豬圓環(huán)病毒2型ORF5蛋白功能分析和ORF4蛋白拮抗細(xì)胞凋亡機(jī)制研究
發(fā)布時(shí)間：2018-07-20 18:20
【摘要】：豬圓環(huán)病毒2型(Porcine circovirus type 2,PCV2)是一種已知的能夠感染哺乳類動(dòng)物的最小病毒,是引起豬圓環(huán)病毒病(Porcine circovirus associated diseases,PCVAD)的病原體。PCV2是一種無囊膜病毒,屬于圓環(huán)病毒科(Circoviridae)的圓環(huán)病毒屬(Circovirus),其基因組是一種單鏈閉合環(huán)狀的雙義的不分節(jié)的DNA。該病毒曾經(jīng)被預(yù)測(cè)含有11個(gè)開放閱讀框,其中4個(gè)已經(jīng)被鑒定能夠編碼該病毒的蛋白。它們分別是ORF1編碼病毒的復(fù)制酶相關(guān)蛋白R(shí)ep和Rep',ORF2編碼病毒的衣殼蛋白Cap,ORF3編碼病毒的促凋亡蛋白ORF3蛋白以及ORF4編碼病毒的抗凋亡蛋白ORF4蛋白(盡管該蛋白的抗凋亡機(jī)制尚不清楚)。序列比對(duì)分析發(fā)現(xiàn)Hamel等曾預(yù)測(cè)的ORF5基因(nt1016-1177)并不存在,這是因?yàn)樵摷俣ǖ牡鞍自谝恍㏄CV2中國分離株中翻譯至第7個(gè)氨基酸就提前終止了。然而,許多學(xué)者在對(duì)不同PCV2毒株序列比較時(shí)發(fā)現(xiàn)該病毒的基因組中有一段基因(nt553-732)高度保守,推測(cè)其很可能編碼病毒的某個(gè)蛋白,甚至還各自為這個(gè)未知蛋白起了名字(如基因序列號(hào)O92288、Q77GS3、Q77S04等)。為了探究該蛋白(本文將其命名為ORF5蛋白)存在與否,其所編碼產(chǎn)物的性質(zhì)和功能,以及ORF4蛋白拮抗細(xì)胞凋亡的分子機(jī)制,開展了研究工作,獲得了以下結(jié)果:(1)基因與蛋白水平驗(yàn)證PCV2 ORF5蛋白的存在。PCV2楊凌株感染豬肺泡巨噬細(xì)胞3D4/2(Porcine alveolar macrophages,PAMs)后,通過RT-PCR和Northern blot的檢測(cè)發(fā)現(xiàn),ORF5基因能夠在PCV2復(fù)制過程中轉(zhuǎn)錄,其轉(zhuǎn)錄本約為180 bp,完全鑲嵌在ORF1的內(nèi)部且與ORF1轉(zhuǎn)錄方向一致。此外,借助制備的鼠抗ORF5蛋白多克隆抗體(包括利用GST-ORF5融合蛋白、化學(xué)合成法得到的ORF5抗原表位多肽以及真核表達(dá)質(zhì)粒pcDNA-ORF5為抗原免疫Balb/c小鼠制備的多克隆抗體),可在ORF5基因轉(zhuǎn)染的PAM中檢測(cè)到Western blot信號(hào),也可在PCV2感染的PAM中檢測(cè)到IFA信號(hào),說明PCV2感染過程中存在ORF5基因編碼的病毒蛋白,即ORF5蛋白。盡管如此,ORF5蛋白在翻譯水平的表達(dá)還需要進(jìn)一步驗(yàn)證,畢竟Western blot未能檢測(cè)到特異性O(shè)RF5蛋白條帶。研究結(jié)果分別從基因與蛋白水平初步驗(yàn)證了PCV2 ORF5基因的轉(zhuǎn)錄和翻譯,為后續(xù)該蛋白性質(zhì)和功能的研究奠定了基礎(chǔ)。(2)PCV2 ORF5蛋白功能的研究。為了分析PCV2 ORF5蛋白的功能,構(gòu)建了缺失ORF5蛋白的PCV2感染性克隆(PCV2Δ),經(jīng)過對(duì)其復(fù)制動(dòng)力學(xué)分析發(fā)現(xiàn)ORF5蛋白并不是pcv2復(fù)制所必需的病毒蛋白。利用拯救出的orf5蛋白缺失體病毒pcv2Δ,重組型病毒rpcv2以及野生型病毒wpcv2分別感染pam后用實(shí)時(shí)熒光定量pcr方法(rt-qpcr)分析orf1、orf2、orf3、orf4和orf5mrna的表達(dá)曲線,結(jié)果發(fā)現(xiàn)orf5基因在病毒感染細(xì)胞后約48h表達(dá)量最高,而且起始密碼子的突變并不影響orf5基因的轉(zhuǎn)錄,提示orf5基因轉(zhuǎn)錄的起始位點(diǎn)可能位于更上游的位置。此外,pcv2Δ感染后的orf1和orf2mrna表達(dá)量較wpcv2和rpcv2都有顯著下降趨勢(shì),表明orf5蛋白的缺失可能會(huì)影響病毒orf1和orf2基因的轉(zhuǎn)錄進(jìn)程。為了進(jìn)一步分析orf5蛋白在pcv2感染過程中的作用,構(gòu)建了orf5蛋白的真核表達(dá)載體pegfp-orf5,轉(zhuǎn)染pam細(xì)胞后檢測(cè)該蛋白對(duì)pam細(xì)胞生理功能的影響,結(jié)果發(fā)現(xiàn)gfp標(biāo)記的orf5蛋白能夠通過蛋白酶體途徑被降解,能夠抑制pam細(xì)胞的增殖,延長pam細(xì)胞周期的s期,定位在內(nèi)質(zhì)網(wǎng)并引起細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激,激活nf-κb并上調(diào)其下游基因的表達(dá)。但是,由于gfp-orf5蛋白的功能可能并不能完全代表orf5蛋白自身的功能,因此有關(guān)orf5蛋白功能的描述還需要進(jìn)一步的證實(shí)。(3)pcv2orf4和orf5蛋白胞內(nèi)互作蛋白的篩選。為了篩選pcv2orf4和orf5這兩個(gè)新型病毒蛋白在宿主細(xì)胞內(nèi)的互作蛋白,構(gòu)建了豬肺泡巨噬細(xì)胞的cdna文庫,分別利用pcv2orf4和orf5蛋白為誘餌蛋白通過酵母雙雜交的方法篩選出了與pcv2orf4蛋白互作的4個(gè)蛋白(fhc,snrpn,cox8a和laminc)和與pcv2orf5蛋白互作的5個(gè)蛋白(gpnmb,cyp1a1,ywhab,znf511和srsf3)。(4)pcv2orf4蛋白拮抗細(xì)胞凋亡機(jī)制的研究。pcv2orf4蛋白曾被推測(cè)為抗凋亡蛋白,但迄今為止關(guān)于該蛋白抗凋亡的具體機(jī)制尚不明確。為了澄清這一問題,選擇上述酵母雙雜交結(jié)果中的重鏈鐵蛋白(fhc)為出發(fā)點(diǎn),先通過gstpull-down和co-ip分別在細(xì)胞外和細(xì)胞內(nèi)驗(yàn)證orf4蛋白與fhc能夠相互結(jié)合,后又用激光共聚焦技術(shù)在細(xì)胞水平證明orf4蛋白與fhc蛋白在hek293t細(xì)胞中存在共定位現(xiàn)象,表明pcv2orf4蛋白與宿主fhc蛋白存在相互作用�？紤]到fhc具有抗凋亡的作用且該作用具有劑量依賴性,首先用過表達(dá)(lv-fhc)和基因干擾(sh-fhc)的方法證實(shí)fhc參與了pcv2誘導(dǎo)的凋亡,然后用過表達(dá)(gfp-orf4)和基因缺失(感染性克隆pcv2Δ)的方法證實(shí)orf4蛋白雖然不能影響宿主細(xì)胞fhc基因的轉(zhuǎn)錄,但卻能夠通過某種方式的蛋白修飾降低細(xì)胞內(nèi)具有生物活性的fhc蛋白的濃度,提示pcv2orf4蛋白很可能通過穩(wěn)定宿主細(xì)胞內(nèi)fhc蛋白水平的恒定而發(fā)揮抗凋亡的作用。綜上所述,本研究初步證實(shí)了pcv2orf5蛋白是病毒復(fù)制的非必需蛋白,但是gfp標(biāo)記的orf5蛋白卻能夠通過延長細(xì)胞周期的s期而抑制細(xì)胞的增殖,能夠定位在內(nèi)質(zhì)網(wǎng)并且可誘導(dǎo)細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激,能夠激活nf-κb并上調(diào)其下游基因;orf5蛋白可與細(xì)胞內(nèi)的gpnmb、cyp1a1、ywhab、znf511和srsf3互作。orf4蛋白能夠與細(xì)胞內(nèi)的fhc、snrpn、cox8a和laminc互作,其中orf4蛋白能夠通過與宿主fhc互作而維持細(xì)胞內(nèi)FHC蛋白水平的恒定,從而發(fā)揮拮抗細(xì)胞凋亡的作用,為病毒的早期復(fù)制提供幫助。
[Abstract]:The porcine circovirus type 2 (Porcine circovirus type 2, PCV2) is the smallest virus that is known to be able to infect mammals, and is the pathogen that causes the swine Circovirus Disease (Porcine circovirus associated diseases, PCVAD).PCV2 is a non capsula virus, belonging to the cycovirus family (Circoviridae) of the genus circovirus (Circovirus), its base. The DNA. virus has been predicted to contain 11 open reading frames, 4 of which have been identified as the proteins that can encode the virus. They are replicin related proteins Rep and Rep'of the ORF1 encoding virus, the capsid protein Cap of the ORF2 encoding virus, and the apoptosis promoting of the ORF3 encoded virus. Protein ORF3 protein and ORF4 encoding the anti apoptotic protein ORF4 protein of the virus (although the anti apoptotic mechanism of the protein is not clear). Sequence alignment analysis found that the ORF5 gene (nt1016-1177), which was predicted by Hamel, did not exist, because the postulate protein was translated to seventh amino acids in some PCV2 Chinese isolates and terminated in advance. However, many scholars have found that one gene (nt553-732) in the genome of the virus is highly conserved when comparing the sequence of different PCV2 strains. It is presumed that it is likely to encode a certain protein of the virus, or even the name of the unknown protein (such as the gene sequence number O92288, Q77GS3, Q77S04, etc.). The properties and functions of the encoded products, as well as the molecular mechanism of ORF4 protein antagonism to cell apoptosis, were named as ORF5 protein. The following results were obtained: (1) gene and protein level verified the presence of PCV2 ORF5 protein in.PCV2 Yangling strain of 3D4/2 (Porcine alveolar macrophages, PAMs) in porcine alveolar macrophages. After the detection of RT-PCR and Northern blot, it was found that the ORF5 gene could be transcribed during the PCV2 replication process, and its transcript was about 180 BP, completely inlaid in the interior of ORF1 and in the same direction as ORF1. In addition, the mouse anti ORF5 protein polyclonal antibody (including the ORF5 antigen table by the use of GST-ORF5 fusion protein and chemical synthesis) The polyclonal and eukaryotic expression plasmid pcDNA-ORF5 is the polyclonal antibody prepared by the antigen immunized Balb/c mice. The Western blot signal can be detected in the PAM transfected ORF5 gene, and the IFA signal can be detected in the PAM infected by PCV2, indicating that the ORF5 gene coding virus protein, that is, the ORF5 protein, is found in the PCV2 infection. The expression of white at the level of translation needs further verification. After all, Western blot failed to detect the specific ORF5 protein bands. The results of the study have preliminarily verified the transcription and translation of the PCV2 ORF5 gene from the gene and protein levels, and laid the foundation for the study of the properties and functions of the protein. (2) the study of the function of the PCV2 ORF5 protein. The function of the PCV2 ORF5 protein was analyzed, and the PCV2 infected clone (PCV2 delta) missing ORF5 protein was