抗B7-H4-scFv/PE38KDEL重组免疫毒素的表达和功能性检测

Expression and functional detection of recombinant immunotoxins anti-B7-H4-scFv/PE38KDEL

目的:构建基于抗B7-H4单链抗体(sc Fv)的重组毒素anti-B7-H4-scFv-PE38KDEL,检测毒素蛋白的抗肿瘤作用。方法:通过重叠延伸PCR(SOE-PCR)技术将anti-B7-H4-scFv基因和毒素PE38KDEL基因进行连接,重组基因克隆到原核表达载体pET28a(+)中表达,蛋白经变复性和镍柱亲和层析(Ni-NTA)纯化后进行Western blot鉴定;间接ELISA和流式分析技术进行特异性鉴定。利用MTT法和皮下移植瘤模型实验,检测毒素对体外、内肿瘤细胞的抑制作用,并对肿瘤组织进行HE染色和免疫组化分析。结果:酶联后得到重组表达载体pET28a-anti-B7-H4-scFv-PE38KDEL,纯化后的毒素蛋白对肿瘤细胞具有一定杀伤力,并且在肿瘤模型实验中能抑制体内肿瘤的生长。结论:成功构建了基于抗B7-H4单链抗体的重组毒素表达体系,经鉴定重组毒素蛋白有良好的生物学功能活性和抗肿瘤活性。

Objective:To construct anti-B7-H4-scFv-PE38KDEL,a recombinant toxin based on anti-B7-H4 single chain antibody(scFv),to detect anti-tumor effect of toxin protein.Methods:The anti-B7-H4-scFv gene was ligated with the toxin PE38KDEL gene by overlapping extension PCR(SOE-PCR).The recombinant gene was cloned into prokaryotic expression vector pET28a(+),and the protein was renatured and purified by chromatography(Ni-NTA),and was identified by Western blot.Indirect ELISA and flow analysis technology were used for specific identification.The inhibitory effects of toxins on tumor cells were detected by MTT assay and subcutaneous xenograft model in vitro and in vivo.HE staining and immunohistochemical analysis were performed on tumor tissues.Results:The recombinant expression vector pET28a-anti-B7-H4-scFv-PE38KDEL was obtained by restriction endonuclease digestion.The purified toxin protein was inoculated on the tumor cells.The tumor growth was inhibited in the tumor model.Conclusion:The recombinant toxin expression system based on anti-B7-H4 single chain antibody was successfully constructed.The recombinant toxin protein had good biological activity and anti-tumor activity.