IFITM3真核表达载体的构建和细胞内定位研究

Construction and Expression of Eukaryotic Expressing Vector of Interfern Induced Transmembrane Protein

目的构建人干扰素诱导的跨膜蛋白3(IFITM3)的真核表达载体并观察其表达产物的细胞内定位,以研究IFITM3的蛋白功能及作用机制。方法提取人外周血淋巴细胞mRNA,行RT-PCR扩增IFITM3编码序列并经琼脂糖凝胶电泳鉴定片段大小,PCR产物及真核表达载体pEGFP-C2经酶切、连接、转化入Top10菌株进行筛选和扩增,重组的pEGFP-C2-IFITM3载体用酶切及DNA测序鉴定其插入序列的正确性,进而转染该重组真核表达载体进入sy5y细胞,倒置荧光共聚焦显微镜观察融合蛋白在细胞内定位。结果琼脂糖凝胶电泳显示经RT-PCR的扩增产物与IFITM3编码区的实际长度相吻合,重组入绿色荧光载体pEGFP-C2的pEGFP-C2-IFITM3经酶切和测序显示实际连入的DNA片段大小、序列及读码框均正确。经真核表达后,重组蛋白定位于细胞膜和在细胞内存在颗粒样聚集。结论成功构建IFITM3的真核表达载体,该载体表达的IFITM3融合蛋白在细胞内定位清晰,为后续的IFITM3功能研究奠定了基础。

Objective To construct eukaryotic expressing vector of interfern induced transmembrane protein （ IFITM3 ） gene and to detect the localization of IFITM3 protein by transfecting into sy5y cell line for its gene function research. Methods mRNA was extracted from peripheral lymphocytes and coding sequence （CDS） of IFITM3 was amplifieated by reverse transcript PCR. The restriction enzymes and T4 ligase were used to insert the amplified DNA sequence into the pEGFP-C2 vector. The enzyme digestion analysis and sequencing were used to confirm the pEGFP-C2-IFITM3 recombinant vector. The localization of IFITM3 was detected by transfecting the recombinant vector into sySy cells. Results CDS of IFITM3 gene was correctly amplified from lymphocytes by RT-PCR. pEGFP-C2-IFITM3 recombinant expression vector was constructed, conformed and further transfected for localization. GFP-IFITM3 fusion protein was localized in cell membrane and part of cell plasma cultured in vitro. Conclusion Eukaryotic expression vector of IF- ITM3 has been successfully constructed and expressed, and provided a good foundation for the further study of the molecular roles of IFITM3.