FcγRIIb基因真核表达质粒的构建与表达

目的构建FcγRIIb基因的真核表达重组质粒pEGFP-FcγRIIb,并观察其在真核细胞中的表达。方法以U937细胞的RNA为模板,通过RT-PCR扩增FcγRIIb基因,并将其定向插入绿色荧光蛋白质粒pEGFP-C1中,构建真核表达重组质粒pEGFP-FcγRIIb。经PCR、限制性核酸内切酶HindⅢ和Sma I的酶切鉴定及DNA序列分析后,用脂质体法将pEGFP-FcγRIIb转染NIH3T3细胞,用荧光显微镜观察pEGFP-FcγRIIb的表达。结果扩增出了963bp的FcγRIIb基因,成功构建了真核表达重组质粒pEGFP-FcγRIIb。在细胞膜与细胞内观察到较强的绿色荧光,表明pEGFP-FcγRIIb成功转入NIH3T3细胞,并在NIH3T3细胞中得到了表达。结论构建的真核重组表达质粒pEGFP-FcγRIIb能够表达FcγRIIb蛋白,为进一步研究FcγRIIb的功能奠定了基础。

The Construction and Expression of Eukaryotic Expression Plasmid of Fc gamma Receptor IIb Gene

Objective To construct the recombinant plasmid of pEGFP-FcγRIIb and to detect the protein expression in eukaryotic cells.Methods The Fc gamma receptor IIb gene was amplified from the total RNA of U937 cells by RT-PCR and then was inserted into the pEGFP-C1 plasmid.The recombinant plasmid was named pEGFP-FcγRIIb and 瓦was analyzed with restriction endonuclease HindⅢ and Sma I digestion,PCR and DNA sequencing techniques.The NIH3T3 cell was transfected by the recombinant plasmid pEGFP-FcγRIIb with lipofection strategy.The expression products of Fc gamma receptor IIb gene were detected by Fluorescence microscope.Results The Fc gamma receptor IIb gene of 963 bp in length was amplified.The recombinant plasmid of Fc gamma receptor IIb gene was successfully constructed.It was found that there was dark green fluorescence on the cell membrane and inside the cells.The results showed that the NIH3T3 cell was transfected by pEGFP-FcγRIIb successfully and the protein was expressed in the cells.Conclusion The recombinant plasmid of pEGFP-FcγRIIb could express FcγRIIb protein which would play an important role in exploring the immunogenic function of Fc gamma receptor IIb.