牦牛六个多能性相关转录因子OSKMNL的克隆和多顺反子慢病毒载体的构建

doi:10.11843/j.issn.0366-6964.2024.04.021

摘要/Abstract

摘要： 旨在克隆牦牛6个多能性相关转录因子Oct4、Sox2、Klf4、c-Myc、Nanog、Lin28(OSKMNL)，构建多顺反子慢病毒载体FUW-teto-OSM-EGFP和FUW-teto-KNL-mCherry。本研究以1头3~5月龄健康雌性牦牛胎儿的生殖嵴组织为研究材料，利用RT-PCR技术克隆牦牛6个多能性相关转录因子OSKMNL的完整编码区序列，并对其进行生物信息学分析；应用无缝克隆技术构建慢病毒载体FUW-teto-OSM-EGFP和FUW-teto-KNL-mCherry；用293T细胞包装慢病毒，将包装好的慢病毒感染293T细胞和牦牛成纤维细胞，通过观察荧光表达和RT-PCR技术检测病毒感染情况(试验分为病毒感染组和空白对照组，每个组设置3个重复)。结果表明，克隆的牦牛OSKMNL基因的编码区大小分别为1 083、963、1 434、1 320、903、618 bp；序列分析发现牦牛OSKMNL氨基酸序列与黄牛的同源性在99%以上，其中Sox2、Klf4、c-Myc与黄牛的同源性为100%；进化树结果显示牦牛与黄牛、瘤牛、水牛的亲缘关系最近，与小鼠的亲缘关系最远；蛋白结构预测发现牦牛的6个转录因子OSKMNL都具有该基因家族相应蛋白功能的结构，比如POU结构域、HOX结构域、HMG结构域、Znf-C2H2结构域、HLH结构域、CSP结构域等；构建了慢病毒载体FUW-teto-OSM-EGFP和FUW-teto-KNL-mCherry；慢病毒感染293T细胞发现试验组表达红色和绿色荧光；慢病毒感染牦牛成纤维细胞发现试验组表达红色和绿色荧光，RT-PCR结果显示预期大小的条带。本研究成功克隆了牦牛的6个多能性相关转录因子OSKMNL；构建了分别携带牦牛3个转录因子的慢病毒载体FUW-teto-OSM-EGFP和FUW-teto-KNL-mCherry；包装的慢病毒能感染牦牛成纤维细胞。有利于推动多能性相关转录因子OSKMNL在牦牛干细胞中的应用，也为后续研究牦牛iPSC做准备。

关键词: 牦牛, 基因克隆, 序列分析, 慢病毒载体

Abstract: The purpose of this study was to clone 6 pluripotency-related transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28 (OSKMNL) of yak and construct polycistronic lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry. In this study, the reproductive crest tissue of a 3-5-month-old healthy female yak fetus was used as the research material. The complete coding region sequences of 6 pluripotent related transcription factors OSKMNL were cloned by RT-PCR and analyzed by bioinformatics. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed by seamless cloning. The lentivirus was packaged with 293T cells, and the packaged lentivirus was infected with 293T cells and yak fibroblasts. The virus infection was detected by fluorescence expression and RT-PCR technique (the experiment was divided into virus infection group and blank control group, each group had 3 repeats). The results showed that the coding region of cloned yak OSKMNL gene was 1 083, 963, 1 434, 1 320, 903 and 618 bp, respectively. Sequence analysis showed that the amino acid sequence of OSKMNL in yak shared more than 99% homology with cattle, and the homology of Sox2, Klf4, c-Myc with cattle was 100%. The results of evolutionary tree showed that yak had the closest genetic relationship with cattle, zebu cattle and buffalo, but the farthest relationship with mice. Protein structure prediction showed that the 6 transcription factors OSKMNL of yak all had the corresponding protein functional structures of the gene family, such as POU domain, HOX domain, HMG domain, Znf-C2H2 domain, HLH domain, CSP domain and so on. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed. 293T cells infected by lentivirus were found to express red and green fluorescence. Yak fibroblasts infected with lentivirus showed red and green fluorescence in the experimental group, and RT-PCR results showed bands with expected size. In this study, 6 pluripotency related transcription factors OSKMNL of yak were successfully cloned. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed, which contain 3 transcription factors of yak, respectively. Lentivirus could infect yak fibroblasts. It is helpful to promote the application of pluripotent related transcription factor OSKMNL in yak stem cells and prepare for the follow-up study of yak iPSC.

Key words: yak, gene cloning, sequence analysis, lentiviral vector

中图分类号:

S823.85

黄显朋, 邢嘉仪, 白媛媛, 姜雨婷, 麻志伟, 付伟, 兰道亮. 牦牛六个多能性相关转录因子OSKMNL的克隆和多顺反子慢病毒载体的构建[J]. 畜牧兽医学报, 2024, 55(4): 1579-1591.

HUANG Xianpeng, XING Jiayi, BAI Yuanyuan, JIANG Yuting, MA Zhiwei, FU Wei, LAN Daoliang. Cloning of Six Pluripotent Related Transcription Factors OSKMNL in Yak and Construction of Polycistron Lentiviral Vector[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1579-1591.

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