牛巨噬细胞天然抗性蛋白Ⅰ基因可变剪接体(NRAMP1-ISO)的克隆及在细胞和组织中的表达  

Cloning of the Bovine Natural Resistance-associated Macrophage Protein Ⅰ Gene Isoform(NRAMP1-ISO) and Its Expression in the Cells and the Tissues

作　　者：程祥[1] 邓捷[1] 孟书燕[1] 来威锋[1] 王华岩[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,杨凌712100

出　　处：《农业生物技术学报》2011年第4期685-691,共7页Journal of Agricultural Biotechnology

基　　金：国家抗病转基因牛新品种培育项目(No.2009ZX08007-008B)资助

摘　　要：巨噬细胞天然抗性蛋白1(NRA MP1)可抑制结核分枝杆菌(Mycobacterium)和布氏杆菌(Brucella)等多种胞内寄生病原菌的感染,提高动物机体的抗病能力。本研究从秦川牛脾脏中克隆了NRA MP1基因的一种可变剪接体NRA MP1-ISO,序列分析表明,NRA MP1-ISO比NRA MP1多了第3个内含子,从而导致编码序列提前终止于第3个内含子,NRA MP1-ISO编码200个氨基酸。为了探索可变剪接体NRA MP1-ISO的表达情况,本研究分别构建了原核表达载体pET-41-NRA MP1-ISO和真核表达载体pEGFP-NRA MP1-ISO。原核表达载体pET-41-NRA MP1-ISO可在不同浓度的IPTG诱导下在大肠杆菌BL21中高效表达。真核表达载体pEGFP-NRA MP1-ISO转染牛成纤维细胞后,EGFP-NRA MP1-ISO融合蛋白分布在细胞核和细胞质中,而正常的NRA MP1蛋白只分布在细胞质的溶酶体膜周围。半定量RT-PCR检测表明,NRA MP1-ISO基因在心、脾脏、肺脏等组织有较高的表达,而在脑、胰、生殖嵴中的表达量相对较低。本研究为进一步研究NRA MP1-ISO基因生物学功能提供了重要信息。Natural resistance-associated macrophage protein 1（NRAMP1） can defense the invasion of the intracellular bacteria such as Mycobacterium and Brucella to improve the ability of resisting disease.In this study we cloned a NRAMP1 isoform from the spleen of Qinchuan Bos Taurus.Sequence analysis showed that NRAMP1 isoform had a third intron in comparison with NRAMP1,which resulted in the protein only containing 200 amino acids because the translation stopped in the third intron.In order to get further study on the expression of NRAMP1-ISO,we constructed the prokaryotic expression vector pET-41-NRAMP1-ISO and the eukaryotic expression vector pEGFP-NRAMP1-ISO.The prokaryotic expression vector pET-41-NRAMP1-ISO could efficiently express in the BL21 Escherichia coli under different concentrations of the IPTG.The eukaryotic expression vector pEGFP-NRAMP1-ISO could both express in the nucleus and the cytoplasm of the bovine fibroblast while NRAMP1 was normally expressed around the lysosome membrane in the cytoplasm.Semi-quantitative RT-PCR showed that NRAMP1-ISO gene was highly expressed in the heart,spleen and lungwhile lowly expressed in the brain,pancreas and the genital ridge.The results provided the useful imformation for further anzlyzing the function of NRAMP1-ISO gene.

关 键 词：牛 巨噬细胞天然抗性蛋白1(NRA MP1) 可变剪接 分子克隆 载体构建 组织表达 

分 类 号：S852.659.6[农业科学—基础兽医学]