论文部分内容阅读
目的探讨非复制型腺病毒介导抗-HBc单链抗体(ScFv)细胞内表达效率,并确定是否具有特异性抗原结合活性。方法将经细菌内同源重组并在293细胞中包装产生的携带抗HBc ScFv基因的非复制型重组腺病毒,按感染复数为10体外转染HepG2细胞,荧光显微镜下观察绿色荧光蛋白的表达；取培养上清液和细胞裂解上清液进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳和Western blot法检测HBc ScFv表达。结果重组腺病毒Ad-ScFv感染HepG2细胞后,荧光显微镜下观察到HepG2细胞中绿色荧光蛋白；十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示培养上清液和细胞裂解物中可见2.7×10~4左右的蛋白条带；Western blot显示有乙型肝炎核心抗原特异性结合活性阳性反应条带出现。结论携带抗-HBc ScFv基因的非复制型重组腺病毒能在真核细胞中有效表达抗-HBc ScFv,并具有特异性抗原结合活性。
Objective To investigate the efficiency of non-replicating adenovirus-mediated intracellular expression of anti-HBc single chain antibody (ScFv) and determine whether it has specific antigen-binding activity. Methods HepG2 cells were transfected into HEpG2 cells by homologous recombination in bacteria and non - replicating recombinant adenovirus carrying anti - HBc ScFv gene in 293 cells. The expression of GFP was observed by fluorescence microscopy. The culture supernatant and cell lysate supernatant were taken for sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot to detect the expression of HBc ScFv. Results The recombinant adenovirus Ad-ScFv was transfected into HepG2 cells and green fluorescent protein (HepG2) cells were observed under a fluorescence microscope. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the supernatant and cell lysate showed 2.7 × 10 ~ 4 protein bands; Western blot showed hepatitis B core antigen specific binding activity positive reaction bands appear. Conclusion The non-replicative recombinant adenovirus carrying anti-HBc ScFv gene can efficiently express anti-HBc ScFv in eukaryotic cells and has specific antigen-binding activity.