犬细小病毒VP2蛋白重组人5型腺病毒的构建  

Construction of recombinant human adenovirus 5 with VP2 protein of canine parvovirus

作　　者：杨洋[1] 宫婷[2] 米立娟[2] 赵敬慧[2] 陈奇[2] 钱方[2] 刘晔[2] 张守峰[2] 扈荣良[2] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2013年第5期599-602,共4页Chinese Journal of Biologicals

基　　金：国家863项目(2011AA10A212)资助

摘　　要：目的构建犬细小病毒(Canine parvovirus,CPV)VP2蛋白重组人5型腺病毒。方法将从CPV中PCR扩增的VP2基因克隆至穿梭质粒pacAd5 CMVK-NpA中,经酶切及测序鉴定,将构建正确的重组穿梭质粒pacAd5-cVP2与骨架质粒pacAd5 9.2-100经PacⅠ酶切线性化,共转染293AD细胞,进行同源重组,包装出重组腺病毒rAd5v-cVP2,按Karber法计算重组腺病毒的病毒滴度(TCID50)。电镜下观察重组腺病毒的形态;RT-PCR及Western blot法检测CPV VP2基因mRNA的转录及蛋白的表达水平。结果重组穿梭质粒pacAd5-cVP2经酶切及测序证明构建正确;包装出的重组腺病毒rAd5v-cVP2的病毒滴度可稳定在108.25TCID50/ml;电镜下观察可见典型的腺病毒外型特征;重组腺病毒感染的293AD细胞可检测到VP2基因的转录和表达。结论已成功构建了CPV VP2蛋白重组人5型腺病毒。Objective To construct a recombinant adenovirus with VP2 protein of canine parvovirus（CPV）.Methods VP2 gene was amplified from CPV by PCR and cloned into shuttle plasmid pacAd5 CMVK-NpA.The constructed recombinant shuttle plasmid pacAd5-cVP2,identified by restriction analysis and sequencing,and backbone plasmid pacAd5 9.2100 were linearized with PacⅠ,then co-transfected to 293AD cells for homologous recombination,and the obtained recombinant adenovirus rAd5v-cVP2 was calculated for tite（rTCID 50）by Karber method,observed for morphology under electron microscope,and determined for mRNA transcription and protein expression of CVP VP2 by RT-PCR and Western blot respectively.Results Restriction analysis and sequencing proved that recombinant shuttle plasmid pacAd5cVP2 was constructed correctly.The titer of rAd5v-cVP2 was as stable as 10 8.25 TCID 50/ml.Typical morphology of adenovirus was observed under electron microscope.Both mRNA transcription and protein expression of VP2 were observed in 293AD cells infected with rAd5v-cVP2.Conclusion The recombinant adenovirus with VP2 protein of CPV was successfully constructed.

关 键 词：细小病毒 犬 VP2基因 腺病毒 人 

分 类 号：Q786[生物学—分子生物学] R373.9[医药卫生—病原生物学]