紫红笛鲷MHC Ⅱα和MHC Ⅱβ多克隆抗体的制备  

Preparation of Polyclonal Antibodies Against MHC Ⅱα and MHC Ⅱβ of Mangrove Red Snapper(Lutjanus argentimaculatus)

作　　者：王天燕[1,2] 常虹[1,2] 余时琛[1,2] 陈璐[1,2] 蔡中华[2] 

机构地区：[1]清华大学生命科学学院,北京100084 [2]清华大学深圳研究生院海洋科学与技术学部,广东深圳518055

出　　处：《现代生物医学进展》2013年第1期1-4,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(41106087)

摘　　要：目的:制备紫红笛鲷主要组织相溶性复合体MHC Ⅱα和MHC Ⅱβ多克隆抗体,为蛋白水平研究紫红笛鲷MHC II分子提供理论和实践依据。方法:从已有的紫红笛鲷cDNA文库菌中分别克隆其MHC Ⅱα和MHC Ⅱβ分子的部分开放阅读框,与PQE-30构建表达载体,转入大肠杆菌E.coli M15以IPTG诱导表达;纯化得到的重组蛋白与弗氏佐剂混合乳化后注射新西兰大白兔制备多克隆抗体,再以酶联免疫吸附(ELISA)和免疫印迹(Western blot)检测所获抗血清的效价及效果。结果:①重组表达和纯化得到紫红笛鲷MHC Ⅱα和MHC Ⅱβ部分肽链。②制备的紫红笛鲷MHC Ⅱα和MHC Ⅱβ兔抗血清效价都大于1:25600,达到预期水平。③以获得的紫红笛鲷MHC Ⅱα和MHC Ⅱβ兔抗血清分别与紫红笛鲷头肾巨噬细胞蛋白进行免疫印迹,显示两种抗血清能分别杂合出各自的目标蛋白,说明制备的多克隆抗体实际应用效果良好。结论:紫红笛鲷MHC Ⅱα和MHC Ⅱβ多克隆抗血清制备成功。Objective： To prepare the polyclonal antibodies against MHC Ⅱα and MHC Ⅱβ of mangrove red snapper. Methods： A partial of MHC Ⅱα and MHC Ⅱβ chain was cloned from the cDNA library of mangrove red snapper, respectively. The PCR products were inserted into the expression vectors pQE30 and transformed into the E.coli M 15. By inducing of IPTG, the recombinant proteins of MHC Ⅱα and MHC Ⅱβ fragments were purified, respectively. The proteins were thoroughly mixed with Freund＇s adjuvant, and injected rabbit. The antiserums were detected by ELISA and Westem blot. Results： （1） The recombinant proteins of MHC Ⅱα and MHC Ⅱβ frag- ments were purified successfully. （2） The antiserums against MHC Ⅱα and MHC Ⅱβ both had a high titer above 1：25600. （3）The westem blot of head kidney macrophages showed the specification of MHC Ⅱα and MHC Ⅱβ antiserums, respectively. Conclusions： The high titer and specific polyclonal antibodies against MHC Ⅱα and MHC Ⅱβ of mangrove red snapper were prepared successfully.

关 键 词：MHC 主要组织相溶性复合体 抗体 鱼 紫红笛鲷 

分 类 号：Q95-33[生物学—动物学] Q75