斑点叉尾天然单链(ScFv)噬菌体抗体库的构建及初步鉴定  

Construction and Preliminary Identification of Single-chain Antiboly Library of Natural Phage in Channel Catfish,Ietalurus punetaus

作　　者：王辉[1] 邹世平[2] 

机构地区：[1]华中农业大学水产学院,武汉430070 [2]中国水产科学研究院长江水产研究所,武汉430223

出　　处：《湖北农业科学》2011年第24期5239-5242,5249,共5页Hubei Agricultural Sciences

摘　　要：以健康未经免疫的斑点叉尾为试验材料,取其新鲜外周血,分离淋巴细胞,提取总RNA,逆转录合成cDNA。PCR扩增重链Fd和轻链cDNA,以纯化的VH、VL基因为模板,以与VH基因3′-端和VL基因-5′端序列互补的Linker—Primer Mix为引物,将VH、VL随机拼接为ScFv。通过酶切连接,依次将PCR产物插入质粒p3MH相应位点,热激法转化大肠杆菌XL1-Blue,加入辅助噬菌体VCSM 13,构建噬菌体单链抗体库。测定库容并酶切鉴定。斑点叉尾天然单链(ScFv)噬菌体抗体库成功构建为进一步筛选特异性抗体及从斑点叉尾病变组织中提取特异性抗原用于免疫治疗奠定了基础。Peripheral blood lymphocytes were isolated from 250 mL blood,which was obtained from 50 non-immunnized healthy channel catfish through their vena caudalis.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were PCR amplified and the amplification products were compacted by Linker—Primer Mix into ScFv.Then the amplification products were ligated successively into the phagemid vector p3MH,then the ligated sample was transformed into competent E.coli XL1-Blue.The transformed cells were infected with VCSM 13 helper phage to yield recombinant phage ScFv antibody.The successful construction of naive channel catfish phage antibody library created favourable conditions for selecting specific phage antibody and abstracting specific antigen.

关 键 词：斑点叉尾 抗体库 噬菌体展示 抗体单链(ScFv) 

分 类 号：Q959.468[生物学—动物学]