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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5.0×107pfu/mL,扩增后文库滴度为3.5×1012pfu/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 construct T7 phage display peptide library of renal cell carcinoma and lay a foundation for the next screening of molecular markers for early diagnosis of renal cell carcinoma. Methods: Trizol method was used to extract total RNA of 31 cases of renal cell carcinoma tissues of various tissue types. After confirming the integrity, the total RNA was mixed according to the equal value of OD value. The kit was used to separate the mRNA and the quality was detected by electrophoresis. After reverse transcription, the end of the flush, the length of the screening, plus linker, double enzyme digestion, remove the extra linker and <300 bp cDNA fragments and other steps, and then T7Select10-3b vector connection, in vitro packaging and amplification of renal cancer T7 Phage display cDNA library. The library quality was identified by plating assays and PCR techniques. Results: The library capacity of the original library was 5.0 × 107pfu / mL. The amplified library titer was 3.5 × 1012pfu / mL, the recombination rate was 95%, and the inserted fragment was 300-2000 bp. CONCLUSION: The high quality cDNA library of renal cell carcinoma, constructed by T7 bacteriophage, was successfully constructed and laid the foundation for the screening of specific markers of renal cell carcinoma for early clinical diagnosis.