表达狂犬病病毒糖蛋白膜外区重组犬2型腺病毒的构建及其免疫原性研究  

Construction and immunogenicity of recombinant Canine adenovirus type 2 with the external part of glycoprotein of Rabies virus

作　　者：王永志[1] 张守峰[1] 扈荣良[1] 王莘[2] 肖跃强[1] 

机构地区：[1]军事医学科学院军事兽医研究所,长春130062 [2]吉林农业大学生物技术学院,长春130118

出　　处：《微生物学报》2005年第2期213-217,共5页Acta Microbiologica Sinica

基　　金：国家自然科学基金 (3 0 170 70 4);国家"863计划"(2 0 0 4AA2 13 10 1)~~

摘　　要：为研制 1种以犬 2型腺病毒为载体的狂犬病疫苗 ,以狂犬病病毒SRV9株基因组为模板 ,通过RT_PCR技术扩增得到狂犬病病毒糖蛋白膜外区序列 ;以其为目的基因构建以CMV为启动子、SV4 0polyA为终止信号的表达盒。将犬 2型腺病毒的E3区克隆入中间质粒中 ,然后对其进行部分缺失 ,并将表达盒克隆入缺失处 ,再以此重组的E3区置换犬 2型腺病毒的E3区。以重组的犬 2型腺病毒基因组转染MDCK细胞 ,最终获得了表达盒分正向和反向插入E3区的重组犬 2型腺病毒。用两株重组腺病毒免疫幼犬 ,均在犬体内产生了针对狂犬病病毒的抗体。The cases of rabies increase greatly in recent years in China and rabies continues to be a serious problem in developing countries due to the reservoirs of Rabies virus in dogs and wildlife vectors. The control of rabies depends on the development of safe,effective,economical vaccines that may be used for preexposure vaccination in animals. For this purpose, the external part of glycoprotein gene of Rabies virus strain SRV 9 (RVG) was amplified by the RT-PCR and cloned into pEGFP-C1 with replacement of the GFP gene. The expression cassette pERVG 2 is composed of CMV promoter, external glycoprotein gene and SV40 early mRNA polyadenylation signal. The expression cassette was released by AseⅠ/MluⅠ double digestion and it was cloned rightwards and leftwards into Canine adenovirus type 2(CAV-2) E3 region plasmid pVAXE3 in which E3 region was deleted partly by SspⅠ/DraⅢ digestion. After the replacement of CAV-2 E3 region in plasmid pCAV-2 which contains CAV-2 genome with the recombinant E3 region, recombinant CAV-2 genome plasmid was obtained. Recombinant CAV-2 genome was released from plasmid by AscⅠ/ClaⅠ digestion,and then transfected into MDCK cells. Two replication-competent recombinant CAV2 expressing Rabies Virus external part of glycoprotein were produced. Vaccination experiment showed that the recombinant viruses can elicit an efficient antibody response in dogs.

关 键 词：犬2型腺病毒 狂犬病病毒 蛋白膜 表达 免疫原性 外区 RT-PCR技术 MDCK细胞 糖 狂犬病疫苗 病毒基因组 重组腺病毒 E3区 基因构建 SV40 启动子 CMV 克隆 缺失 幼犬 抗体 

分 类 号：S852.65[农业科学—基础兽医学]