人胃癌细胞定向克隆表达cDNA文库的构建及鉴定

Construction and Idenification of Directional Expressing cDNA Library from Human Gastric Cancer MGC-803 Cell

目的：克隆胃癌的相关基因。方法：提取人胃癌细胞总RNA，分离其mRN。以NatI／oligo（dT）12-18为引物合成双链。cDNA，除去小于400bp的cDNA片段后，连接EcoRⅠ人工接头，经NotⅠ酶酶切，插入定向克隆表达载体λExcellNotI／EcoRI／CIP，体外包装后转染大肠杆菌NM522，构建了人胃癌细胞MGC－803定向克隆表达。cDNA文库。结果：文库含1．2×106个重组子，重组率为96．7％。用PCR技术鉴定，文库中插入cDNA的平均大小为1kbc结论：该文库适于筛选各种丰度mRNA的cDNA克隆。

Purpose: Cloning of the associated antigen of gastric cancer. Metheds: Total RNA and mRNA was extracted from the gastric cancer cell strain MGC-803. The cDNA was synthesized through reveme transcription by using Not I/oligo(dt) 12 - 18 as primer. The cDNA fragments smaller than. oeoebp were removend and the remaining cDNA was connected with Ecor I adaptors which was then digested with Not I and ligated to dimetional expressing vector λ Excel Not I/EcoR I/CIP. The recombinat cDNA packaged in vitro was used to infect NM522. Eventually, the directonal expressing cDNA library for human gastric cancer MGC-803 cell was constructed. Result: The library consisted of 1 .2 ×10° recombinats. The recombination rate was 96.7%. The average size of the inserts was 1kb. Conclusion: The library was suitable for screening all abundant InRNA for cDNA clone.