摘要
采用PCR方法从重组质粒pMD18_T/PP中扩增出FMDV的聚蛋白(PP)编码基因,再亚克隆至腺病毒穿梭质粒中,形成重组穿梭质粒rpAd_CMV/PP;将获得的重组穿梭质粒与腺病毒骨架载体通过在大肠杆菌内质粒间同源重组获得重组腺病毒质粒rpAd/PP。将腺病毒载体线性化后用脂质体介导转染293细胞从而获得含有口蹄疫病毒PP编码基因的重组腺病毒。通过倒置显微镜观测,可见明显的细胞病变,利用荧光显微镜可观测到报告基因绿色荧光蛋白的表达,并在电镜下观察到FMDV的空衣壳。结果证明已成功获得了含有口蹄疫病毒PP编码基因的重组腺病毒rAd/PP,并成功表达组装FMDV空衣壳,为FMDV腺病毒活载体疫苗的研究奠定了基础。
The gene coding for the polyprotein （PP） of foot-and-mouth disease virus （FMDV）was obtained by PCR from recombinant plasmid rpMDIS-T/PP. The PCR product was digested with Xba [ and Not ] and inserted into the cloning site of the adenovirus shuttle vector pAdTrack-CMV, previously digested with the same enzymes. This recombinant shuttle plasmid was designated rpAd- CMV/PP. The recombinant adenovirus vector rpAd/PP was obtained by homologous recombination of plasmid rpAd-CMV/PP and adenovirus skeletal vector pAdeasy-1 in E. coli. Plasmid rpAd/PP was linearized by Pme I and transformed into 293 competent cells to pack the adenovirus using liposome mediated gene transfer method and, as a result, the recombinant adenovirus rAd/PP that contained the polyprotein coding gene was obtained. Obvious CPE could be observed under an inverted microscope, the green fluorescence protein expression can be detected under fluorescence microscope and the empty capsid of FMDV was observed under electron microscope. These results indicated that the recombinant adenovirus rAd/PP expressed the PP protein and that this protein could be assembled into the empty capsid of FMDV. The recombinant adenovirus obtained in this study can be used for further research for making FMDV recombinant adenovirus vaccine.
出处
《微生物学报》
CAS
CSCD
北大核心
2006年第2期223-226,共4页
Acta Microbiologica Sinica
基金
国家"863计划"(2004AA213091)
国家"973"前期研究专项(2004CCA00500)~~
关键词
FMDV
聚蛋白
编码基因
同源重组
重组腺病毒
空衣壳
Foot-and-mouth disease
Polyprotein（PP）
Homologous recombination
Recombinant adenovirus
Empty capsid
作者简介
张兴旺（1973-），男，河北东光人，硕士，主要从事分子病毒学研究。Tel：86-931-8281605；E-mail：ZXW2002@xinhuanet．com
通讯作者。Tel：86-931-8342682；E-mail：Liujixing@hotmail，com
