人源噬菌体抗体对Peroxiredoxin Ⅰ高表达肺腺癌细胞增殖的抑制作用  

Inhibitory effect of novel internalized fully human phage antibody fragments on proliferation of lung adenocarcinoma cell line overexpressing peroxiredoxin Ⅰ

作　　者：罗弋[1] 庞华[2] 李淑杰[1] 曹辉[1] 李少林[1] 樊春波[1] 

机构地区：[1]重庆医科大学放射医学教研室,重庆400016 [2]重庆医科大学附属第一医院核医学科,重庆400016

出　　处：《癌症》2009年第10期1061-1066,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金项目(No.30370422)~~

摘　　要：背景与目的:研究表明过氧化物酶Peroxiredoxin Ⅰ(Prx Ⅰ)与癌症的发展有密切关系。我们已通过噬菌体展示技术构建了肺腺癌相关的人源单链抗体库。本研究对该库进行筛选,得到抗Prx Ⅰ肺腺癌单链抗体,并检测其对肺腺癌细胞A549增殖的抑制作用。方法:PCR法检测TG1中scFv基因插入率,1%琼脂糖凝胶电泳鉴定Sfi Ⅰ和Not Ⅰ双酶切质粒的结果,以A549细胞及在肺癌中高表达的抗氧化蛋白Prx Ⅰ为靶抗原分别对抗体库进行3轮筛选富集。将阳性克隆用IPTG诱导表达并进行检测。放射性核素计数法测定细胞单链抗体内摄水平,MTT法及流式细胞术检测单链抗体对A549细胞的增殖抑制和凋亡情况,免疫印迹法检测抗体作用A549细胞后Prx Ⅰ的表达水平。结果:scFv基因插入率为77%,双酶切鉴定检测到目的条带。在亲和筛选过程中,肺腺癌单链抗体得到富集,收获率逐轮提高,第6轮为第1轮的180倍。ELISA法检测到在随机选取的10个克隆中,有6个与A549细胞呈阳性反应,阳性率60%。SDS-PAGE及ELISA检测证实得到人源抗PrxI肺腺癌单链抗体。被A549细胞内摄的单链抗体介导了细胞的凋亡以及细胞内Prx Ⅰ蛋白表达水平的下降。结论:从噬菌体抗体库中筛选获得具有较高特异性的抗Prx Ⅰ肺腺癌单链抗体。单链抗体与肺腺癌细胞有特异性亲和力,并能有效抑制其增殖。Background and Objective： Previous researches have implicated the close relationship between peroxiredoxin I （Prx I） and cancer progression. A lung adenocarcinoma-related human phage antibody library has been constructed by using phage display techniques. This study was to screen out the single chain variable fragment （scFv） antibodies from the library against a lung adenocarcinoma cell line overexpressing Prx I and to analyze their antiproliferation ability. Methods： The insertion ratio of scFv gene was identified by polymerase chain reaction （PCR）. The products were digested by Sfi I and Not I, and analyzed on 1% agarose gel. Three rounds of panning against lung adenocarcinoma cell line A549 and Prx I were performed separately, and the positive clones were chosen for soluble expression. The internalization of radiolabeled scFv fragments was then quantified. The proliferation and apoptosis of A549 cells were detected by MTT assay and flow cytometry （FCM）. The protein expression of Prx I in A549 cells was analyzed by Western blot. Results： The insertion ratio of scFv gene was 77% （23/30） and enzyme digestion showed the target products. The sixth phage harvest yielded 180 times as much as that of the first one, Positive reactions with A549 cells were detected in six （60%） of ten random clones. The human scFv fragments against Prx I of lung adenocarcinoma were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and enzyme-linked immunosorbent assay （ELISA）. The internalized scFvs mediated cell apoptosis and Prx I expression down-regulation. Conclusions. The scFv fragments against Prx I of lung adenocarcinoma are acquired by screening the phage antibody library. The soluble antibodies have specific avidity and inhibitory effect on proliferation of human lung adenocarcinoma cells.

关 键 词：肺腺癌 噬菌体抗体库 单链抗体 PEROXIREDOXIN I 增殖抑制 

分 类 号：R392[医药卫生—免疫学]