中文摘要：

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（DE3）;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-XhoI和NcoI-EcoRI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。

英文摘要：

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-SA3 with 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-SA3 could 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 HepG2 cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.