猪轮状病毒NSP4及其viroporin调控内质网应激和细胞凋亡的分子机制研究
发布时间：2018-08-14 18:59
【摘要】：轮状病毒(Rotavirus,RV)是引起幼龄动物和婴幼儿急性肠胃炎的一种常见病原体,特别是在发展中国家,每年可造成约45万名5岁以下儿童死亡。猪轮状病毒(Porcine rotavirus,PoRV)是引起仔猪病毒性腹泻的主要病原体之一,仔猪感染猪轮状病毒后迅速发生腹泻,严重者常常因脱水而死亡,对畜牧业的发展危害很大。RV非结构蛋白NSP4是一种病毒肠毒素,由175个氨基酸组成,NSP4片段(47-91 aa)具有viroporin的结构特征,NSP4及其viroporin在轮状病毒致病过程中发挥着重要作用。研究发现NSP4(47-91aa)突变体能在内质网形成一个跨膜的水孔,破坏膜的内稳态而增加胞质钙浓度。NSP4及其viroporin可能与轮状病毒诱导的内质网应激和细胞凋亡有关,对轮状病毒NSP4及其viroporin的深入研究,为进一步阐明PoRV致病机理奠定基础,具有重要的公共卫生意义。本研究用猪轮状病毒NSP4及其viroporin真核表达质粒转染细胞,首先检测凋亡因子的变化,确定其引起凋亡,然后检测了内质网应激因子的变化,以探究NSP4及viroporin与内质网应激和细胞凋亡之间的关系,具体研究结果如下:(1)RT-PCR扩增得到猪轮状病毒的NSP4基因和NSP4基因片段(47-91 aa),通过引物设计在它们C端引入Flag标签,并将其连接到pcDNA3.1(+)构建真核表达质粒pcDNA3.1-NSP4-Flag和pcDNA3.1-viroporin-Flag。双酶切鉴定和测序结果与预期结果一致,说明本研究成功构建了NSP4和NSP4片段(47-91 aa)的真核表达质粒。(2)将重组质粒NSP4-GFP和viroporin-GFP转染猪小肠上皮细胞(SIEC),通过荧光倒置显微镜观察和Western blot检测,结果表明NSP4和NSP4片段(47-91 aa)成功表达。DNA ladder是细胞凋亡的特征之一,抽提转染NSP4-GFP和viroporin-GFP的SIEC细胞的DNA,用10 g/L琼脂糖凝胶电泳分析,结果显示NSP4和NSP4片段(47-91aa)所在泳道出现DNA ladder现象,说明NSP4和NSP4片段(47-91 aa)能诱导SIEC细胞凋亡。为进一步确定NSP4和NSP4片段(47-91 aa)能调控哪些凋亡相关过程,通过荧光定量PCR分析了Bax和Bcl-2的表达,发现NSP4和NSP4片段(47-91 aa)能促进Bax的表达,抑制Bcl-2的表达。Western blot检测发现NSP4和NSP4片段(47-91aa)能促进caspase-3、caspase-8和caspase-9的切割,说明NSP4和NSP4片段(47-91 aa)能够诱导细胞的凋亡。(3)为进一步确定内质网应激是否参与了NSP4和NSP4片段(47-91 aa)诱导的细胞凋亡,检测了GRP78、CHOP、caspase-12、ATF6、ATF4和IERα等多种关键因子的表达。荧光定量PCR和Western blot检测发现NSP4和NSP4片段(47-91 aa)能在mRNA水平和蛋白水平上调GRP78和CHOP的表达。caspase-12、ATF6、ATF4和IERα常参与内质网介导的细胞凋亡,荧光定量PCR和Western blot检测发现NSP4和NSP4片段(47-91 aa)能上调caspase-12、ATF6、ATF4和IERα的表达。这些结果说明NSP4和NSP4片段(47-91 aa)能诱导内质网应激,并且能诱导内质网应激介导的细胞凋亡。综上所述,本研究成功构建了猪轮状病毒NSP4及其viroporin的带Flag标签的真核表达载体;通过实时荧光定量PCR和Western blot检测了NSP4及其viroporin对细胞凋亡的影响,并进一步研究发现该凋亡与内质网应激有密切关系,提示内质网应激也是NSP4诱导凋亡的途径之一,且viroporin有可能是其关键区域。本研究为轮状病毒致腹泻的机制研究提供了新的理论依据。
[Abstract]:Rotavirus (RV) is a common pathogen causing acute gastroenteritis in young animals and infants. Especially in developing countries, it can cause about 450 thousand children under 5 years of age to die each year. Porcine rotavirus (PoRV) is one of the main pathogens causing * * * virus diarrhea in piglets. Piglets infected with porcine rotavirus. RV nonstructural protein NSP4 is a viral enterotoxin consisting of 175 amino acids. NSP4 fragment (47-91 aa) has the structural characteristics of viroporin. NSP4 and its viroporin play an important role in the pathogenesis of rotavirus. (47-91aa) mutant can form a transmembrane pore in the endoplasmic reticulum, destroy the membrane homeostasis and increase the cytoplasmic calcium concentration. NSP4 and its viroporin may be related to rotavirus-induced endoplasmic reticulum stress and cell apoptosis. Further study on rotavirus NSP4 and its viroporin will lay a foundation for further elucidating the pathogenesis of PoRV. * in this study, we used porcine rotavirus NSP4 and viroporin eukaryotic expression plasmid to transfect cells. First, we detected the changes of apoptotic factors, determined the apoptosis, and then detected the changes of endoplasmic reticulum stress factors, so as to explore the relationship between NSP4 and viroporin and endoplasmic reticulum stress and apoptosis. *: (1) RT-PCR amplified the NSP4 gene and NSP4 gene fragment (47-91 AA) of porcine rotavirus, and introduced Flag tag at the C end by