猪传染性胃肠炎病毒非结构蛋白3a 和3b 融合表达及对细胞周期的影响

摘要

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 B1 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-expressing cells compared with the control cells by qRT-PCR assay. At the same time, the protein level of Cyclin A was significantly increased and Cyclin B1 level was down-regulated in TGEV 3b-GFP expressing cells by western blot assay. TGEV nsp 3a had no influence on cell cycle. The mRNA level of GRP78 was higher in 3a-GFP expressing cells than the control cells by qRT-PCR assay, and the expression level of GRP78 was up-regulated by TGEV nsp 3a compared with the control cells by western blot assay. Moreover, TGEV nsp 3b had no influence on GRP78 expression in IEC.[Conclusion]The expressed nsp 3a and 3b of TGEV distributed in the whole transfected cells. Nsp 3a induced ER stress by up-regulated GRP78. Nsp 3b caused cell cycle arrested at G2/M phase by up-regulated Cyclin A and down-regulated Cyclin B1.%【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 pEGFP-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35kD 的3a-GFP和54kD 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 mRNA 水平高于对照组细胞（IEC 和 IEC-GFP），同时 Western blotting 分析结果显示表达3b 蛋白的细胞中 Cyclin A 蛋白水平表达量高于对照组，并且 Cyclin B1的表达量低于对照组细胞，差异显著；TGEV 非结构蛋白3a 对细胞周期没有影响。实时荧光定量 PCR 分析结果显示，表达 TGEV 非结构蛋白3a 的细胞中 GRP78的 mRNA 水平高于对照组， Western blotting 分析 GRP78的蛋白水平高于对照组，差异显著，说明非结构蛋白3a 可以使 GRP78表达水平的上调；TGEV 非结构蛋白3b 对 GRP78的表达没有影响。【结论】TGEV 陕西株的非结构蛋白3a 和3b 在 IEC 细胞中成功表达，3a 蛋白可以引起细胞内质网应激反应，3b 蛋白通过使细胞周期蛋白 Cyclin A 表达上调并且使 Cyclin B1表达下调引起细胞周期阻滞于 G2/M 期。