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当前位置: 首页 > 文章 > 抗HepG2核糖体展示抗体库的构建 安徽农业科学 2009,37 (13) 80-82
Position: Home > Articles > Construction of Anti-HepG2 Ribosome Display Library Journal of Anhui Agricultural Sciences 2009,37 (13) 80-82

抗HepG2核糖体展示抗体库的构建

作  者：
周雷;毛伟平;冯娟;刘洪云;魏传静;李文秀;周凤云
单  位：
南京师范大学生命科学学院生化与生物制药研究所
关键词：
核糖体展示;单链抗体;肝癌;抗体库
摘  要：
[目的]构建鼠源抗HepG2核糖体展示抗体库。[方法]以培养的HepG2肝癌细胞4次免疫Balb/c小鼠,取脾脏分离总RNA,逆转录合成第一条链,PCR扩增重链可变区和轻链k,经linker DNA连接形成VH/k核糖体展示抗体库。将VH/k与pMD18-T载体的连接产物转化E.coli TG1,蓝白斑筛选,挑取白色菌落,提取质粒,PCR鉴定,并测序。[结果]VH和k链得到正确的扩增,长度分别为399和647bp,VH/k连接产物正确,连接产物长度为1 093 bp。[结论]所采用的引物及反应条件能够扩增出目的基因,成功构建了鼠源抗HepG2核糖体展示抗体库,为进行核糖体展示体外筛选抗HepG2特异性单链抗体奠定基础。
译  名：
Construction of Anti-HepG2 Ribosome Display Library
作  者：
ZHOU Lei et al(Institute of Biochemistry and Biological Preparations,College of Life Science,Nanjing Normal University,Nanjing,Jiangsu 210046)
关键词：
Ribosome display;Single chain antibody;Hepatoma;Antibody library
摘  要：
[Objective]The aim was to construct anti-HepG2 ribosome display library. [Method] Balb/c mice were immunized 4 times with HepG2.The total RNA was isolated from the spleens of immunized mice.The cDNA was generated by reverse transcription.Variable region of heavy chain and k chain genes(VH and k) were amplified separately,and anti-HepG2 VH/k chain ribosome display library was constructed by assembling VH and k into VH/k chain with a specially constructed linker DNA by PCR.The VH/k chain was ligated into pMD18-T vector and the ligated sample was transformed into competent E.coli TG1.white clone was picked and the plasmid DNA was purfied.The plasmid DNA was identified with PCR and sequenced.[Results]VH and k were amplified correctly and the length were 399 and 647 bp respectively.VH/k was ligated correctly and the length was 1 093 bp.[Conclusion]The primers and reaction condition were able to generate required genes,VH/k chain ribosome display library was constructed correctly.It was the basis for screening single chain antibody specific for HepG2 by ribosome display technique.

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