primer design, and connected it to pcDNA3.1 (+) to construct eukaryotic expression plasmid pcDNA3.1-NSP4-Flag and pcDNA3.1-viroporin-Flag. double enzyme digestion. The results of identification and sequencing were consistent with the expected results. The recombinant plasmid NSP4 and NSP4 fragment (47-91 AA) were constructed. (2) recombinant plasmid NSP4-GFP and viroporin-GFP were transfected into porcine intestinal epithelial cells (SIEC *), and observed by fluorescence inverted microscope and Western blot. The results showed that NSP4 and NSP4 fragments (47-91 AA) were expressed as one of the characteristics of apoptosis. DNA of SIEC cells with P4-GFP and viroporin-GFP was analyzed by 10 g/L agarose gel electrophoresis. The results showed that DNA ladder appeared in the swimming lanes of NSP4 and NSP4 fragments (47-91aa), indicating that NSP4 and NSP4 fragments (47-91aa) could induce apoptosis of SIEC cells. The expression of Bax and Bcl-2 was analyzed by photoquantitative PCR. NSP4 and NSP4 fragments (47-91 aa) could promote the expression of Bax and inhibit the expression of Bcl-2. Western blot analysis showed that NSP4 and NSP4 fragments (47-91 aa) could promote the cleavage of caspase-3, Caspase-8 and caspase-9, indicating that NSP4 and NSP4 fragments (47-91 aa) could induce cell apoptosis. The expression of GRP78, CHOP, caspase-12, ATF6, ATF4 and IERalpha was detected. Fluorescence quantitative PCR and Western blot analysis showed that NSP4 and NSP4 fragments (47-91 aa) could up-regulate the expression of GRP78 and CHOP at mRNA and protein levels. The expression of caspase-12, ATF6, ATF4 and IERalpha was up-regulated by NSP4 and NSP4 fragments (47-91 aa) by fluorescence quantitative PCR and Western blot. These results suggest that NSP4 and NSP4 fragments (47-91 aa) can induce ER stress and ER stress-mediated apoptosis. We have successfully constructed a Flag * tagged eukaryotic expression vector for porcine rotavirus NSP4 and its viroporin. The effects of NSP4 and its viroporin on apoptosis were detected by real-time fluorescence quantitative PCR and Western blot. Further studies showed that the apoptosis was closely related to endoplasmic reticulum stress, suggesting that ER stress is also NSP4. Viroporin may be one of the key apoptotic pathways. This study provides a new theoretical basis for the mechanism of rotavirus-induced diarrhea.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651
本文编号：2183816
[Abstract]:Rotavirus (RV) is a common pathogen causing acute gastroenteritis in young animals and infants. Especially in developing countries, it can cause about 450 thousand children under 5 years of age to die each year. Porcine rotavirus (PoRV) is one of the main pathogens causing * * * virus diarrhea in piglets. Piglets infected with porcine rotavirus. RV nonstructural protein NSP4 is a viral enterotoxin consisting of 175 amino acids. NSP4 fragment (47-91 aa) has the structural characteristics of viroporin. NSP4 and its viroporin play an important role in the pathogenesis of rotavirus. (47-91aa) mutant can form a transmembrane pore in the endoplasmic reticulum, destroy the membrane homeostasis and increase the cytoplasmic calcium concentration. NSP4 and its viroporin may be related to rotavirus-induced