constructed. After the analysis of its replication kinetics, it was found that ORF5 protein was not the necessary viral protein for PCV2 replication. Using the saved ORF5 protein deletion somatic virus PCV2 Delta, the recombinant virus rpcv2 and the wild type virus wpcv2 infected PAM. The expression curves of ORF1, ORF2, ORF3, ORF4 and orf5mrna were analyzed by real time fluorescence quantitative PCR method (RT-qPCR). The results showed that the ORF5 gene expressed the highest 48h expression after the virus infected cells, and the mutation of the starting codon did not affect the transcription of the ORF5 gene, suggesting that the starting site of the ORF5 gene transcription may be located in a more upstream position. Besides, PCV. The expression of ORF1 and orf2mrna after 2 delta infection was significantly lower than that of wpcv2 and rpcv2, indicating that the deletion of ORF5 protein may affect the transcriptional process of the ORF1 and ORF2 genes. In order to further analyze the role of ORF5 protein in the PCV2 infection process, the eukaryotic expression carrier of the ORF5 protein was constructed, and then the PAM cells were detected. The effect of the protein on the physiological function of PAM cells shows that the GFP labeled ORF5 protein can be degraded through the proteasome pathway, which can inhibit the proliferation of PAM cells, prolong the S phase of the PAM cell cycle, locate the endoplasmic reticulum and induce the endoplasmic reticulum stress, activate the nf- kappa B and regulate the expression of the downstream genes. However, due to the gfp-orf5 protein. The function may not fully represent the function of ORF5 protein itself, so the description of the function of ORF5 protein needs further confirmation. (3) screening of intercellular proteins of pcv2orf4 and ORF5 proteins. In order to screen the interacting proteins of the two new viral proteins of pcv2orf4 and ORF5 in the host cells, the porcine alveolar macrophages were constructed. The cDNA library, using pcv2orf4 and ORF5 protein as bait protein, screened 4 proteins interacting with pcv2orf4 protein (FHC, SNRPN, cox8a and laminc) and the 5 proteins interacting with pcv2orf5 protein (gpnmb, CYP1A1, ywhab, laminc and pcv2orf5). (4) study on the mechanism of apoptosis in antagonistic cells. 4 protein has been presumed to be an anti apoptotic protein, but so far the specific mechanism about the anti apoptosis of the protein is not clear. In order to clarify this problem, we choose the heavy chain ferritin (FHC) in the results of the yeast two hybrid results as the starting point. First, gstpull-down and co-Ip are used to verify that the ORF4 protein can interact with FHC in the fine cell and in the cells respectively. In addition, the co localization of ORF4 protein and FHC protein in HEK293T cells was demonstrated by laser confocal technique at the cell level, indicating the interaction between the pcv2orf4 protein and the host FHC protein. Considering the anti apoptosis effect of FHC and the dose dependent effect, the first use of expression (lv-fhc) and gene interference (sh-fhc) The method confirmed that FHC was involved in PCV2 induced apoptosis, and then using the method of overexpression (gfp-orf4) and gene deletion (infectious clone PCV2 delta), it was proved that ORF4 protein could not affect the FHC gene transcription of the host cell, but it could reduce the concentration