猪轮状病毒非结构蛋白NSP4亚细胞定位的研究.doc

文档介绍：
猪轮状病毒非结构蛋白 NSP4 亚细胞定位的
研究#
高婉俊,王静,林鸷,张彦明**
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(西北农林科技大学动物医学院,陕西杨凌 712100)
摘要:【目的】采用猪轮状病毒的靶细胞猪小肠黏膜上皮细胞,探究非结构蛋白 NSP4 在该
细胞的亚细胞定位情况。【方法】克隆出非结构蛋白 NSP4 编码区基因,双酶切之后将其连
入真核表达载体 pEGFP-N1 和 pEGFP-C1 上,构建了重组质粒 NSP4-EGFP 和 EGFP-NSP4,
对重组质粒进行 PCR、双酶切和测序鉴定。将内质网定位载体导入永生化猪小肠黏膜上皮
细胞,筛选出稳定表达的细胞株。将重组质粒导入稳定表达内质网定位载体的细胞株,在激
光共聚焦显微镜下观察,明确非结构蛋白 NSP4 的亚细胞定位。【结果】NSP4-EGFP 融合
蛋白定位于细胞的内质网以及内质网以外的细胞质,EGFP-NSP4 融合蛋白定位于细胞的内
质网和细胞核。【结论】明确了猪轮状病毒非结构蛋白 NSP4 在永生化猪小肠黏膜上皮细胞
中的定位情况。
关键词:猪轮状病毒;永生化猪小肠黏膜上皮细胞;非结构蛋白 NSP4;亚细胞定位
Study on the subcellular localization of porcine rotavirus
non-structural protein 4
Gao Wanjun, Wang Jing, Lin Zhi, Zhang Yanming
(Department of veterinary medicine, Northwest A&F University, ShaanXi YangLing 712100)
Abstract: 【Objective】Immortalized swine intestinal mucosal epithelial cells are target cells of
porcine rotavirus, the purpose of this study is to explore the subcellular localization of porcine
rotavirus non-structural protein 4 in this cells. 【Method】Porcine rotavirus non-structural protein
4 gene was cloned and double digested, then we connected it to eukaryotic expression vectors
pEGFP-N1 and pEGFP-C1 to construct the binant plasmids NSP4-EGFP and EGFP-NSP4.
The binant plasmids were identified by PCR, double digestion and sequencing. Endoplasmic
reticulum positioning carrier was transfected into immortalized swine intestinal mucosal epithelial
cells, then we established stable expression cell lines. The binant plasmids were transfected
into this cell lines, then we used confocal laser scanning microscopy to observe the subcellular
localization of porcine rotavirus non-structural protein 4 in cells. 【Result】NSP4-EGFP fusion
protein was located to endoplasmic reticulum and other cytoplasm, EGFP-NSP4 fusion protein
was located to endoplasmic reticulum and nucleus. 【Conclusion】We confirmed the subcellular
localization of porcine rotavirus non-structural protein 4 in immortalized swine intestinal mucosal
epithe