猪FcγRⅠ真核表达载体的构建及稳定表达细胞系的建立

Construction of Eukaryotic Expression Plasmid of poFcγRⅠ and Establishment of Stable Transfected Cell Line

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(300μg.mL-1)和G418(400μg.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。

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.