人源性大肠癌抗原基因噬菌体表达文库的构建及鉴定

Construction and identification of the cDNA phage expression library for human colorectal cancer antigens

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500p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.coli XL1-B1ue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Sfi Ⅰ酶切鉴定插入的cDNA片段大小.结果构建成含2.39×106pfu/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600～4000bp,平均长约1400bp.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 a cDNA phage expression library for human colorectal carcinoma antigens. Methods After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphosrylated λTriplEx2 phage vector. The recombinant phage were then packaged in vitro by MaxPlax TM Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue. Titer measurement was performed so as to determine the capacity of the library. SfiⅠrestriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA. Results The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39×10 6 pfu/ml bacteriophages with a recombination rate of 97.5% and the lenth of the inserted cDNA fragment ranged from 600 to 4 000 bp with an average of 1 400 bp. Conclusion The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening.