南方医科大学学报
南方醫科大學學報
남방의과대학학보
JOURNAL OF SOUTHERN MEDICAL UNIVERSITY
2010年
1期
30-34
,共5页
罗弋%庞华%李淑杰%曹辉%李少林%樊春波%王洁
囉弋%龐華%李淑傑%曹輝%李少林%樊春波%王潔
라익%방화%리숙걸%조휘%리소림%번춘파%왕길
噬菌体抗体库%单链抗体%Peroxiredoxin Ⅰ%肺腺癌
噬菌體抗體庫%單鏈抗體%Peroxiredoxin Ⅰ%肺腺癌
서균체항체고%단련항체%Peroxiredoxin Ⅰ%폐선암
phage antibody library%single chain variable fragment%peroxiredoxin Ⅰ%lung adenocarcinoma
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.
目的 構建人源噬菌體抗體庫,從中篩選齣抗Peroxiredoxin Ⅰ(Prx Ⅰ)肺腺癌人源單鏈抗體.方法 提取肺癌患者癌徬淋巴結組織,通過RT-PCR擴增齣重鏈可變區基因(V_H)和輕鏈可變區基因(V_L),再經剪切-重疊-延伸PCR(SOE-PeR)將V_H和V_L連接得劍單鏈抗體(ScFv);將雙酶切後的ScFv基因片段剋隆入噬菌體錶達載體pCANTAB5E,得到初級噬菌體抗體庫.PCR檢測TG1中ScFv基因插入率;1%瓊脂糖凝膠電泳鑒定SfiⅠ和NotⅠ雙酶切質粒的結果;以肺腺癌細胞株D549及在肺癌中高錶達的抗氧化蛋白PrxⅠ為靶抗原對抗體庫進行篩選富集.將暘性剋隆用IPTG誘導錶達.用SDS-PAGE及Western blotting鑒定該抗體;用ELISA法、免疫細胞化學法鑒定該抗體與人肺腺癌細胞的結閤特異性.結果 成功構建噬菌體單鏈抗體庫.ScFv基因插入率為77%,雙酶切鑒定檢測到目的條帶.在親和篩選過程中,肺癌單鏈抗體得到富集.收穫率逐輪提高,第6輪為第1輪的180倍.隨機選取10箇剋隆,通過ELISA法檢測劍其中6箇與A549細胞呈暘性反應,暘性率為60%.SDS-PAGE及Western blotting證實抗體得到可溶錶達.ELISA及免疫細胞化學檢測錶明抗體能相對特異地與肺腺癌細胞結閤.結論 成功構建肺腺癌人源噬菌體抗體庫,併從中篩選得到能相對特異地與高錶達Prx Ⅰ的肺腺癌細胞D549結閤的單鏈抗體.
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.