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目的研发能诱导有效免疫应答的丙型肝炎病毒(HCV)基因疫苗候选者及探索磁性纳米材料作为基因载体的应用前景。方法选择5个HCV保守性T/B细胞识别的抗原表位基因,克隆入pcDNA3.1(+),鉴定重组质粒,测定其细胞水平的表达以及在小鼠体内诱导的免疫反应。同时合成壳聚糖(CTS)修饰的Fe3O4磁性纳米微粒(CTS-Fe3O4),采用MTT法检测对人胚肾细胞株HEK-293和小鼠成纤维细胞株3T3的细胞毒性作用,研究在外加磁场作用下,磁性材料作为基因投递系统的应用。结果成功构建了HCV多表位基因疫苗pcDNA3.1(+)-MA,RT-PCR和间接免疫荧光实验证实了重组质粒在细胞内的表达,疫苗中的B表位能被81%HCV阳性血清样本所识别,磁性材料浓度在1mmol/L内无细胞毒性。小鼠体内免疫实验初步显示出pcDNA3.1(+-)MA能有效诱导体液和细胞免疫应答反应,并且磁性纳米材料介导的免疫实验组诱导的免疫应答反应更强(P<0.05)。结论本研究构建的HCV多表位基因疫苗可成为有前景的HCV候选疫苗之一。壳聚糖修饰的Fe3O4纳米材料可以作为一种优良的基因靶向投递载体,并有免疫佐剂的作用。
Objective To develop candidate hepatitis C virus (HCV) vaccine candidates that induce an effective immune response and to explore the potential applications of magnetic nanomaterials as gene vectors. Methods Five epitopes recognized by HCV conservative T / B cells were selected and cloned into pcDNA3.1 (+). The recombinant plasmids were identified and their cellular levels were measured. The immune responses induced in mice were also analyzed. At the same time, CTS-modified Fe3O4 magnetic nanoparticles (CTS-Fe3O4) were synthesized. The cytotoxicity of CTS-Fe3O4 on human embryonic kidney cell line HEK-293 and mouse fibroblast cell line 3T3 was tested by MTT assay. The role of magnetic materials as gene delivery systems. Results The HCV multi - epitope gene vaccine pcDNA3.1 (+) - MA was successfully constructed. The expression of the recombinant plasmid was confirmed by RT - PCR and indirect immunofluorescence. The B epitope in vaccine could be detected by 81% HCV positive sera Samples identified, magnetic material concentration within 1mmol / L without cytotoxicity. In vivo experiments in mice showed that pcDNA3.1 (+ -) MA can effectively induce humoral and cellular immune responses, and immune response induced by magnetic nanomaterials was stronger (P <0.05). Conclusion The HCV multi-epitope gene vaccine constructed in this study can be one of the promising HCV vaccine candidates. Chitosan-modified Fe3O4 nanomaterials can be used as an excellent gene-targeted delivery vector and have an immune adjuvant effect.