猪细胞周期蛋白A基因的克隆及其功能研究  

Cloning and Functional Analysis of Cyclin A Gene in Pig

作　　者：范丽[1] 唐青海[1] 张彦明[1] 刘伟[1] 仝刚[1] 

机构地区：[1]西北农林科技大学动医学院,杨凌712100

出　　处：《畜牧兽医学报》2010年第4期469-477,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金项目(30972186)

摘　　要：本研究旨在克隆猪细胞周期蛋白A基因(CyclinA),并在猪脐静脉血管内皮细胞(SUVEC)中表达,以验证其功能。以人的CyclinA基因为信息探针,经电子克隆得到猪CyclinA基因,通过RT-PCR和DNA测序验证电子克隆结果并对其作生物信息学分析,用RT-PCR、Westernblot研究它在SUVEC中的表达情况。应用激光共聚焦显微镜分析它在细胞中的亚细胞定位,并通过流式细胞仪分析细胞周期以及用MTS法测定细胞的增殖能力。结果如下:核酸测序证明RT-PCR产物同电子克隆结果相符。该cDNA包含1299bp的完整开放阅读框(ORF),共编码432个氨基酸,经NCBIBLAST分析,该基因定位于猪的8号染色体上。Westernblot结果显示CyclinA基因编码蛋白的相对分子质量大小约为40ku,亚细胞定位研究表明该蛋白定位于细胞核中。经流式细胞仪分析表明稳定表达该基因的细胞,其G1期的细胞数量比对照组细胞增加了15%～20%,而S期的细胞数量比对照组细胞减少了约18%;MTS法检测显示细胞株SUVEC-CycAGFP的增殖活性明显高于对照组细胞。作者成功克隆到新基因猪源细胞周期蛋白A,并验证了其生物学功能,为今后开展病毒感染对猪细胞周期蛋白A影响的研究奠定了基础。The study was aimed at cloning porcine Cyclin A gene and expressing it in swine umbilicus veins endothelial cell（SUVEC）to verify its functionality.The human Cyclin A gene was utilized as the informational probe to e-clone the pigs Cyclin A gene.Reverse transcription polymerase chain reaction（RT-PCR）was used to identify the products in silico cloned,and the bioinformatics methods was used to analyze structure characteristics of the gene,RT-PCR,Western blot were employed for investigating its expression in SUVEC.Subcellular localization of Cyclin A was analyzed by confocal microscope,and the flow cytometry was used to analyze the cell cycle,and MTS was used to detect the proliferation of the cells.The results proved that the product of RT-PCR was consistent with that of silico cloning.This cDNA contains the complete open reading frame（ORF）of 1 299 bp coding 432 amino acid residues,and NCBI BLAST analysis indicated that the gene is located in swine on chromosome 8.Western blot manifested that molecular size of Cyclin A protein was about 40 kDa,and subcellular localization showed that this protein was localized in nuclear.The flow cytometry analysis showed that cell lines SUVEC-CycAGFP of G1 phase cells increased by 15% to 20%,while those in S phase decreased about 18% compared with that of the control cells.MTS assay showed that the proliferative activity of cell lines SUVEC-CycAGFP was significantly higher than that of the control groups.Our results indicate that Cyclin A gene of pig was successfully cloned and its biological function was also confirmed,which will provide a foundation for further research on the impact of virus infection on the Cyclin A.

关 键 词：猪细胞周期蛋白A基因 克隆 猪脐静脉血管内皮细胞 真核表达 细胞周期 MTS 

分 类 号：S852.23[农业科学—基础兽医学] Q78[农业科学—兽医学]