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目的构建人源性大肠癌抗原基因cDNA噬菌体表达文库。方法从人大肠癌组织中提取总RNA,用RT-PCR合成cDNA第1链,用LD-PCR合成cDNA第2链,除去小于500 bp的小片段,与去磷酸化的λTriplEx2噬菌体左、右臂连接,体外包装,构建成cDNA噬菌体表达文库。转化E.coliXL1-Blue感受态细胞,滴定文库的滴度,确定文库容量大小;用ITPG诱导和X-gal显色测定重组率;用SfiⅠ酶切鉴定插入的cDNA片段大小。结果构建成含2.39×106 pfu/ml重组噬菌体的人大肠癌抗原基因cDNA噬菌体表达文库,重组率为97.5%,插入cDNA片段大小范围从600~4 000 bp,平均长约1 400 bp。结论成功构建高质量人源性大肠癌抗原基因cDNA噬菌体表达文库,适合于大批量筛选cDNA克隆的大肠癌相关抗原基因。
Objective To construct cDNA phage expression library of human colorectal cancer antigen gene. Methods Total RNA was extracted from human colorectal cancer tissue. The first strand of cDNA was synthesized by RT-PCR. The second strand of cDNA was synthesized by LD-PCR. Small fragments of less than 500 bp were removed and ligated with the left and right arms of dephosphorylated λTriplEx2 phage Connected, packaged in vitro and constructed into cDNA phage expression library. The competent cells were transformed into competent cells of E.coli XL-1 and the titer of the library was titrated to determine the size of the library. The recombination rate was determined by ITPG induction and X-gal staining. The size of the inserted cDNA was identified by SfiI digestion. Results The cDNA phage expression library of human colorectal cancer antigen was constructed with recombinant phage containing 2.39 × 106 pfu / ml. The recombination rate was 97.5%. The inserted cDNA fragments ranged from 600 to 4 000 bp in length with an average length of 1 400 bp. Conclusion The cDNA phage expression library of high quality human colorectal cancer antigen gene was successfully constructed and suitable for large scale screening of colorectal cancer related antigen genes of cDNA clones.