豬輪狀病毒NSP4及其viroporin調(diào)控內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞凋亡的分子機(jī)制研究
發(fā)布時(shí)間：2018-08-14 18:59
【摘要】：輪狀病毒(Rotavirus,RV)是引起幼齡動(dòng)物和嬰幼兒急性腸胃炎的一種常見病原體,特別是在發(fā)展中國家,每年可造成約45萬名5歲以下兒童死亡。豬輪狀病毒(Porcine rotavirus,PoRV)是引起仔豬病毒性腹瀉的主要病原體之一,仔豬感染豬輪狀病毒后迅速發(fā)生腹瀉,嚴(yán)重者常常因脫水而死亡,對畜牧業(yè)的發(fā)展危害很大。RV非結(jié)構(gòu)蛋白NSP4是一種病毒腸毒素,由175個(gè)氨基酸組成,NSP4片段(47-91 aa)具有viroporin的結(jié)構(gòu)特征,NSP4及其viroporin在輪狀病毒致病過程中發(fā)揮著重要作用。研究發(fā)現(xiàn)NSP4(47-91aa)突變體能在內(nèi)質(zhì)網(wǎng)形成一個(gè)跨膜的水孔,破壞膜的內(nèi)穩(wěn)態(tài)而增加胞質(zhì)鈣濃度。NSP4及其viroporin可能與輪狀病毒誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞凋亡有關(guān),對輪狀病毒NSP4及其viroporin的深入研究,為進(jìn)一步闡明PoRV致病機(jī)理奠定基礎(chǔ),具有重要的公共衛(wèi)生意義。本研究用豬輪狀病毒NSP4及其viroporin真核表達(dá)質(zhì)粒轉(zhuǎn)染細(xì)胞,首先檢測凋亡因子的變化,確定其引起凋亡,然后檢測了內(nèi)質(zhì)網(wǎng)應(yīng)激因子的變化,以探究NSP4及viroporin與內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞凋亡之間的關(guān)系,具體研究結(jié)果如下:(1)RT-PCR擴(kuò)增得到豬輪狀病毒的NSP4基因和NSP4基因片段(47-91 aa),通過引物設(shè)計(jì)在它們C端引入Flag標(biāo)簽,并將其連接到pcDNA3.1(+)構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.1-NSP4-Flag和pcDNA3.1-viroporin-Flag。雙酶切鑒定和測序結(jié)果與預(yù)期結(jié)果一致,說明本研究成功構(gòu)建了NSP4和NSP4片段(47-91 aa)的真核表達(dá)質(zhì)粒。(2)將重組質(zhì)粒NSP4-GFP和viroporin-GFP轉(zhuǎn)染豬小腸上皮細(xì)胞(SIEC),通過熒光倒置顯微鏡觀察和Western blot檢測,結(jié)果表明NSP4和NSP4片段(47-91 aa)成功表達(dá)。DNA ladder是細(xì)胞凋亡的特征之一,抽提轉(zhuǎn)染NSP4-GFP和viroporin-GFP的SIEC細(xì)胞的DNA,用10 g/L瓊脂糖凝膠電泳分析,結(jié)果顯示NSP4和NSP4片段(47-91aa)所在泳道出現(xiàn)DNA ladder現(xiàn)象,說明NSP4和NSP4片段(47-91 aa)能誘導(dǎo)SIEC細(xì)胞凋亡。為進(jìn)一步確定NSP4和NSP4片段(47-91 aa)能調(diào)控哪些凋亡相關(guān)過程,通過熒光定量PCR分析了Bax和Bcl-2的表達(dá),發(fā)現(xiàn)NSP4和NSP4片段(47-91 aa)能促進(jìn)Bax的表達(dá),抑制Bcl-2的表達(dá)。Western blot檢測發(fā)現(xiàn)NSP4和NSP4片段(47-91aa)能促進(jìn)caspase-3、caspase-8和caspase-9的切割,說明NSP4和NSP4片段(47-91 aa)能夠誘導(dǎo)細(xì)胞的凋亡。(3)為進(jìn)一步確定內(nèi)質(zhì)網(wǎng)應(yīng)激是否參與了NSP4和NSP4片段(47-91 aa)誘導(dǎo)的細(xì)胞凋亡,檢測了GRP78、CHOP、caspase-12、ATF6、ATF4和IERα等多種關(guān)鍵因子的表達(dá)。熒光定量PCR和Western blot檢測發(fā)現(xiàn)NSP4和NSP4片段(47-91 aa)能在mRNA水平和蛋白水平上調(diào)GRP78和CHOP的表達(dá)。caspase-12、ATF6、ATF4和IERα常參與內(nèi)質(zhì)網(wǎng)介導(dǎo)的細(xì)胞凋亡,熒光定量PCR和Western blot檢測發(fā)現(xiàn)NSP4和NSP4片段(47-91 aa)能上調(diào)caspase-12、ATF6、ATF4和IERα的表達(dá)。這些結(jié)果說明NSP4和NSP4片段(47-91 aa)能誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激,并且能誘導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激介導(dǎo)的細(xì)胞凋亡。綜上所述,本研究成功構(gòu)建了豬輪狀病毒NSP4及其viroporin的帶Flag標(biāo)簽的真核表達(dá)載體;通過實(shí)時(shí)熒光定量PCR和Western blot檢測了NSP4及其viroporin對細(xì)胞凋亡的影響,并進(jìn)一步研究發(fā)現(xiàn)該凋亡與內(nèi)質(zhì)網(wǎng)應(yīng)激有密切關(guān)系,提示內(nèi)質(zhì)網(wǎng)應(yīng)激也是NSP4誘導(dǎo)凋亡的途徑之一,且viroporin有可能是其關(guān)鍵區(qū)域。本研究為輪狀病毒致腹瀉的機(jī)制研究提供了新的理論依據(jù)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.651
本文編號：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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.651
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 周晗;喬薪媛;葛俊偉;李一經(jīng);;豬輪狀病毒NSP4蛋白135、136和138位點(diǎn)氨基酸突變對毒力影響的研究[J];中國預(yù)防獸醫(yī)學(xué)報(bào);2013年02期
，本文編號：2183816
本文鏈接：http://sikaile.net/yixuelunwen/dongwuyixue/2183816.html
最近更新
教材專著
