联合应用噬菌体展示技术与重组cDNA表达文库血清学分析技术筛选肺癌早期诊断相关抗原  

Combination of phage display and SEREX for screening early lung cancer associated antigens

作　　者：袁娜[1] 辛国宏[2] 左笑笑[1] 黄尚科 王莹[1] 侯磊[1] 秦天洁[1] 赵新汉[1] 

机构地区：[1]西安交通大学医学院第一附属医院肿瘤内科,陕西西安710061 [2]河北省人民医院肿瘤三科,河北石家庄050051

出　　处：《浙江大学学报（医学版）》2014年第4期388-396,共9页Journal of Zhejiang University(Medical Sciences)

基　　金：陕西省“13115”科技创新工程重大科技专项项目(2009ZDKG73);教育部长江学者和创新团队发展计划(PCSIRT:1171)

摘　　要：目的：筛选有效的人肺癌早期诊断相关抗原，以提高肺癌的早期诊断水平。方法：构建人早期肺癌cDNA T7噬菌体展示文库；筛选出差异性噬菌体克隆，测序后进行生物信息学分析；最后利用重组cDNA表达文库血清学分析（SEREX）技术对8个差异性噬菌体克隆构建肺癌相关抗原微阵列，分别与肺癌患者和正常人的血清反应，评价各个抗原单独应用与联合应用诊断肺癌的价值。结果：构建的肺癌T7噬菌体展示文库的滴度为3．71×10^6pfu／ml，噬菌体数为1．11×10^6pfu。cDNA文库的重组率超过90％。筛选出9个差异性噬菌体克隆，测序后发现A42与A83抗原的基因序列相同。生物信息学分析发现8个抗原的基因均为已知基因，除了A54以外，其余与肿瘤均有明确的关系。8个抗原与肺癌患者血清的阳性反应率均高于与正常人血清的阳性反应率，差异均具有统计学意义（均P〈0．05）。在特异性不低于60％的情况下，各个抗原单独用于肺癌诊断时的灵敏性均低于70％。各个抗原的曲线下面积均低于0．8。而复合抗原诊断肺癌的灵敏性为90．8％，特异性为94．1％，曲线下面积高达0．969。结论：本研究成功构建了库容量大、重组率高、代表性好的人肺癌T7噬菌体展示文库，并筛选到8个肺癌相关抗原。由这8个抗原组成的复合抗原诊断肺癌的灵敏性与特异性均在90％以上，诊断价值较高。Objective： To screen out effective lung cancer associated antigens for early diagnosis in order to improve the level of early diagnosis. Methods： A T7 phage display cDNA library of human early lung cancer was developed. And then differential phage clones were picked out to be sequenced and bioinformatically analyzed. With the 8 screened differential phage clones a lung cancer associated antigen microarray was established to evaluate the single or combined roles of all the selected antigens in the diagnosis of lung cancer by the reaction of the antigens plus serum from normal subjects and patients with lung cancer, respectively. Results： The titer of the constructed cDNA library was 3. 71 × 10^6 pfu/ml and the number of phage was 1. 11 × 10^6 pfu, with a recombination rate of cDNA library over 90%. Nine differential phage clones were initially screened out, but the genes of two antigens （A42 and A83） were found the same. Bioinformatics analyses showed that the genes of the 8 antigens were known before and they were all proven to be related with tumor except A64. The positive reaction rates of the 8 antigens with serum from lung cancer patients were significantly higher than that with serum from normal subjects （Ps 〈 0. 05 ）. When keeping specificity no less than 60% , the sensitivity of each antigen in predicting lung cancer alone was under 70% and the areas under curve （AUC） of the antigens were all under 0. 8. However, when all the antigens were combined to detect lung cancer, the sensitivity and specificity was 90. 8% and 94. 1 % , respectively, and AUC reached up to 0.969. Conclusion： A T7 phage display cDNA library with a good quality of capacity, recombination rate and representativeness of human early lung cancer was successfully developed, and 8 lung cancer associated antigens were screened out. A combination of the 8 antigens can greatly improve their value to diagnose lung cancer with a higher sensitivity and specificity （both above 90% ）.

关 键 词：肺肿瘤/诊断 细菌噬菌体 DNA 互补/遗传学 抗原 肿瘤/分析 重 组 遗传 早期诊断 

分 类 号：R734.2[医药卫生—肿瘤]