摘要
为制备猪伪狂犬病病毒(PRV)gE糖蛋白的单克隆抗体(MAb),本研究将PRV JM株gE蛋白主要抗原表位区域(aa52~aa238)的基因序列经密码子优化后由公司合成后克隆于pET-32a载体中构建重组表达质粒pET-32a-gE,经原核表达及纯化获得重组gE蛋白(rgE),采用SDS-PAGE鉴定rgE的表达及纯化效果,并经BCA法测定rgE的浓度。将其免疫BALB/c小鼠。取3次免疫小鼠的脾脏制备脾细胞,与小鼠骨髓瘤细胞(SP2/0)融合。通过ELISA筛选杂交瘤细胞株,经western blot和间接免疫荧光试验(IFA)鉴定MAb的反应性,利用MAb亚类鉴定试剂盒检测其重链和轻链类型。以截短的gE合成多肽作为抗原,采用间接ELISA方法鉴定MAb识别的抗原表位,并利用MAb通过western blot和IFA鉴定PRV野毒株JM及gE基因缺失株PRV JMΔgE/TK中gE蛋白的表达水平。结果显示,经间接ELISA方法筛选到1株稳定分泌gE蛋白的单克隆细胞株,命名为4C6B。Western blot和IFA均表明本研究筛选的MAb能够特异性识别PRV感染PK-15细胞中表达的gE蛋白;亚类鉴定结果显示MAb重链为IgM型,轻链为κ链。间接ELISA结果显示MAb能够识别gE蛋白中的一个线性B细胞表位131ACHPDLVLGRACVPEAPEMG^(150)。利用制备的MAb腹水进行PRV野毒和gE基因缺失株的鉴定,结果显示该MAb能够有效鉴别PRV JM野毒株与gE缺失株。本研究制备的PRV gE蛋白的4C6B MAb可以用于特异性鉴别PRV JM野毒株和gE基因缺失株,同时也可以为PRV检测方法的建立、PRV基因缺失疫苗的构建以及gE蛋白的生物学功能研究提供生物材料。
To prepare monoclonal antibodies(MAbs)against the gE glycoprotein of pseudorabies virus(PRV),this study constructed a recombinant expression plasmid pET-32a-gE containing the gene sequence of the major antigenic epitope region(aa52-aa238)of the gE protein of PRV-JM strain.The recombinant gE protein was expressed and purified,and then immunized into BALB/c mice.The spleens of mice immunized three times were collected to prepare splenocytes,which were fused with mouse myeloma cells(SP2/0).A hybridoma cell line stably secreting anti-PRV gE MAb was obtained through ELISA screening.The reactivity of the MAb was identified by western blot and indirect immunofluorescence assay.Heavy and light chain identities were determined using a MAb subclass identification kit.The antigenic epitope recognized by the MAb was identified by indirect ELISA with a truncated gE synthetic peptide as the antigen.The MAb was used to detect gE protein expression in wild-type PRV and the gene-deleted virus PRV JMΔgE/TK strain.The results showed that a monoclonal cell line stably secreting the anti-gE MAb was successfully obtained using the indirect ELISA method,which was named 4C6B.Both western blot and IFA indicated that the MAb specifically recognized gE protein expressed in PRV-infected PK-15 cells.Subclass identification revealed the heavy chain was IgM and the light chain wasκ.Indirect ELISA showed the MAb recognized a linear B cell epitope 131ACHPDLVLGRACVPEAPEMG^(150).The MAb-prepared ascites was used to identify wild-type PRV JM strain and gE gene deleted strain.The results showed that the 4C6B MAb can specifically distinguish PRV JM wild-type and gE gene-deleted strains.In summary,the 4C6B MAb against PRV gE protein prepared in this study can be used to specifically identify PRV JM wild-type strain and gE gene-deleted strain.It can also provide materials for the establishment of PRV diagnostic methods,the construction of gene-deleted vaccine,and the research on gE protein biological functions.
作者
俞机敏
刘宏扬
刘云飞
王尚辉
宋厚辉
张朝霞
翁长江
YU Ji-min;LIU Hong-yang;LIU Yun-fei;WANG Shang-hui;SONG Hou-hui;ZHANG Zhao-xia;WENG Chang-jiang(College of Animal Science and Technology&College of VeterinaryMedicine of ZhejiangA&F University,Hangzhou 311300,China;Division of Fundamental Immunology,State Key Laboratory for Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Heilongjiang Provincial Key Laboratory of Veterinary immunology,Harbin 150069,China)
出处
《中国预防兽医学报》
北大核心
2025年第8期832-838,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
中国农业科学院科学中心项目(CAAS-CSLPDCP-202401)。
关键词
猪伪狂犬病病毒
单克隆抗体
gE蛋白
表位鉴定
pseudorabies virus
monoclonal antibody
gE protein
epitope identification
