人大肠癌抗原基因cDNA噬菌体表达文库的构建和鉴定

Construction and identification of cDNA library for colorectal carcinoma antigens

目的:为获得大肠癌的特异性抗原基因,构建人大肠癌抗原基因的cDNA噬菌体表达文库。方法:从2株人大肠癌细胞CCL-187和CX-1中提取总RNA,分离纯化mRNA,并以此为模板合成第1链cDNA,用置换法合成第2链cD-NA。双链cDNA经末端削平、EcoRⅠ接头连接、XhoⅠ酶切、过柱分级分离,除去<400bp片段。收集符合需要的cDNA片段与噬菌体ZAPExpress载体连接,体外包装后获得人大肠癌抗原基因的cDNA噬菌体表达文库。结果:构建的人大肠癌抗原基因的cDNA噬菌体文库,原始库容量为2.3×109pfu/L,重组率97%。重组子插入cDNA片段平均大小1kb以上。结论:成功地构建高质量的人大肠癌抗原基因的cDNA噬菌体表达文库,为大肠癌的特异性抗原筛选奠定了基础。

AIM： To construct a cDNA phage expression library of colorectal carcinoma antigens for screening colorectal carcinoma specific-antigens. METHODS： The total RNA was extracted from two colorectal carcinoma cell lines CCL-187 and CX-1 . The mRNA from total RNA was isolated to synthesize the first and second strand cDNA. The ds-cDNA termini were blunted with pfu DNA polymerase. The blunted cDNAs were ligated with EcoR I adapters and then digested by Xho I. Small cDNA molecules（ 〈400 bp） were removed through size fraction. After the cDNAs were inserted into ZAP expression vector, the ligated products were packaged in vitro and the bacteriophage particles infected the host strains XL1-Blue MRF＇. RESULTS： The efficiency of the primary was 2.3 × 10^9 pfu/L, while the recombination rate reached to 97%. The average length of the inserted fragment was over 1 kb. CONCLUSION： The quality of colorectal carcinoma cDNA phage expression library is excellent and helpful to screen colorectal carcinoma specificantigens.