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。
[Objective]The aim was to construct anti-HepG2 ribosonre display library. [ Methed] Balb/c nfiee 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 PCR. The VH/k chain was tigated into pMD18-T vector and the ligated sample was transformed into competent E. colt TGI. white elone was picked and the plasmid DNA was puttied. The plasmid DNA was identified with PCR and sequeneed. [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 reaetiou 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.
出处
《安徽农业科学》
CAS
北大核心
2009年第13期5886-5888,共3页
Journal of Anhui Agricultural Sciences
基金
江苏省教育厅自然科学基金(08KJD350002)
关键词
核糖体展示
单链抗体
肝癌
抗体库
Ribosome display
Single chain antibody
Hepatoma
Antibody library
