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目的 用重组人腺病毒表达经修饰的轮状病毒 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肽的 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 2 93细胞 ,点杂交 ,酶谱分析筛选重组病毒。结果 VP4全基因 2 36 2 bp未发现突变。间接免疫荧光试验表明 ,重组腺病毒所表达的外源蛋白具有特异性和较强抗原性 ,蛋白印迹及免疫沉淀试验证明 ,表达的蛋白相对分子质量大于野生型 VP4蛋白 ,符合糖化后应有的相对分子质量 ,且这种糖基化蛋白可被特异性酶所消化。结论 提高重组蛋白的稳定性、抗原性 ,进行目的基因改造也许是一条有效的途径。
Objective To express the modified rotavirus VP4 gene with recombinant human adenovirus. Methods The full-length VP4 gene was amplified by RT-PCR. The signal sequence was introduced into the 5 ’end of the gene. The VP4 gene with signal peptide sequence was inserted into the plasmid containing human cytomegalovirus promoter. Then, The VP4 gene of the signal peptide is transferred to a human adenovirus type 5 transfer vector. A total of 293 cells were co-transfected by homologous recombination method, and the recombinant viruses were screened by enzyme-linked immunosorbent assay. Results No mutation was found in 2 36 2 bp of VP4 gene. Indirect immunofluorescence assay showed that the foreign protein expressed by the recombinant adenovirus was specific and strong antigenicity. Western blot and immunoprecipitation showed that the relative molecular mass of the expressed protein was larger than that of the wild-type VP4 protein, Relative molecular mass, and this glycosylated protein can be digested by specific enzymes. Conclusion To improve the stability and antigenicity of the recombinant protein, it may be an effective way to transform the target gene.