endoplasmic reticulum stress and cell apoptosis. Further study on rotavirus NSP4 and its viroporin will lay a foundation for further elucidating the pathogenesis of PoRV. * in this study, we used porcine rotavirus NSP4 and viroporin eukaryotic expression plasmid to transfect cells. First, we detected the changes of apoptotic factors, determined the apoptosis, and then detected the changes of endoplasmic reticulum stress factors, so as to explore the relationship between NSP4 and viroporin and endoplasmic reticulum stress and apoptosis. *: (1) RT-PCR amplified the NSP4 gene and NSP4 gene fragment (47-91 AA) of porcine rotavirus, and introduced Flag tag at the C end by primer design, and connected it to pcDNA3.1 (+) to construct eukaryotic expression plasmid pcDNA3.1-NSP4-Flag and pcDNA3.1-viroporin-Flag. double enzyme digestion. The results of identification and sequencing were consistent with the expected results. The recombinant plasmid NSP4 and NSP4 fragment (47-91 AA) were constructed. (2) recombinant plasmid NSP4-GFP and viroporin-GFP were transfected into porcine intestinal epithelial cells (SIEC *), and observed by fluorescence inverted microscope and Western blot. The results showed that NSP4 and NSP4 fragments (47-91 AA) were expressed as one of the characteristics of apoptosis. DNA of SIEC cells with P4-GFP and viroporin-GFP was analyzed by 10 g/L agarose gel electrophoresis. The results showed that DNA ladder appeared in the swimming lanes of NSP4 and NSP4 fragments (47-91aa), indicating that NSP4 and NSP4 fragments (47-91aa) could induce apoptosis of SIEC cells. The expression of Bax and Bcl-2 was analyzed by photoquantitative PCR. NSP4 and NSP4 fragments (47-91 aa) could promote the expression of Bax and inhibit the expression of Bcl-2. Western blot analysis showed that NSP4 and NSP4 fragments (47-91 aa) could promote the cleavage of caspase-3, Caspase-8 and caspase-9, indicating that NSP4 and NSP4 fragments (47-91 aa) could induce cell apoptosis. The expression of GRP78, CHOP, caspase-12, ATF6, ATF4 and IERalpha was detected. Fluorescence quantitative PCR and Western blot analysis showed that NSP4 and NSP4 fragments (47-91 aa) could up-regulate the expression of GRP78 and CHOP at mRNA and protein levels. The expression of caspase-12, ATF6, ATF4 and IERalpha was up-regulated by NSP4 and NSP4 fragments (47-91 aa) by fluorescence quantitative PCR and Western blot. These results suggest that NSP4 and NSP4 fragments (47-91 aa) can induce ER stress and ER stress-mediated apoptosis. We have successfully constructed a Flag * tagged eukaryotic expression vector for porcine rotavirus NSP4 and its viroporin. The effects of NSP4 and its viroporin on apoptosis were detected by real-time fluorescence quantitative PCR and Western blot. Further studies showed that the apoptosis was closely related to endoplasmic reticulum stress, suggesting that ER stress is also NSP4. Viroporin may be one of the key apoptotic pathways. This study provides a new theoretical basis for the mechanism of rotavirus-induced diarrhea.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.651
【参考文献】
相关期刊论文 前1条
1 周晗;乔薪媛;葛俊伟;李一经;;猪轮状病毒NSP4蛋白135、136和138位点氨基酸突变对毒力影响的研究[J];中国预防兽医学报;2013年02期
，本文编号：2183816
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