重组腺病毒表达轮状病毒SA11毒株VP4蛋白及其糖基化

用重组人腺病毒表达经修饰的轮状态病毒ＶＰ４基因。方法ＲＴ－ＰＣＲ扩增全长ＶＰ４基因,在基因５’末端引入信号肽序列,将带信号肽顺序的ＶＰ４基因插入到含人巨细胞病毒启动子质粒中,然后克隆带巨细胞病毒启动子和信号ＶＰ４基因到人腺病毒５型转移载体上。用同源重组方法共转染２９３细胞,点杂交,酶谱分析筛选重组病毒。

Expression and glycosylation of rotavirus strain SA11 VP4 protein in a recombinant adenovirus

Objective A modified VP4 gene of rotavirus SA11 strain was expressed by a recombinant human adenovirus. Methods A whole VP4 gene was obtained with PCR and induced the signal peptide at the gene N terminal.The chimera gene was cloned into pCMV plasmid that consist of human cytomagolovirus promoter and then cloned the gene to transfer human adenovirus type 5 vector.Homologues recombinant was performed by co transfection to 293 cell line with recombinant plasmid and viral genome using CaPO 4 precipitation. Results VP4 gene is 2 362 base pair in length mutation was not found in whole VP4 gene sequence.Expressed product in recombinant adnovirus was confirmed to be specific and more antigenicity by indirect immunofluorescence assay.Both Western blot and immunoprecipitation assays showed that expressed protein molecular weight was higher than wild type VP4 protein and that modified product was corresponding to a glycosylation of VP4 protein. Conclusions It may be a effective method to modify interested gene for enhancing stability,antigenicity and immunogenicity of expressive product.