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犬冠状病毒S糖蛋白基因重组犬2型腺病毒的构建及其免疫原性研究 被引量：3

Construction of recombinant Canine adenovirus type 2 expressing Canine coronavirus spike glycoprotein and its immunogenicity
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摘要 首次构建了能表达犬冠状病毒纤突糖蛋白(CCVS1)的重组犬2型腺病毒(CAV-2)。用RT-PCR方法从CCVDXMV株细胞培养物中扩增出编码S糖蛋白A、B、C和D4个抗原位点的基因片段S1,将其克隆到pVAX1中,然后将含有CCVS1基因的完整表达盒(CMV-S1-PolyA)进一步定向克隆到含有CAV-2E3区的穿梭质粒pVAXE3中,构建出pVAX△E3S1。通过SalⅠ+NruⅠ双酶切pVAX△E3S1回收含有目的基因的表达盒,将其克隆入含有CAV-2全基因组的骨架质粒pPoly2-CAV-2中,获得重组质粒pCAV-2-CCV-S1。ClaⅠ+AscⅠ酶切pCAV-2-CCV-S1释放重组基因组,转染MDCK细胞,获得了重组病毒CAV-2-S1。该重组病毒在MDCK细胞上能产生典型的腺病毒细胞病变。通过mRNA水平和Westernblot检测,证实重组病毒能表达CCVS1蛋白。动物免疫试验表明,该重组病毒可以有效地诱导免疫犬产生抗CCV和CAV-2抗体。 In order to construct a recombinant Canine adenovirus type 2 (CAV-2) expressing the spike glycoprotein of Canine coronavirus (CCV), the S1 gene fragment of CCV strain DXMV, encoding major antigenic region A, B, C and D of S protein, was amplified by RT-PCR and cloned into pVAX1 vector. The complete S1 expression cassette was subcloned into the shuttle vector pVAXE3, then further cloned into the backbone vector pPoly2-CAV2 containing complete genome of CAV-2.To gain the recombinant Canine adenovirus, the recombinant plasmid pCAV-2-CCV-S1 was linearized by ClaⅠ/AscⅠ to release recombinant genome, and then transfected into MDCK cell. The recombinant virus CAV-2-S1 was gained through 4 passages in MDCK, which showed classical CPE of CAV-2.The expressed S1 protein of CCV, which was identified by RT-PCR and Western blot, can be specifically recognized by polyclonal antibody against CCV. The immunization in dogs indicated that the recombinant CAV-2 could effectively induce the specific antibodies against CCV and CAV.
作者 乔军 夏咸柱 杨松涛 胡桂学 谢之景 闫芳 高玉伟 黄耕
机构地区 中国人民解放军军事医学科学院军事兽医研究所 中国人民解放军军事医学科学院军事兽医研究所 吉林农业大学动物科技学院
出处 《微生物学报》 CAS CSCD 北大核心 2005年第4期588-592,共5页 Acta Microbiologica Sinica
基金 国家自然科学基金资助项目(30000123) 全军"十五"医药卫生重点项目(Z2001095)~~
关键词 犬冠状病毒 纤突糖蛋白 重组犬2型腺病毒 Canine coronavirus, Spike Glycoprotein, Recombinant Canine adenovirus type 2
分类号 S852.65 [农业科学—基础兽医学]
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参考文献18

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二级参考文献28

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微生物学报
2005年 第4期

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