上皮组织特异性表达N-LMP1和CR2转基因猪胚胎成纤维细胞的构建与鉴定

Construction and identification of transgenic porcine embryonic fibroblasts expressing epithelium-specific N-LMP1 and CR2

目的构建上皮组织特异性表达鼻咽癌来源潜伏膜蛋白1(N-LMP1)和人补体受体2(CR2)真核表达载体,转染猪胚胎成纤维细胞并筛选整合有N-LMP1和CR2基因的细胞克隆,为构建与EBV感染相关的猪鼻咽癌模型奠定基础。方法通过直接合成ED-L2启动子和N-LMP1,从人B淋巴细胞中的RNA经RT-PCR扩增出CR2,将上述3个片段逐个连接到真核表达载体pN1上,构建上皮组织特异性表达N-LMP1和CR2的载体pN1-EDL2-N-LMP1-CR2;用脂质体转染猪胚胎成纤维细胞,经药物G418筛选和PCR鉴定阳性克隆。结果成功构建上皮组织特异性表达N-LMP1和CR2的真核表达载体pN1-ED-L2-N-LMP1-CR2,并成功整合到猪胚胎成纤维细胞的基因组中,获得了整合有目的基因N-LMP1和CR2的猪胚胎成纤维细胞克隆。结论获得了上皮组织特异性表达N-LMP1和CR2的猪胚胎成纤维细胞克隆,为通过细胞核移植方法获得表达N-LMP1和CR2转基因猪提供了供体细胞。

Objective To construct an eukaryotic expression vector and identify the integration of nasopharyngeal carcinoma-derived oncogene latent membrane protein 1 （N-LMP1） and CR2 gene in porcine embryonic fibroblasts, and to provide a basis for construction of EBV-infection associated swine nasopharyngeal carcinoma model. Methods ED-L2 and N-LMP1 were synthesized directly. CR2 was amplified by RT-PCR from human B lymphocytes. The three fragments mentioned above were subcloned one by one into the eukaryotic expression vector pN1, and then the vector was transfected into porcine embryonic fibroblasts using the liposome reagent, according to the manufacturer＇s protocol. The cells were selected with G418 antibiotic and identified by PCR amplification. Results N-LMP1 and CR2 epithelium-specific expression vector was successfully constructed and integrated into the genome of the porcine fibroblasts, and clone cells integrating the N-LMP1 and the CR2 genes were obtained. Conclusions The porcine fibroblast clones integrating N-LMP1 and CR2 are obtained and they should be of great value for the construction of N-LMP1 and CR2 transgenic swine via cell nuclear transfer.