CAJ | 학술논문

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K lf4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1434 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K lf4在BFF和BSEC中都有表达，pMX介导的外源K lf4能在BFF中表达。
Krüppel양인자4(Klf4)재조공세포증식、분화화배태발육중유중요작용，야시생산유도다능간세포(iPSC)중요적전록인자지일。본연구대수우Klf4진행극륭화표체연구，위생산수우iPSC화연구기재체세포중편정중적궤리전정기출。채용RT-PCR극륭병분석수우K lf4편마구서렬(CDS)；mRNA수평화단백수평검측Klf4재수우태인성섬유세포(BFF)화위상피세포(BSEC)중적표체정황；구건표체수우Klf4적역전록병독재체(pMX-Klf4)、병독감염BFF、면역형광기술분석외원Klf4재BFF중적표체정황。결과표명：수우CDS전장1434 bp，안기산서렬여우、저、인화서상응안기산서렬적상사성분별위99%、97%、92%화92%，단백결구보수；내원K lf4재BFF화BSEC중도유표체，pMX개도적외원K lf4능재BFF중표체。
Krüppel-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 K lf4 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.