水牛转录因子 c-Myc 克隆和表达研究

The Cloning and Expression of Buffalo Transcription Factor c-Myc

原癌基因c-Myc是诱导多能干细胞(iPSC)生产中重要的转录因子之一。水牛c-Myc的克隆和表达,可以为下一步研究其功能和使用特异性转录因子生产水牛iPSC奠定基础。采用RT-PCR扩增水牛c-Myc编码区(CDS),RT-PCR和免疫荧光技术检测内源c-Myc在水牛胎儿成纤维细胞(BFF)和胃上皮细胞(BSEC)中的表达情况;构建表达c-Myc的逆转录病毒载体(pMX-c-Myc),病毒法转入BFF中,免疫荧光技术分析外源c-Myc在BFF中的表达情况。结果表明:c-Myc在水牛BFF和BSEC中都有表达;水牛c-Myc的CDS全长1 320 bp,氨基酸序列与牛、猪、人和鼠相应蛋白的同源性分别为98%、96%、91%和86%,与小鼠c-Myc蛋白的结构相似;逆转录病毒载体介导的c-Myc能在BFF中表达。

Oncogene c-Myc is an important transcriptional factor for generation of induced pluripotent stem cells （iPSC）. The cloning and expression of buffalo c-Myc will provide a good foundation to investigate the functions of c- Myc and generate buffalo iPSC. The coding sequence （CDS） of c-Myc was amplified by RT-PCR. Whether c-Myc expressed in buffalo fetal fibroblasts （BFF） or buffalo stomach epithelial cells （BSEC） was identified by RT-PCR and immunofluorescence assay. The pMX retrovirus vector based c-Myc （pMX-c-Myc） was constructed and transfected into BFF by retrovirus infection, the expression of c-Myc was detected by immunofluorescence. The results showed that c-Myc expressed both in BFF and BSEC; the CDS length of c-Myc was 1320bp, and the amino acids exhibited high homology with bovine （98%）, pig （96%）, human （91%） and mouse （86%）; the protein structure of the buffalo c- Myc was similar to that of mouse; pMX retrovirus based c-Myc could expressed in the BFF.