摘要
为评价重组腺病毒融合表达猪瘟病毒(CSFV)E0和E2蛋白的免疫保护效果,本研究以CSFV基因组为模板,应用RT-PCR扩增E0和E2蛋白的编码基因,通过pET-32a载体将E0和E2基因串联,形成pET-E0-E2重组质粒。用KpnⅠ和NotⅠ双酶切pET-E0-E2得到E0-E2融合基因,定向亚克隆于穿梭载体pAdTrack-CMV中,采用"两步转化法"在细菌内同源重组,构建携带E0-E2基因的重组腺病毒转移载体质粒pAdEasy-E0-E2,经PacⅠ酶切线性化后转染人胚胎肾细胞(HEK293),成功包装出重组腺病毒(rAd-E0-E2),PCR和western blot检测表明,E0-E2基因已重组于腺病毒基因组中并获得表达。将rAd-E0-E2接种于小鼠和猪,并通过ELISA进行抗体检测。另外,将rAd-E0-E2经肌肉2次(间隔7d)免疫接种6周龄～7周龄猪,3周后用103TCID50CSFV石门株攻毒。结果表明,rAd-E0-E2免疫组6/7头存活,各组织器官带毒时间不超过10d,而非重组腺病毒rAd-CMV免疫组和空白对照组全部死亡,各组织器官均能分离到CSFV。结果提示,rAd-E0-E2能使免疫猪抵抗CSFV强毒攻击,为进一步研制猪瘟基因工程疫苗提供了实验依据。
A recombinant adenovirus rAd-E0-E2 fusion-expressing the E0 and E2 gene of atypical classical swine fever virus （CSFV） was constructed by cloning CSFV E0 and E2 genes into the adenoviral shuttle plasmid pAdTrack-CMV and transfected into 293 cells. The integration of E0-E2 gene in the recombinant adenovirus genome was confirmed by PCR, and the expression of E0-E2 protein in 293 cells was detected by western blot. Pigs were administered with rAd-E0-E2 intramascularly two times at a 7 days interval followed by challenge with 10 3 TCID50 of CSFV Shimen strain 3 weeks later. The results showed that 6/7 of the pigs vaccinated with rAd-E0-E2 were protected, and the persistence of CSFV in organs was less than 10 days. None of the pigs vaccinated with the human adenovirus （rAd-CMV） and DMEM survived. These results demonstrated that the recombinant adenovirus rAd-E0-E2 constructed in this study could induce effective protection in pigs against CSFV challenge, which provided a reference for the development of CSF genetic engineering vaccine.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2010年第6期419-423,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(30960285)
云南省教育厅(08C0072)
云南省科技厅(K2100390)
关键词
重组腺病毒
猪瘟病毒
E0-E2基因
保护效果
recombinant adenovirus
classical swine fever virus
E0-E2 gene
protection effect
作者简介
作者简介：杨玉艾（1978-）．女，陕西咸阳人，博士，讲师，主要从事临床兽医学研究．
通信作者：E-mail：sunyongke@126．com.
