山羊痘DNA疫苗pVAX1-P32的构建及其安全性评估  

Construction and safety evaluation of the DNA vaccine pVAX1-P32 against goat pox

作　　者：殷俊磊[1] 朱时杰[1] 周碧君[1,2] 文明[1,2] 杨颖[1] 李霞云[1] 樊翠华[1] 

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省动物疫病研究室,贵州贵阳550025

出　　处：《中国兽医科学》2010年第10期1052-1056,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30940054);贵州省优秀科技教育人才省长专项基金项目(黔省专合字[2006]12号)

摘　　要：为了构建山羊痘病毒(GPV)P32基因真核表达质粒pVAXl-P32,探讨pVAXl-P32重组表达质粒作为DNA疫苗在小鼠体内的组织分布和生物安全性。以pMD18-T-P32/LD为模板,通过PCR扩增GPVP32基因片段,并定向插入真核表达载体pVAX1中,构建重组表达质粒pVAX1-P32,将其转入大肠杆菌DH5α中,筛选阳性克隆进行双酶切与PCR鉴定。大量提取纯化重组质粒pVAX1-P32,经肌肉注射免疫小鼠,于免疫后不同时间剖杀小鼠,分别采集心肌、肝、脾、肺、肾、脑、十二指肠、腿肌、血液等组织样品,抽提总DNA,利用PCR分析pVAX1-P32在小鼠组织内的动态分布及其与宿主细胞染色体的整合情况;同时,以PCR技术检测免疫小鼠粪便,分析pVAX1-P32重组表达质粒在外界环境中的释放情况。结果表明,真核表达重组质粒pVAX1-P32的双酶切鉴定及PCR扩增结果与预期结果相符;肌肉注射小鼠后,pVAX1-P32迅速分布到小鼠其他组织器官中,同时,未发现pVAX1-P32与宿主细胞染色体有整合现象;粪便中也均未检测到目的基因。表明,成功构建了真核表达重组质粒pVAX1-P32,且其作为疫苗对动物和环境是安全的。To construct the recombinant expression plasmid pVAX1-P32 encoding the P32 protein of goatpox virus and evaluate the biosafety and tissue distribution in mice as a DNA vaccine,the P32gene was amplified from the plasmid pMD18-T-P32/LD by PCR and inserted into the vector pVAX1.This recombinant plasmid pVAX1-P32 was identified by endonuclease digestion and PCR.It was extracted and purified from DH5α in large scale for DNA vaccine and was intramuscularly injected on the legs of mice.The mice were killed at the different time points after immunization and the DNA was extracted from cardiac muscle, liver,spleen,lung,kidney,brain,duodenum,leg muscle and blood.The circumstance of integrate into the chromosome,the distribution and the residual time of recombinant plasmid pVAX1-P32 in the tissue of mice were analyzed by PCR.At the same time,the feces of mice were tested by PCR to investigate the release of pVAX1-P32 in the external environment.The results showed that the recombinant plasmid pVAX1-P32 was constructed successfully and the P32 gene was detected by PCR from all tissues,no pVAX1-P32 was integrated into the chromosome of mice,and the P32 gene was not detected in the feces.It was safe to mice and surrounding environment as a vaccine.

关 键 词：山羊痘病毒 P32基因 DNA疫苗 真核表达载体 安全性 

分 类 号：S852.659.1[农业科学—基础兽医学]