人肺癌相关抗原的基因克隆

Molecular cloning of a novel human lung cancer associated antigen cDNA

目的克隆人肺癌相关抗原的基因。方法用λｇｔ１１Ｓｆｉ－Ｎｏｔ定向克隆载体构建人肺鳞癌细胞系Ｌ－７８的ｃＤＮＡ文库，并以抗人肺癌单抗ＡＬＴ－０４为探针进行了筛选。对名为ｈｌｃ－１４的ｃＤ－ＮＡ克隆作了测序分析与进一步研究。用重组的ｐＸＪ４１－ｈｌｃ１４质粒转染ＮＩＨ３Ｔ３细胞，通过免疫组化法对克隆基因进行了表达检测与鉴定，还观察了转染细胞的软琼脂集落形成能力。结果建成１．４×１０６ｐｆｕ／ｍｌ人肺癌ｃＤＮＡ文库，经过四轮免疫学筛选获得６个ｃＤＮＡ克隆，最大的ｃＤＮＡ克隆为ｈｌｃ－１４，长９５４ｂｐ。对该克隆的测序结果与基因库资料比较，未发现同源序列。ｈｌｃ－１４ｃＤＮＡ转染的ＮＩＨ３Ｔ３细胞有明显的肺癌相关抗原表达，并且在软琼脂中的集落形成能力增强。结论ｈｌｃ－１４是一个新的肺癌相关抗原的ｃＤＮＡ序列。这一序列的发现为阐明肺癌的发病机理。

Objective To clone the gene of human lung cancer associated antigen. Methods The human lung squamous carcinoma cell line L 78 cDNA expression library was constructed with λgt 11 Sfi Not directional cloning vector and screened with the anti human lung cancer McAb ALT 04. The cDNA clone named hlc 14 was sequenced and further studied. NIH3T3 cells were transfected with the recombinant pXJ41 hlc14 plasmid, and the expression of hlc 14 cDNA was detected and characterized immunohistochemically. Soft agar colony formation assay of the transfectants was performed. Results A human lung cancer λgt 11 cDNA library of 1.4×10 6 pfu/ml was constructed. Six cDNA clones were isolated following 4 rounds of immunoscreening. The largest cDNA clone hlc 14 contained 954bp with no homology to any known gene when screened through GenBank. In the NIH3T3 cells transfectant of hlc 14 cDNA, the expression of the lung cancer associated antigen was demonstrated. The ability of colony formation of the transfectant in the soft agar was enhanced. Conclusion The hlc 14 clone is a novel human lung cancer associated antigen cDNA. It provides clues for the study of carcinogenesis mechanism of lung cancer and a novel target gene for the gene therapy of lung cancer.