【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。更多还原

【Abstract】 [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.更多还原