猪I类白细胞抗原单克隆抗体的制备及其在非洲猪瘟病毒感染机制研究中的初步应用  

Preparation of SLA I monoclonal antibody and its preliminary application in the study of the infection mechanism of African swine fever virus

作　　者：王可欣 Wedu Tesfagaber 王婉 尹丽 孔惠 张振江 李芳 高彩霞[1] 步志高[1] 赵东明 WANG Ke-xin;WELDU Tesfagaber;WANG Wan;YIN Li;KONG Hui;ZHANG Zhen-jiang;LI Fang;GAO Cai-xia;BU Zhi-gao;ZHAO Dong-ming(State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2025年第1期90-95,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省自然科学基金杰出青年基金项目(JQ2023C005)。

摘　　要：猪I类白细胞抗原(SLA I)是机体内至关重要的免疫相关蛋白,是启动特异性细胞毒性T细胞(CTL)免疫应答的核心分子。在机体内,SLA I负责将内源性抗原肽递呈至细胞膜表面,由CD8+T细胞识别,启动机体适应性免疫应答。为了制备SLA I的单克隆抗体(MAb),本研究将SLA I-1*04:01等位基因胞外区序列与麦芽糖结合蛋白(MBP)序列连接,在大肠杆菌原核表达系统中表达,获得可溶性重组蛋白MBP-SLA I。经亲和层析纯化后,采用MBP-SLA I免疫BALB/c小鼠3次,采用ELISA检测小鼠血清抗体水平,对血清抗体效价在110000以上的小鼠进一步冲击免疫后,分离小鼠脾脏B淋巴细胞,与SP2/0骨髓瘤细胞融合。采用ELISA方法筛选阳性的杂交瘤细胞进行3次克隆纯化,最终筛选得到稳定分泌SLA I抗体的杂交瘤细胞株MAb-1A11。经ELISA检测纯化后的MAb-1A11效价可达13200,经小鼠MAb抗体亚类鉴定试剂盒鉴定其重链为IgG2b,轻链为κ。采用western blot检测该抗体的特异性,结果显示,MAb-1A11能够识别猪肺泡巨噬细胞(PAM)表达的内源性SLA I,在42 ku处出特异性条带,而对HEK293T细胞中表达的同源物人白细胞抗原无交叉识别作用。利用MAb-1A11为一抗,采用western blot进一步检测非洲猪瘟病毒(ASFV)HLJ/18-6GD株感染的PAM中SLA I的表达水平,结果显示,随着ASFV感染时间的延长,SLAI表达水平出现轻微下降。本研究制备了SLAI的MAb,并初步将其应用于ASFV感染机制的研究中,为深入了解SLAI所受调控的机制提供了可靠工具。Swine leukocyte antigen class I(SLA I)is a crucial immune-related protein in the body and it is the core molecule that initiates specific CTL immune responses.SLAⅠis responsible for presenting endogenous antigen peptides to the cell membrane surface within the body.Then SLAⅠ-peptides ligands were recognized by CD8+T cells,thereby activating the adaptive immune response.In this study,the extracellular region of SLA-1*04:01 was fused to maltose-binding protein(MBP)and expressed in the E.coli prokaryotic system,obtaining the soluble recombinant protein MBP-SLA-1.After purification by affinity chromatography,MBP-SLA I was used to immunize BALB/c mice.Following three immunizations,the immune effects were evaluated by ELISA.Mice with serum antibody titers exceeding 110000 were selected for booster immunization.Subsequently,mice spleen B lymphocytes were isolated and fused with SP2/0 myeloma cells.The screening process was performed by ELISA,and the positive hybridoma cells were subcloned by three times.Consequently,the hybridoma cell line MAb-1A11,which stably secreted SLAⅠantibody,was screened.The purified MAb-1A11 titer can reach 13200,and the subtype is IgG2b,κ.The specificity of this antibody was detected with western blot,and the results showed that MAb-1A11 was able to successfully recognize endogenous SLAⅠin porcine alveolar macrophages(PAM),and could not cross-recognize the homologue HLA expressed in HEK293T cells.Furthermore,MAb-1A11 was used as a primarily antibody to examine the expression level of SLAⅠin PAM infected with the African swine fever virus(ASFV)HLJ/18-6GD using western blot.The results showed there was a slight decrease in the expression level of SLAⅠwith the time went on during ASFV infection.In this study,the SLAⅠMAb was prepared and preliminarily applied to the study of ASFV infection mechanisms.It provided a reliable tool for gaining a deeper understanding of the regulatory mechanisms affecting SLA I expression.

关 键 词：猪白细胞抗原I 单克隆抗体 WESTERNBLOT 非洲猪瘟病毒 

分 类 号：S852.6[农业科学—基础兽医学]