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

中国畜牧兽医

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

莫永正¹，罗满林¹，陈瑞爱¹^，2

（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）

收稿日期:2013-09-16 出版日期:2014-03-20 发布日期:2014-05-15
通讯作者: 罗满林，教授，博士生导师。E-mail: LuomLin2010@163.com
作者简介:莫永正(1986—），男，广西人，硕士生，研究方向：动物传染病学。
基金资助:
广东省科学技术部产研合作科学基金（2011A090200117）。

Construction and Identification of Recombinant Adenovirus Expression Vector Carrying the Fusion Genes ORF2 and T Lymphocyte Epitopes of Porcine Circovirus Type 2

MO Yong-zheng¹, LUO Man-lin¹, CHEN Rui-ai¹^,2

(1.College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China;2.Guangdong Dahuanong Animal Health Products Co., Ltd., Xinxing 527400, China)

Received:2013-09-16 Online:2014-03-20 Published:2014-05-15

摘要/Abstract

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10^12.3 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。

关键词: 猪圆环病毒2型; ORF2; T细胞表位; 腺病毒表达系统

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 10^12.3 TCID₅₀/mL. The expression of the fusion protein was verified by Western blotting and indirect immunofluorescence assay (IFA).

Key words: porcine circovirus type 2; ORF2; T cell epitope; adenoviral expression system

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.

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]. .

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