CTS-Fe_3O_4介导丙型肝炎病毒多表位基因疫苗的研究

Investigation of HCV Multi-epitope Gene Vaccine Loaded by CTS-Fe_3O_4

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 In order to develop a promising HCV gene vaccine candidate to induce effective immune response and explore the application of magnetic nanoparticles as gene delivery system.Methods The DNA fragment containing multi-epitope antigen gene of HCV with five conserved mimotopes was synthesized and cloned into plasmid pcDNA3.1（＋）.The Fe3O4 modified with chitoson was prepared and the cytotoxicity of the magnetic material was detected in vitro.Analysis of recombinant plasmid in vitro expression,and its immunogenicity loaded by CTS-Fe3O4 in mice were evaluated in detail.Results The HCV multi-epitope gene vaccine pcDNA3.1（＋）-MA was successfully constructed and recognized by 81% HCV positive sera.There was no cytotoxicity of CTS-Fe3O4 when its concentration was equal or less than 1 mmol/L.Both the antibody production and T-cell activity were induced.Conclusion It was believed that DNA encoding MA was an attractive approach for the therapeutic and prophylactic vaccines against HCV and the Fe3O4 modified with chitoson showed excellent target,safety and adjuvant effect as gene carrier.