CAJ | 학술논문

目的构建人源噬菌体抗体库,并从中筛选出抗肺癌人源单链抗体.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-PCR)将V_H和V_L连接得到单链抗体(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.以肺腺癌细胞株A549为抗原对抗体库进行4轮筛选富集,鉴定抗体库性能.将得到的阳性克隆用IPTG诱导表达并进行检测.结果成功构建噬菌体单链抗体库.经筛选富集,噬菌体收获率得到增加,第4轮是第1轮的115倍.随机选取10个克隆,通过ELISA法检测到其中7个与A549细胞呈阳性反应,阳性率为70%.SDSPAGE及ELISA检测证实得到人源抗肺癌单链抗体.结论成功构建人源单链抗体噬菌体库,从中获得具有较高特异性的抗人肺癌单链抗体.
목적 구건인원서균체항체고,병종중사선출항폐암인원단련항체.방법 제취폐암환자암방림파결조직,통과RT-PCR확증출중련가변구기인(V_H)화경련가변구기인(V_L),재경전절-중첩-연신PCR(SOE-PCR)장V_H화V_L련접득도단련항체(ScFv).장쌍매절후적ScFv기인편단극륭입서균체표체재체pCANTAB5E,득도초급서균체항체고.이폐선암세포주A549위항원대항체고진행4륜사선부집,감정항체고성능.장득도적양성극륭용IPTG유도표체병진행검측.결과 성공구건서균체단련항체고.경사선부집,서균체수획솔득도증가,제4륜시제1륜적115배.수궤선취10개극륭,통과ELISA법검측도기중7개여A549세포정양성반응,양성솔위70%.SDSPAGE급ELISA검측증실득도인원항폐암단련항체.결론 성공구건인원단련항체서균체고,종중획득구유교고특이성적항인폐암단련항체.
Objective To construct a human phage single chain-antibody library, and to sieve out the antibody ScFv against lung cancer from the library. Methods Total RNA was abstracted from lymph node tissue of the lung cancer, and was used to amplify V_H and V_L gene by RT-PCR. V_H and V_L were joined by a DNA linker by SOE-PCR to form the single chain variable fragment ( ScFv) gene. ScFv gene was coloned into the phage vector pCANT-AB5E. Panning against lung cancer cell line A549 was performed and positive clones were chosen for soluble expression. Results A recombination phage single chain-antibody library was constructed. After 4 rounds panning, the number of eluted phages increased by 115 times. Positive reactions to A549 were detected in 7 of 10 random clones. The human ScFvs against lung cancer were produced and confirmed by SDS-PAGE and ELISA analysis. Conclusion ScFvs against lung cancer were acquired by the construction of phage single chain-antibody library. The soluble ScFvs has specificall avidity to human lung cancer cells.