摘要
目的:寻找人源性大肠癌相关抗原基因.方法:构建3个以入Tripl Ex2噬菌体作载体的大肠癌抗原cDNA表达文库,用自体或异体大肠癌患者血清应用SEREX方法进行免疫筛选.用平板法扩增阳性克隆噬菌体,提取纯化DNA,用SfiI酶切和PCR鉴定插入片段的大小.结果:构建成3个大肠癌抗原cDNA噬菌体表达文库,滴度分别为2.39×106nfu/L,2.07×106nfu/L和1.86×106nfu/L,插入片段长度为0.5-4kb,平均分别为1.4kb,1.6kb和1.3kb.筛选发现4个阳性克隆,插入cDNA片段大小分别为2.4 kb,1.8 kb,2.3 kb和2.2 kb.结论:用SEREX方法筛选大肠癌抗原cDNA表达文库,可获得有价值的大肠癌的重组抗原基因克隆,将有利于大肠癌的早期诊断和重组疫苗的研究.
AIM: To screen and identify the human colorectal carcinoma associated antigen genes. METHODS: Three human colorectal carcinoma cDNA phage expression libraries were constructed. They were screened from autologous and allogeneic sera of colorectal cancer patients by SEREX (serological identification of antigens by recombinant expression cloning). The sera were pre-absorbed by the extract E.coli XL1-blue. Four different serum-reactive cDNA clones were isolated by immunoscreening from a colon cancer-derived cDNA expression library. Positive clones were amplified by plate culture, the purified lambda phage DNA was cut by Sfi Ⅰ restriction endonucleases and amplified with PCR in order to identify the insert size of cDNA by electrophresis. RESULTS: Three cDNA phage expression libraries were constructed. The titer of library was 2.39×10~6 nfu/L, 2.07× 10~6 nfu/L and 1.86×10~6 nfu/L respectively, The range of the fiagment length of exogenously inserted cDNA was between 0.5-4 kb, the average was 1.4 kb, 1.6 kb and 1.3 kb, respectively. Four gene clones were obtained by SEREX screening, the length of their insert fragments was 2.4 kb, 1.8 kb, 2.3 kb and 2.2 kb, respectively. CONCLUSION: To screen and identify human colorectal carcinoma cDNA phage expression libraries by SEREX is a useful method to search for human colorectal carcinoma associated antigen genes.It is important for early diagnisis and research of recombinant vaccine for colorectal cancer.
出处
《世界华人消化杂志》
CAS
2003年第9期1378-1381,共4页
World Chinese Journal of Digestology
基金
国家自然科学基金
No.30171053
