈化兰[2] 张秀英[1] CHEN Yan;WANG Fei-fei;YANG Huan-liang;QIAO Chuan-ling;CHEN Hua-lan;ZHANG Xiu-ying(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;Animal Influenza Key Laboratory of the Ministry of Agriculture and Rural Affairs/State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]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/农业农村部动物流感重点开放实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第6期633-638,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然基金(C2018072);中央公益性基金基本科研业务费专项(1610302017001)。

摘　　要：为制备H3N2亚型猪流感病毒(SIV)血凝素(HA)蛋白的特异性单克隆抗体(MAb)并对其抗原表位进行鉴定,本研究采用RT-PCR方法扩增H3N2亚型SIV的HA基因,将其克隆至真核表达载体pCAGGs中,获得重组质粒pCAGGs-HA,以重组质粒pCAGGs-HA免疫BALB/c小鼠,取其脾淋巴细胞与骨髓瘤细胞SP2/0融合,采用血凝抑制(HI)试验和ELISA方法筛选出1株稳定分泌抗SIV HA1蛋白MAb的杂交瘤细胞株(3F8),经ELISA检测结果显示其诱导小鼠产生的腹水效价为1:10^(6);HI试验检测结果显示其效价为12 log2。利用噬菌体随机7肽库对纯化的抗H3N2亚型SIV HA1蛋白的MAb 3F8进行了4轮筛选,经DNA测序得到3个7肽序列,分别是"HPRRRRV"(E1)、"WPFHHHR"(E2),"IERGRTS"(E3),3个7肽序列经比对显示与H3N2 SIV HA1蛋白有部分同源区,为HA蛋白的模拟表位,分别位于HA1蛋白序列的157、158位(E1)、196、199、200位(E2)、276、279、282位(E3),即位于流感病毒HA蛋白5个抗原位点中的B位点与C位点,且均位于HA1段上,与制备的抗HA1段蛋白的MAb鉴定结果一致。本研究制备了SIV H3N2亚型的HA蛋白MAb,并利用噬菌体短肽库技术获得该SIV HA模拟表位,为进一步设计具有广谱保护性的SIV表位疫苗提供了数据支持。This study was aimed to prepare monoclonal antibody(MAb)against hemagglutinin(HA)protein of H3N2 swine influenza virus(SIV)and to identify its epitope.HA gene of the SIV was amplified by RT-PCR and then cloned into the eukaryotic expression vector pCAGGS(pCAGGS-HA).BALB/c mice were immunized with pCAGGS-HA plasmid,and splenocytes from the immunized mice were fused with SP2/0 myeloma cells.Positive hybridoma clones were screened by hemagglutination inhibition(HI)test and Enzyme Linked Immunosorbent Assay(ELISA).A hybridoma cell line(3 F8)stably secreting monoclonal antibody against the SIV HA1 protein was obtained.The titers of ascites induced by 3 F8 MAb were 1:10^(6)and 12 log2 detected by ELISA and HI test,respectively.Phage random 7 peptide library was used to screen the epitopes reacting with the purified 3 F8 MAb.After the fourth screening,twenty phage clones were identified by using ELISA.Three heptapeptide sequences were obtained"HPRRRRV"(E1),"WPFHHHR"(E2)and"IERGRTS"(E3)which did not completely match the sequence of SIV HA protein.Mimotopes located at sites 157 and 158 of B antigenic region,and at sites 196,199,200,276,279,and 282 of C antigenic region of HA protein.All of the mimotopes located in the HA1 chain.In this study,we obtained the simulated epitopes of SIV HA protein by using the phage display library technology,which laid a theoretical support for the further design of a broad-spectrum protective SIV epitope vaccine.

关 键 词：H3N2 猪流感病毒 单克隆抗体 噬菌体展示 抗原表位 

分 类 号：S852.65[农业科学—基础兽医学]