肾癌T7噬菌体展示肽库构建的实验研究  

Construction of renal cell carcinoma T7 phage-peptide library

作　　者：陈贻玲[1] 吴玲玲[2] 赵晋丰[2] 曹广文[2] 邓松华[1] 

机构地区：[1]安徽医科大学病理生理教研室,安徽合肥230032 [2]第二军医大学流行病学教研室,上海200433

出　　处：《中华肿瘤防治杂志》2009年第22期1757-1760,1764,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：安徽省自然科学基金(080413136)

摘　　要：目的：构建肾细胞癌T7噬菌体展示肽库,为下一步筛选肾癌早期诊断分子标志群打下基础。方法：用传统Trizol方法分别抽取31例涵盖各种组织类型肾癌组织标本的总RNA,确定完整性后再根据OD值等量混合总RNA,用试剂盒完成mRNA的分离并电泳检测其质量,经反转录、末端平齐、片段长短筛选、加接头、双酶切、去除多余接头和〈300 bp的cDNA片段等步骤,再与T7Select10-3b载体连接、体外包装并扩增得到肾癌T7噬菌体展示cDNA文库。通过铺平板测定和PCR技术鉴定所建文库质量。结果：建成原始文库的库容量为5.0×10^7pfu/mL,扩增后文库滴度为3.5×10^12pfu/mL,重组率为95%,插入片段为300-2 000 bp。结论：成功用T7噬菌体构建了高质量的肾癌cD-NA文库,为筛选可用于临床早期诊断的肾癌特异标志奠定基础。OBJECTIVE： To establish a renal cell carcinoma phage-peptide library which can be used as a valuable source in screening biomarkers for early diagnosis.METHODS： Total RNA was extracted separately from 31 renal cell carcinoma tissue samples according to a standard Trizol protocol.These samples contained all sorts of renal cell carcinoma.Equal amounts of total RNA from the 31 tissues were pooled together after the integrity of total RNA being confirmed.mRNA was isolated from total RNA and the agarose gel electrophoresis showed the range of mRNA size.Then reverse transcription,end modification,ligation to directional EcoR Ⅰ/Hind Ⅲ linkers,EcoR Ⅰ/Hind Ⅲ digesting,eliminating excess linkers and small fragments which were less than 300 bp were performed.After cDNA was ligated with T7Select10-3b EcoR Ⅰ/Hind Ⅲ vector arms,the renal cell carcinoma phage display cDNA library was constructed by package reaction in vitro and plate proliferation.The plaque assay and PCR were used to evaluate the library.RESULTS： The complexity of primary phage peptide library was 5.0×107 pfu/mL and the amplified library had a titer of 3.5×1012 pfu/mL.PCR identification showed that recombination ratio was 95% with the range of 300-2 000 bp inserts.CONCLUSION： The T7 phage display cDNA library from renal cell carcinoma is construted with high quality,and can be used to find the clear specific biomarkers for early detection of renal cell carcinoma.

关 键 词：肾肿瘤 细菌噬菌体T7 肽库 

分 类 号：R737.11[医药卫生—肿瘤]