应用改建的人5型腺病毒载体表达成人腹泻轮状病毒第5基因编码的内壳蛋白  

Expression of gene 5 of adult diarrhea rotavirus with reconstructed adenovirus type 5 vector

作　　者：彭瑚[1] 洪涛[1] 王长安[1] 张炳华[1] 屈建国[1] 

机构地区：[1]中国预防医学科学院病毒学研究所,北京100052

出　　处：《中华实验和临床病毒学杂志》1996年第4期325-329,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家863高科技生物技术领域资助课题

摘　　要：应用前期工作克隆的腺病毒晚期表达盒对引进的5型腺病毒(Adenovirus type 5,Ad5)载体进行改建,首先将成人腹泻轮状病毒(Adult Diarrhea rotavirus,ADRV)第5基因克隆入表达盒中,而后将该表达盒插入到Ad5载体的E3区,最后重组质粒与Ad5 DNA EcoR I大片段共转染A549细胞,经打点杂交和Southern转印筛选出重组病毒空斑。酶联免疫吸附试验(ELISA)检测重组病毒感染细胞上清和沉淀的抗原滴度分别为1:8,1:64。Western转印显示在分子量44 000处有一条转印带。ADRV第5基因在改建的Ad5载体上的表达为口服腺病毒基因工程疫苗的研究积累了实验经验,为ADRV诊断试剂的研制提供了便利条件。An adenovirus type 5 (Ad5) vector pBRAd5 E3 has been reconstructed that contains an adenovirus late expression cassette. The gene 5 of Adult diarrhea rotavirus (ADRV), encoding group specific protein VP6, was cloned in the adenovirus late expression cassette. The cassette was then inserted in the E3 region of Ad5 vector and the recombinant plasmid was cotransfeeted with EcoRI digested AdS DNA fragment into A549 cells. The recombinant plaques were selected by dot blot and southern blot hybridization. The gene 5 of ADRV was expresed in adenovirus recombinants and the antigen titer of infected supernatant and cell lysate were 1:8 and 1:64 respectively by ELISA method. The expressed protein detected by Western blot showed a molecular weight of 44KD, being identical to those of ADRV VP6 derived from virions. Expression of gene 5 of ADRV not only indicates the reconstructed Ad5 vector working well, but also accumulates necessary experimen--tal experiences for adenovirus based oral vaccine and developing ADRV diagnostic assavs.

关 键 词：腺病毒 轮状病毒 病毒基因 病毒抗原 

分 类 号：R373[医药卫生—病原生物学]