CAJ | 학술논문

目的:探讨人源抗肝癌单链抗体SA3与增强型绿色荧光蛋白(EGFP-SA3)的融合表达、纯化、复性及其体内靶向性.方法:将SA3,EGFP基因,插入pET-25b(+),构建EGFP-SA3/pET-25b(+)原核表达载体,测序鉴定后转化大肠杆菌BL21 (DF3)；IPTG诱导融合蛋白EGFP-SA3的表达,复性、纯化后经SDS-PAGE鉴定；EGFP-SA3与HepG2细胞经体外孵育后在荧光显微镜下观察单链抗体SA3与肝癌细胞的结合作用；然后通过尾静脉将其注射入荷肝癌裸鼠体内观察EGFP-SA3的体内靶向作用.结果:SA3,EGFP基因分别插入载体pET-25b(+)后,重组表达载体EGFP-SA3/pET-25b(+)分别行NcoI-Xho I和Nco I-Eco RI双酶切,结果发现在750 bp左右出现一条带,与EGFP基因大小一致,在1.5 kb左右处出现一条带,与EGFP和SA3两个基因片段的总大小一致,DNA测序结果证实融合表达载体EGFP-SA3/pET-25b(+)构建成功；重组表达载体EGFP-SA3/pET-25b(+)经IPTG诱导表达后,SDS-PAGE显示融合蛋白EGFP-SA3分子质量为58 kD,主要以包涵体的形式存在；纯化、复性后,免疫荧光检测结果显示SA3与HepG2细胞能特异性地靶向结合；注射了EGFP-SA3的荷肝癌小鼠的肿瘤部位有绿色荧光发出,显示EGFP-SA3具有良好的肿瘤特异性.结论:融合蛋白EGFP-SA3与HepG2细胞有较强的结合能力,单链抗体SA3有望用于肝癌的分子诊断和靶向治疗.
목적:탐토인원항간암단련항체SA3여증강형록색형광단백(EGFP-SA3)적융합표체、순화、복성급기체내파향성.방법:장SA3,EGFP기인,삽입pET-25b(+),구건EGFP-SA3/pET-25b(+)원핵표체재체,측서감정후전화대장간균BL21 (DF3)；IPTG유도융합단백EGFP-SA3적표체,복성、순화후경SDS-PAGE감정；EGFP-SA3여HepG2세포경체외부육후재형광현미경하관찰단련항체SA3여간암세포적결합작용；연후통과미정맥장기주사입하간암라서체내관찰EGFP-SA3적체내파향작용.결과:SA3,EGFP기인분별삽입재체pET-25b(+)후,중조표체재체EGFP-SA3/pET-25b(+)분별행NcoI-Xho I화Nco I-Eco RI쌍매절,결과발현재750 bp좌우출현일조대,여EGFP기인대소일치,재1.5 kb좌우처출현일조대,여EGFP화SA3량개기인편단적총대소일치,DNA측서결과증실융합표체재체EGFP-SA3/pET-25b(+)구건성공；중조표체재체EGFP-SA3/pET-25b(+)경IPTG유도표체후,SDS-PAGE현시융합단백EGFP-SA3분자질량위58 kD,주요이포함체적형식존재；순화、복성후,면역형광검측결과현시SA3여HepG2세포능특이성지파향결합；주사료EGFP-SA3적하간암소서적종류부위유록색형광발출,현시EGFP-SA3구유량호적종류특이성.결론:융합단백EGFP-SA3여HepG2세포유교강적결합능력,단련항체SA3유망용우간암적분자진단화파향치료.
Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.