摘要
目的 构建人源性大肠癌cDNA噬菌体表达文库。方法 从人大肠癌组织中提取总RNA ,RT PCR反转录合成cDNA第 1链 ,用长距离PCR技术合成cDNA第 2链 ,除去小于 5 0 0bp的小片段后与λTriplEx2噬菌体连接 ,体外包装 ,构建成cDNA噬菌体表达文库。转染E .coliXL1 Blue大肠杆菌 ,滴定文库的滴度 ,确定文库容量大小 ,测定重组率。用EXTagTM酶做PCR和SfiⅠ酶切鉴定插入的cDNA片段大小。结果 构建成含 2 .39× 10 6pfu/ml重组子的人大肠癌cDNA噬菌体表达文库 ,重组率为 97.5 %。插入的外源cDNA片段大小为 5 0 0bp～ 4kb ,平均长约 1.4kb。 结论 成功构建高质量人源性大肠癌cDNA噬菌体表达文库 ,适合于免疫筛选cDNA克隆的人大肠癌相关抗原基因。
Objective To construct a human colorectal carcinoma cDNA phage expression library. Methods Total RNA was extracted from the tissues of human colorectal carcinoma. The single-strand and double-strand of cDNA were synthesized through reverse transcription PCR and long distance PCR. cDNA fragments smaller than 500 bp were removed, and the remaining cDNAs were combined with the dephophrylated λTriplEx2 phage vector. The recombinant cDNAs were packaged in vitro with MaxPlax TM Packaging Extract, and then a small portion of packaged phage was used to infect E.coli XL1-Blue for determining the titration and the percentage of recombinant clones. EX Tag TM Extrue PCR and Sfi Ⅰ restriction endonucleases were used to identify the size of inserted cDNA. Results The human colorectal carcinoma cDNA phage library consisting of 2.39×10 6 pfu/ml bacteriophages was constructed, and the recombinant percentage was 97.5%. The range of the fragment lenth of the inserted cDNA was between 500 bp and 4 kb, with the average of 1.4 kb. Conclusion The successful construction of the human colorectal carcinoma cDNA phage library may have an important role in the searching for human colorectal carcinoma associated antigen genes through immunoscreening cDNA clones.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2003年第10期599-602,共4页
Chinese Journal of Digestion
基金
国家自然科学基金 (3 0 1710 5 3 )
