犬弓首蛔虫黏蛋白2的原核表达及多克隆抗体的制备  

Prokaryotic Expression and Polyclonal Antibody Preparation of Toxocara canis Mucin-2

作　　者：贾红菓 谭纯 王冰楠 梅四鹏 孙雪 杨晓伟 周荣琼 JIA Hongguo;TAN Chun;WANG Bingnan;MEI Sipeng;SUN Xue;YANG Xiaowei;ZHOU Rongqiong(College of Veterinary Medicine,Southwest University,Rongchang Chongqing 402460,China)

机构地区：[1]西南大学动物医学院,重庆荣昌402460

出　　处：《西南大学学报（自然科学版）》2023年第1期66-73,共8页Journal of Southwest University(Natural Science Edition)

基　　金：重庆自然科学基金项目(cstc2019jcyj-msxm2338);中央高校基本科研业务费资助项目(XDJK2020RC001).

摘　　要：根据犬弓首蛔虫(Toxocara canis)黏蛋白2的基因组数据(GenBank:AF167707),以T.canis成虫的基因组DNA为模板扩增Tc-muc-2基因并进行序列分析;同时构建Tc-muc-2/pCold TF原核表达载体,采用ITPG诱导表达,对重组蛋白进行纯化并制备多克隆抗体.结果显示Tc-muc-2基因全长序列为549 bp,共编码183个氨基酸;多重序列比对发现犬弓首蛔虫的黏蛋白均具有SKhT保守结构域,且含有不同串联重复序列组成的黏蛋白结构域;种系发育分析结果显示与猪蛔虫进化关系较近;SDS-PAGE结果表明重组蛋白Tc-MUC-2相对分子质量为7.5×10^(4),以可溶性形式表达;利用Ni-NTA亲和层析柱纯化蛋白,以250 mmol/L咪唑进行洗脱时可获得高纯度目的蛋白;将纯化后的重组蛋白免疫新西兰大白兔以制备多克隆抗体,间接ELISA结果显示其多克隆抗体效价大于1∶512000,表明重组蛋白的免疫原性较好,SDS-PAGE结果显示纯化后的抗体纯度高于95%,Western Blot结果显示抗体能与Tc-MUC-2蛋白特异性结合,表明抗体特异性高.The Toxocara canis mucin-2(Tc-muc-2)gene was amplified with the adult worm as template according to the genomic data of Tc-muc-2(GenBank:AF167707)and analyzed.The prokaryotic expression vector Tc-muc-2/pCold TF was constructed and induced by IPTG.The recombinant protein was purified and polyclonal antibody was prepared.The results showed that the full-length sequence of the Tc-muc-2 gene was 549 bp,which encoded 183-amino-acid.Multiple alignment found that the mucins of T.canis contained a conserved SKhT domain and a mucin domain which composed of different tandem repeats.The phylogenetic tree analysis suggested that Tc-MUC-2 was closely related to the sequence of Ascaris suum.SDS-PAGE analysis showed that the molecular weight of the recombinant protein Tc-MUC-2 was about 7.5×10^(4) and in soluble form.The protein was purified by Ni-NTA affinity chromatography column by eluting with 250 mmol/L imidazole.The purified recombinant protein was used to immunize the New Zealand white rabbits to prepare polyclonal antibody.Indirect ELISA result showed the titer of the polyclonal antibody was higher than 1∶512000,indicating that the recombinant protein had good immunogenicity.The SDS-PAGE result showed that the purity of antibody was higher than 95%,and the Western Blot result showed that antibody could specifically recognize Tc-MUC-2,indicating that the antibody had good specificity.

关 键 词：犬弓首蛔虫 黏蛋白2 克隆 原核表达 多克隆抗体 

分 类 号：S852.7[农业科学—基础兽医学]