人食管癌细胞cDNA表达文库的构建和鉴定

Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells

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 a cDNA phage expression library for human esophageal cancer cells. Methods After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand eDNA were synthesized through reverse transcription. With the undesirable eDNA fragments removed, the remaining eDNA （linked with EcoR I aptamer and phosphorylated its 5＇end） combined with the carrier of T7 Selectl0-3b. The recombinant phage were packaged in vitro for preliminary eDNA library. PCR was used to identify the size of inserted eDNA. Results The constructed original eDNA phage expression library for human esophageal cancer cells was consisted of 2.01 × 106 pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted eDNA fragments were range from 300 bp to 1 500 bp. Conclusion The eDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen eDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone （SEREX）.