CAJ | 학술논문

目的构建人源噬菌体抗体库,从中筛选出抗Peroxiredoxin Ⅰ(Prx Ⅰ)肺腺癌人源单链抗体.方法提取肺癌患者癌旁淋巴结组织,通过RT-PCR扩增出重链可变区基因(V_H)和轻链可变区基因(V_L),再经剪切-重叠-延伸PCR(SOE-PeR)将V_H和V_L连接得剑单链抗体(ScFv);将双酶切后的ScFv基因片段克隆入噬菌体表达载体pCANTAB5E,得到初级噬菌体抗体库.PCR检测TG1中ScFv基因插入率;1%琼脂糖凝胶电泳鉴定SfiⅠ和NotⅠ双酶切质粒的结果;以肺腺癌细胞株D549及在肺癌中高表达的抗氧化蛋白PrxⅠ为靶抗原对抗体库进行筛选富集.将阳性克隆用IPTG诱导表达.用SDS-PAGE及Western blotting鉴定该抗体;用ELISA法、免疫细胞化学法鉴定该抗体与人肺腺癌细胞的结合特异性.结果成功构建噬菌体单链抗体库.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.随机选取10个克隆,通过ELISA法检测剑其中6个与A549细胞呈阳性反应,阳性率为60%.SDS-PAGE及Western blotting证实抗体得到可溶表达.ELISA及免疫细胞化学检测表明抗体能相对特异地与肺腺癌细胞结合.结论成功构建肺腺癌人源噬菌体抗体库,并从中筛选得到能相对特异地与高表达Prx Ⅰ的肺腺癌细胞D549结合的单链抗体.
목적 구건인원서균체항체고,종중사선출항Peroxiredoxin Ⅰ(Prx Ⅰ)폐선암인원단련항체.방법 제취폐암환자암방림파결조직,통과RT-PCR확증출중련가변구기인(V_H)화경련가변구기인(V_L),재경전절-중첩-연신PCR(SOE-PeR)장V_H화V_L련접득검단련항체(ScFv);장쌍매절후적ScFv기인편단극륭입서균체표체재체pCANTAB5E,득도초급서균체항체고.PCR검측TG1중ScFv기인삽입솔;1%경지당응효전영감정SfiⅠ화NotⅠ쌍매절질립적결과;이폐선암세포주D549급재폐암중고표체적항양화단백PrxⅠ위파항원대항체고진행사선부집.장양성극륭용IPTG유도표체.용SDS-PAGE급Western blotting감정해항체;용ELISA법、면역세포화학법감정해항체여인폐선암세포적결합특이성.결과 성공구건서균체단련항체고.ScFv기인삽입솔위77%,쌍매절감정검측도목적조대.재친화사선과정중,폐암단련항체득도부집.수획솔축륜제고,제6륜위제1륜적180배.수궤선취10개극륭,통과ELISA법검측검기중6개여A549세포정양성반응,양성솔위60%.SDS-PAGE급Western blotting증실항체득도가용표체.ELISA급면역세포화학검측표명항체능상대특이지여폐선암세포결합.결론 성공구건폐선암인원서균체항체고,병종중사선득도능상대특이지여고표체Prx Ⅰ적폐선암세포D549결합적단련항체.
Objective To construct a human phage antibody library and screen the single chain variable fragment (ScFv) antibudies to peroxiredoxin Ⅰ (Prx Ⅰ) of lung adenocarcinoma. Methods The total RNA was isolated from the lymph nodes of lung cancer patients to amplify V_H and V_L genes by RT-PCR. V_H and V_L were linked with a DNA linker by SOE-PCR to construct the single chain variable fragment gene. The ScFvs were eoloned into the phage vector pCANTAB5E. The insert ratio of the ScFv antibody library was identified by PCR, and the products were digested by SfiⅠ/NotⅠ and analyzed with 1% agarose gel electrophoresis. Three rounds of panning against lung adenoearcinorna cell line A549 and Prx Ⅰ were performed, and the positive clones were identified for soluble expression. The soluble antibodies were identified by SDS-PAGE and Western blotting, and ELISA and immunocytochemistry were used to characterize the activity of the antibodies. Results A recombination phage antibody library was constructed. The insert ratio of ScFv gene was 77% (23/30), and enzyme digestion identified the target product. The sixth phage harvest resulted in a yield 180 folds of that of the first one. Positive reactions to A549 cells were detected in 6 of 10 random clones, with a positivity rate of 60%. The soluble human ScFvs against Prx Ⅰ of lung adenocarcinoma were expressed in E. coli HB2151 and confirmed by SDS-PAGE and Western blotting. ELISA and immunocytochemistry demonstrated a relative specific affinity of the soluble antibodies to A549 cells. Conclusion ScFv antibodies against lung adenocarcinoma have been acquired by phage display antibody library technique, and the soluble antibodies have a relative avidity specific to human lung adenocarcinoma A549 cells overexpressing PrxⅠ.