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目的构建人食管癌细胞cDNA噬菌体表达文库。方法从食管癌细胞中提取总RNA,利用磁珠吸附法分离其中的mRNA,逆转录合成cDNA第一链,再通过碱基互补配对原则合成第二链,对双链cDNA进行末端修饰,连接EcoRⅠ适配子,磷酸化EcoRⅠ适配子5′端,除去不合要求的cDNA片段,将符合要求的与载体(噬菌体T7Select10-3b)连接,然后进行体外包装建成初步的cDNA文库,并对文库进行鉴定。结果食管癌细胞cDNA原始表达文库的滴度为2.01×106 pfu/mL,重组率高达100%,插入片段的大小范围在300~1 500bp之间。结论本实验成功构建了人食管癌T7噬菌体展示cDNA表达文库,并经过了初步鉴定,为下一步利用重组表达cDNA克隆的血清学分析技术(SEREX技术)鉴定食管癌相关抗原奠定基础。
Objective To construct cDNA phage expression library of human esophageal cancer cells. Methods The total RNA was extracted from esophageal cancer cells, and the mRNA was separated by magnetic beads adsorption. The first strand of cDNA was reverse transcribed. The second strand was synthesized by base pairing. The double strand cDNA was modified by EcoRⅠ Aptamers were used to phosphorylate the 5 ’end of the EcoRI adapter to remove unwanted cDNA fragments. The desired cDNA fragments were ligated with the vector (phage T7Select10-3b) and then packaged in vitro to construct a preliminary cDNA library. The library was identified . Results The titer of the cDNA expression library of esophageal cancer cells was 2.01 × 106 pfu / mL, the recombination rate was as high as 100%, and the size of the insert ranged from 300 to 1 500 bp. Conclusions The cDNA library of T7 phage display human esophageal cancer was successfully constructed and preliminary identified. This study laid the foundation for the identification of esophageal cancer related antigen using serological analysis (SEREX technique) of recombinant cDNA cloning in the next step.