重组腺病毒表达轮状病毒SA11毒株VP4蛋白及其糖基化  

Expression and Glycosylation of Rotavirus Strain SA11 VP4 Protein in a Recombinant Adenovirus

作　　者：孙茂盛[1] 昝云红[1] 马雁冰[1] 张光明[1] 杜秋江[1] 戴长柏[1] 

机构地区：[1]中国医学科学院

出　　处：《中国医学科学院学报》2000年第1期52-56,共5页Acta Academiae Medicinae Sinicae

摘　　要：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 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.

关 键 词：轮状病毒 重组腺病毒 糖基化作用 SA11毒株 

分 类 号：R373.9[医药卫生—病原生物学]