猪FcγRⅠ真核表达载体的构建及稳定表达细胞系的建立

畜牧兽医学报 ›› 2010, Vol. 41 ›› Issue (7): 909-914.

猪FcγRⅠ真核表达载体的构建及稳定表达细胞系的建立

史平玲¹，邬成业²，乔松林¹，张改平¹*，王爱萍²，肖治军¹，祁艳华²，卜丹²

1.河南省农业科学院农业部动物免疫学重点开放实验室/河南省动物免疫学重点实验室，郑州 450002；2.郑州大学生物工程系，郑州 450001

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-07-20 发布日期:2010-07-20
通讯作者: 张改平

Construction of Eukaryotic Expression Plasmid of poFcγRⅠ andEstablishment of Stable Transfected Cell Line

SHI Ping-ling¹, WU Cheng-ye², QIAO Song-lin¹, ZHANG Gai-ping¹* ,WANG Ai-ping², XIAO Zhi-jun¹, QI Yan-hua², BU Dan²

1. Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; 2. Department of Bioengineering, Zhengzhou University, Zhengzhou 450001, China

Received:1900-01-01 Revised:1900-01-01 Online:2010-07-20 Published:2010-07-20

摘要/Abstract

摘要： 本研究旨在建立稳定表达猪FcγRⅠ的Marc-145细胞系。从猪肺泡巨噬细胞中提取总RNA, 应用RT-PCR技术获得猪FcγRⅠ和γ链cDNA，并分别构建PIREShyg3-γ和pcDNA3.1-FcγRⅠ真核表达质粒；用脂质体共转染Marc-145细胞，经潮霉素B(300 μg·mL^-1)和G418(400 μg·mL^-1)共筛选获得稳定表达猪FcγRⅠ的细胞系；运用RT-PCR、玫瑰花环和流式细胞术对细胞系进行鉴定。结果表明成功构建了猪FcγRⅠ和γ链真核表达载体，建立了稳定表达猪FcγRⅠ的细胞系，且表达于转染细胞表面的poFcγRⅠ受体分子能与猪IgG特异结合。本研究为进一步研究奠定了基础。

关键词: 猪FcγRⅠ受体, 共转染, Marc-145细胞

Abstract: The objective of the present study was to establish the Marc-145 cell line that stably express porcine FcγRⅠ (poFcγRⅠ). The total RNA was extracted from porcine alveolar macrophage (PAM). poFcγRⅠ and γ-chain cDNAs were cloned by RT-PCR, then they were inserted into the eukaryotic expression vectors pcDNA3.1 (+) and PIREShyg3 respectively. The Marc-145 cell line was stably transfected with pcDNA3.1-poFcγRⅠ and PIREShyg3-γ plasmids by Lipofectamine2000, then the co-transfected cells were selected by Hygromycin B (300 μg·mL^-1) and G418 (400 μg·mL^-1). The expression of poFcγRⅠ on transfected cells was verified through RT-PCR, rosetting test and FCM. The eukaryotic expression vectors were confirmed successfully constructed and the Marc-145 cell line with stable poFcγRⅠ expression was obtained. The Marc-145 cell transfected with the poFcγRI cDNA were able to bind porcine IgG.

Key words: poFcγRⅠ receptor, co-transfection, Marc-145 cell line

史平玲;邬成业;乔松林;张改平;王爱萍;肖治军;祁艳华;卜丹. 猪FcγRⅠ真核表达载体的构建及稳定表达细胞系的建立[J]. 畜牧兽医学报, 2010, 41(7): 909-914.

SHI Ping-ling;WU Cheng-ye;QIAO Song-lin;ZHANG Gai-ping;WANG Ai-ping;XIAO Zhi-jun;QI Yan-hua;BU Dan. Construction of Eukaryotic Expression Plasmid of poFcγRⅠ andEstablishment of Stable Transfected Cell Line[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2010, 41(7): 909-914.

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