水牛转录因子Klf4基因编码区序列克隆及在真核细胞中表达研究

Cloning the Coding Sequence of Buffalo Transcription Factor Klf4 and Expression in Eukaryotic Cells

Krüppel样因子4(Klf4)在调控细胞增殖、分化和胚胎发育中有重要作用,也是生产诱导多能干细胞(iPSC)重要的转录因子之一。本研究对水牛Klf4进行克隆和表达研究,为生产水牛iPSC和研究其在体细胞重编程中的机理奠定基础。采用RT-PCR克隆并分析水牛Klf4编码区序列(CDS);mRNA水平和蛋白水平检测Klf4在水牛胎儿成纤维细胞(BFF)和胃上皮细胞(BSEC)中的表达情况;构建表达水牛Klf4的逆转录病毒载体(pMX-Klf4)、病毒感染BFF、免疫荧光技术分析外源Klf4在BFF中的表达情况。结果表明:水牛CDS全长1 434 bp,氨基酸序列与牛、猪、人和鼠相应氨基酸序列的相似性分别为99%、97%、92%和92%,蛋白结构保守;内源Klf4在BFF和BSEC中都有表达,pMX介导的外源Klf4能在BFF中表达。

Krtippel-like transcription factor 4 （Klf4） is one of the defined factors for iPSC generation. Klf4 plays an important role in regulation of cell proliferation, cell differentiation, embryos development. The cloning and expres- sion of buffalo Klf4 will provide a good foundation to generate buffalo iPSC and investigate the function of Klf4 in the reprogramming process. The coding sequence （CDS） of Klf4 was amplified by RT-PCR and analyzed by bio- informatics; mRNA and protein expression of Klf4 were detected in buffalo fetal fibroblasts （BFF） and buffalo stomach epithelial cells （BSEC）; retroviral vector containing Klf4 was reconstructed, and transfected into BFF, the expression of Klf4 was detected by immunofluorescence. The results showed that the CDS length of Klf4 was 1 434 bp, and the amino acids sequence exhibited high homology with bovine, pig, human and mouse, the percentage of similarity was 99%, 97%, 92% and 92% respectively; Klf4 expressed both in BFF and BSEC, pMX retrovirus based Klf4 could be expressed in the BFF.