| 猪圆环病毒2型ORF2与T细胞表位基因重组腺病毒表达载体的构建及鉴定 |
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| 引用本文： | 莫永正,罗满林,陈瑞爱.猪圆环病毒2型ORF2与T细胞表位基因重组腺病毒表达载体的构建及鉴定[J].中国畜牧兽医,2014,41(3):58-64. |
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| 作者姓名： | 莫永正 罗满林 陈瑞爱 |
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| 作者单位： | （1.华南农业大学兽医学院，广东广州 510642；2.广东大华农动物保健品股份有限公司，广东新兴 527400） |
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| 基金项目： | 广东省科学技术部产研合作科学基金（2011A090200117）。 |
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| 摘 要： | 利用腺病毒表达系统表达猪圆环病毒2型（porcine circovirus type 2，PCV2）的ORF2基因与T细胞表位（T cell epitope，TCE）基因，表达的融合蛋白具有反应原性，为研制PCV2新型疫苗奠定基础。以pMD18-T-ORF2、pMD18-T-TCE为模板，采用PCR方法扩增目的基因ORF2和TCE，以多肽接头（Gly4Ser）3为连接子，运用重叠延伸PCR技术将2段基因通过连接子（Gly4Ser）3进行融合连接。将融合基因定向克隆至腺病毒转移载体pShuttle-CMV构建重组质粒pShuttle-CMV-ORF2-TCE，将该重组质粒用PmeⅠ酶线性化后电转化大肠杆菌BJ5183感受态细胞（内含pAdEasy-1骨架质粒）进行同源重组获得重组腺病毒质粒pAd-ORF2-TCE。PacⅠ酶线性化pAd-ORF2-TCE质粒后转染AD293细胞包装病毒，重组腺病毒经3轮噬斑纯化后获得重组腺病毒rAd-ORF2-TCE，病毒滴度为1012.3 TCID50/mL。Western blotting及间接免疫荧光试验（indirect immunofluorecent assay，IFA）结果表明融合蛋白得到正确表达。
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| 收稿时间： | 2013-09-16 |
Construction and Identification of Recombinant Adenovirus Expression Vector Carrying the Fusion Genes ORF2 and T Lymphocyte Epitopes of Porcine Circovirus Type 2 |
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| MO Yong-zheng,LUO Man-lin,CHEN Rui-ai.Construction and Identification of Recombinant Adenovirus Expression Vector Carrying the Fusion Genes ORF2 and T Lymphocyte Epitopes of Porcine Circovirus Type 2[J].China Animal Husbandry & Veterinary Medicine,2014,41(3):58-64. |
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| Authors: | MO Yong-zheng LUO Man-lin CHEN Rui-ai |
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| Institution: | (1.College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;2.Guangdong Dahuanong Animal Health Products Co., Ltd., Xinxing 527400, China) |
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| Abstract: | This study was aimed to obtain expression of fusion gene （ORF2-TCE） of porcine circovirus type 2 (PCV2) in adenoviral expression system and develop PCV2 subunit vaccine. The target genes ORF2 and T cell epitope (TCE) were obtained by PCR with pMD18-T-ORF2 and pMD18-T-TCE as templates, respectively. The fusion gene ORF2-TCE was successfully obtained by overlap extension PCR method with the ORF2-linker and linker-TCE fragments as templates. The fusion gene was cloned into the transfer vector pShuttle-CMV. The PmeⅠ-linearized plasmid vector pShuttle-CMV-ORF2-TCE was transformed into Escherichia coli bacteria competent cell BJ5183, containing the pAdEasy-1 vector by electroporation. The recombinant plasmid named pAd-ORF2-TCE was obtained and identified by PCR and PacⅠenzyme digestion. To produce the recombinant adenovirus rAd-ORF2-TCE, AD293 cells were transfected with PacⅠ-linearized plasmid pAd-ORF2-TCE. The recombinant virus named rAd-ORF2-TCE were obtained and purified through vital plaque by three times and the viral titer was 1012.3 TCID50/mL. The expression of the fusion protein was verified by Western blotting and indirect immunofluorescence assay (IFA). |
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| Keywords: | porcine circovirus type 2 ORF2 T cell epitope adenoviral expression system |
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