摘要
【目的】克隆及真核表达猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)陕西分离株的3a和3b基因,研究表达蛋白在细胞中的分布及其对细胞周期的影响。【方法】利用软件Primer 5.0参照Genbank公布的序列设计两对分别针对TGEV非结构蛋白基因3a和3b的特异性引物。采用RT-PCR方法从TGEV陕西分离株克隆3a和3b基因,再将其与真核表达载体p EGFP-N1连接,构建真核表达质粒p3a-EGFP-N1和p3b-EGFP-N1。利用脂质体转染法将重组质粒转染猪小肠黏膜上皮细胞(intestinal epithelial cells,IEC),采用激光共聚焦显微镜检测转染细胞内融合蛋白的表达分布情况。通过RT-PCR检测细胞中目的基因的转录情况,Western blotting检测细胞中融合蛋白的表达情况。利用流式细胞仪检测表达蛋白对细胞周期的影响,采用实时荧光定量PCR检测融合蛋白的表达对内质网应激蛋白标志性分子GRP78和细胞周期蛋白Cyclin A的表达的影响,通过Western blotting试验检测细胞中蛋白表达后GRP78、Cyclin A和Cyclin B1表达量的变化情况。【结果】成功克隆出完整的TGEV陕西分离株的3a及3b基因,经过测序鉴定,3a基因的大小为213bp,3b基因的大小为732bp;经过与不同来源的TGEV毒株进行对比分析,3a基因的核苷酸序列与其他毒株同源性为97.4%—100%,氨基酸同源性为98.6%—100%;3b基因的核苷酸序列与其他毒株同源性为98.3%—99.9%,氨基酸同源性为100%。重组表达载体转染IEC细胞后,经Western blotting分析IEC分别表达出分子质量约为35k D的3a-GFP和54k D的3b-GFP的融合蛋白,与预期结果相一致。激光共聚焦显微镜观察到融合蛋白3a-GFP和3b-GFP在IEC细胞的细胞核和细胞质中均有表达,流式细胞仪分析TGEV非结构蛋白3b的表达能够使G2/M期的细胞增多,实时荧光定量PCR分析显示细胞周期蛋白Cyclin A的m RNA水平高于对照组细胞(IEC和IEC-GFP),同时Western blotting分析结果显示表�
[ Objective ] Non structural protein （nsp） 3a and 3b genes of porcine transmissible gastroenteritis virus （TGEV） were cloned and expressed in eukaryotic cell. Expression of nsp 3a and 3b proteins and their influence on cell cycle were studied. [Method] Two pairs of primers used to amplify TGEV SX strain＇s nsp 3a and 3b genes were designed according to the archived TGEV strain nucleotide by Primer 5.0. TGEV SX strain＇s nsp 3a and 3b genes were cloned by RT-PCR, and ligated into eukaryotic expression vector pEGFP-N1, respectively. The recombinant plasmids were named as p3a-EGFP-N1 and p3b-EGFP-N1. The recombinant plasmids p3a-EGFP-N1 and p3b-EGFP-N1 were transfected into swine intestinal epithelial cells （IEC） using lipofectamine 2000. Expression of nsps 3a and 3b were confirmed by confocal microscopy. Transcription of TGEV 3a and 3b genes were analyzed by RT-PCR and the expression of nsp 3a and 3b was analyzed by Western blotting assay. The effects of proteins on cell cycle were investigated by flow cytometry assay. Transcription level of cyclin A and GRP78 were analyzed by qRT-PCR assay, and the expression level of GRP78, Cyclin A and Cyclin B 1 were analyzed by Western blotting assay. [ Result ] The DNA fragments of 3a gene （213bp） and 3b gene （732bp） of TGEV SX strain were cloned successfully. The 3a gene of SX strain shared 97.4%- 100% homology of nucleotide sequences and 98.6%-100% homology of amino acid. 3b gene shared 98.3%-99.9% homology of nucleotide sequences and 100% homology of amino acid with those of TGEV strains. Western blotting assay showed that 3a-GFP and 3b-GFP fusion proteins were in agreement with the predicted MW of 35kD and 54kD, respectively. The confocal microscopy analysis showed that 3a-GFP and 3b-GFP fusion proteins were distributed in the whole cell. Flow cytometry assay showed that the percentage of cells in G2/M phase was more than the control cells （IEC and IEC-GFP） in TGEV nsp 3b expression cells. The transcription level of Cyclin A was higher in 3b-GFP
出处
《中国农业科学》
CAS
CSCD
北大核心
2015年第2期352-361,共10页
Scientia Agricultura Sinica
基金
西北农林科技大学基本科研业务费专项资金项目资助计划(QN2012017)
