O型FMDV P1-2A基因与猪IFN-γ基因共表达质粒的构建及其在BHK-21细胞中的表达

doi:10.7668/hbnxb.2010.05.002

华北农学报 ›› 2010, Vol. 25 ›› Issue (5): 11-15. doi: 10.7668/hbnxb.2010.05.002

所属专题： 畜牧；生物技术；

O型FMDV P1-2A基因与猪IFN-γ基因共表达质粒的构建及其在BHK-21细胞中的表达

吴国华, 张强, 颜新敏, 侯俊玲, 赵志荀, 崔力凡, 李健, 朱海霞, 朱彩珠

中国农业科学院, 兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 农业部畜禽病毒学重点开放实验室, 甘肃, 兰州, 730046

收稿日期:2010-08-03 出版日期:2010-10-28
作者简介:吴国华(1977-),男,江西赣州人,助理研究员,博士,主要从事病原分子生物学及免疫学诊断的研究.
基金资助:
国家科技支撑项目(2006BAD06A11);甘肃省重大科技项目(092NKDA032)

Construction of Co-expressing Plasmid of FMDV P1-2A Gene with Porcine IFN-γ Gene and Expression in BHK-21 Cells

WU Guohua, ZHANG Qiang, YAN Xinmin, HOU Junling, ZHAO Zhixun, CUI Lifan, LI Jian, ZHU Haixia, ZHU Caizhu

Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Lanzhou 730046, China)

Received:2010-08-03 Published:2010-10-28

摘要/Abstract

摘要： 为了构建O型FMDV P1-2A基因与猪IFN-γ基因共表达质粒并在BHK-21细胞中进行表达,本研究利用聚合酶链式反应(PCR)从阳性质粒pGEM-P12A克隆到P1-2A基因,将扩增产物纯化、双酶切后连接到pBudCE-IFN载体,构建重组质粒pBudCE-IFN-P1-2A.重组表达质粒鉴定后,在脂质体介导下转染BHK-21细胞,用RT-PCR和直接免疫荧光试验分别检测P1-2A 和IFN-γ在BHK-21细胞中的表达.RT-PCR检测显示,重组表达质粒转染细胞后P1-2A 和IFN-γ基因成功转录出mRNA;免疫荧光试验表明目的基因在BHK-21细胞中获得了表达.结果证明,本研究成功构建了重组质粒pBudCE-IFN-P1-2A并在体外进行了表达,这为研制抗FMDV DNA疫苗和IFN-γ的佐剂作用奠定了坚实基础.

关键词: FMDV, P1-2A基因, IFN-&gamma, 基因, 共表达

Abstract: To construct the co-expressing plasmid of FMDV P1-2A gene with porcine IFN-γ gene and express in BHK-21 cells. A P1-2A gene of foot-and-mouth disease virus(FMDV) was amplified by PCR from the plasmid pGEM-P12A and subcloned into pBudCE-IFN vector for sequencing after purification and digestion. A recombinant plasmid pBudCE-IFN-P1-2A was constructed and transformed into BHK-21 cells with liposome for in vitro expression. 48 hours after transfection,the mRNAs and expressed proteins of P1-2A and IFN-γ were detected by RT-PCR and direct immunofluorescence assay,respectively. The results showed that P1-2A and IFN-γ were successfully transcripted and expressed in BHK-21 cells. It was proved that the recombinant plasmid pBudCE-IFN-P1-2A was successfully constructed and expressed in vitro,providing a solid basis for development of DNA vaccine against FMDV

Key words: FMDV, P1-2A, IFN-γ, Co-expression

中图分类号:

吴国华, 张强, 颜新敏, 侯俊玲, 赵志荀, 崔力凡, 李健, 朱海霞, 朱彩珠. O型FMDV P1-2A基因与猪IFN-γ基因共表达质粒的构建及其在BHK-21细胞中的表达[J]. 华北农学报, 2010, 25(5): 11-15. doi: 10.7668/hbnxb.2010.05.002.

WU Guohua, ZHANG Qiang, YAN Xinmin, HOU Junling, ZHAO Zhixun, CUI Lifan, LI Jian, ZHU Haixia, ZHU Caizhu. Construction of Co-expressing Plasmid of FMDV P1-2A Gene with Porcine IFN-γ Gene and Expression in BHK-21 Cells[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2010, 25(5): 11-15. doi: 10.7668/hbnxb.2010.05.002.

http://www.hbnxb.net/CN/Y2010/V25/I5/11

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O型FMDV P1-2A基因与猪IFN-γ基因共表达质粒的构建及其在BHK-21细胞中的表达

Construction of Co-expressing Plasmid of FMDV P1-2A Gene with Porcine IFN-γ Gene and Expression in BHK-21 Cells

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O型FMDV P1-2A基因与猪IFN-γ基因共表达质粒的构建及其在BHK-21细胞中的表达

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