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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。
Objective: To investigate the fusion expression, purification, renaturation and in vivo targeting of human anti-hepatoma single chain antibody SA3 and enhanced green fluorescent protein (EGFP-SA3). METHODS: The SA3 and EGFP genes were inserted into pET-25b (+) to construct EGFP-SA3/pET-25b (+) prokaryotic expression vector. After sequencing, the recombinant plasmid was transformed and transformed into E. coli BL21 (DE3); the fusion protein EGFP-SA3 was induced by IPTG. After being expressed, renatured and purified, they were identified by SDS-PAGE. After in vitro incubation of EGFP-SA3 and HepG2 cells, the binding of single-chain antibody SA3 to hepatoma cells was observed under a fluorescence microscope; then injected into the hepatoma bearing nude mice through the tail vein. In vivo targeting of EGFP-SA3 was observed in vivo. RESULTS: After SA3 and EGFP genes were inserted into the vector pET-25b (+) respectively, the recombinant expression vector EGFP-SA3/pET-25b (+) was double digested with NcoI-XhoI and NcoI-EcoRI, respectively, and the results were found to occur around 750 bp. One band, which is identical to the EGFP gene size, shows a band around 1.5 kb, which is consistent with the total size of the EGFP and SA3 gene fragments. DNA sequencing confirmed the successful construction of the fusion expression vector EGFP-SA3/pET-25b(+). The recombinant expression vector EGFP-SA3/pET-25b(+) was induced by IPTG. SDS-PAGE showed that the fusion protein EGFP-SA3 had a molecular weight of 58 kD, mainly in the form of inclusion bodies; after purification, renaturation, immunity. The results of fluorescence detection showed that the combination of SA3 and HepG2 cells could specifically target the binding; the tumor site of the hepatoma-injected mice injected with EGFP-SA3 showed green fluorescence, showing that EGFP-SA3 has good tumor specificity. CONCLUSION: The fusion protein EGFP-SA3 has strong binding ability to HepG2 cells, and the single-chain antibody SA3 is expected to be used for molecular diagnosis and targeted therapy of liver cancer.