of the bioactive FHC protein in the cell by some kind of protein modification, suggesting pcv2orf4. Proteins are likely to play an anti apoptotic role by stabilizing the FHC protein level in the host cells. To sum up, this study has preliminarily confirmed that the pcv2orf5 protein is a non essential protein for viral replication, but the GFP labeled ORF5 protein can inhibit cell proliferation by prolonging the S phase of the cell cycle and can be located in the endoplasmic reticulum and can be located in the endoplasmic reticulum. And it can induce endoplasmic reticulum stress to activate nf- kappa B and up-regulate its downstream genes; ORF5 protein can interact with gpnmb, CYP1A1, ywhab, znf511 and srsf3 in cells to interact with FHC, SNRPN, cox8a, and srsf3 in cells. It can play a role in antagonizing cell apoptosis and provide help for early replication of the virus.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65
，
本文編號(hào)：2134359
[Abstract]:The porcine circovirus type 2 (Porcine circovirus type 2, PCV2) is the smallest virus that is known to be able to infect mammals, and is the pathogen that causes the swine Circovirus Disease (Porcine circovirus associated diseases, PCVAD).PCV2 is a non capsula virus, belonging to the cycovirus family (Circoviridae) of the genus circovirus (Circovirus), its base. The DNA. virus has been predicted to contain 11 open reading frames, 4 of which have been identified as the proteins that can encode the virus. They are replicin related proteins Rep and Rep'of the ORF1 encoding virus, the capsid protein Cap of the ORF2 encoding virus, and the apoptosis promoting of the ORF3 encoded virus. Protein ORF3 protein and ORF4 encoding the anti apoptotic protein ORF4 protein of the virus (although the anti apoptotic mechanism of the protein is not clear). Sequence alignment analysis found that the ORF5 gene (nt1016-1177), which was predicted by Hamel, did not exist, because the postulate protein was translated to seventh amino acids in some PCV2 Chinese isolates and terminated in advance. However, many scholars have found that one gene (nt553-732) in the genome of the virus is highly conserved when comparing the sequence of different PCV2 strains. It is presumed that it is likely to encode a certain protein of the virus, or even the name of the unknown protein (such as the gene sequence number O92288, Q77GS3, Q77S04, etc.). The properties and functions of the encoded products, as well as the molecular mechanism of ORF4 protein antagonism to cell apoptosis, were named as ORF5 protein. The following results were obtained: (1) gene and protein level verified the presence of PCV2 ORF5 protein in.PCV2 Yangling strain of 3D4/2 (Porcine alveolar macrophages, PAMs) in porcine alveolar macrophages. After the detection of RT-PCR and Northern blot, it was found that the ORF5 gene could be transcribed during the PCV2 replication process, and its transcript was about 180 BP, completely inlaid in the interior of ORF1 and in the same direction as ORF1. In addition, the mouse anti ORF5 protein polyclonal antibody (including the ORF5 antigen table by the use of GST-ORF5 fusion protein and chemical synthesis) The polyclonal and eukaryotic expression plasmid pcDNA-ORF5 is the polyclonal antibody prepared by the antigen immunized Balb/c mice. The Western blot signal can be detected in the PAM transfected ORF5 gene, and the IFA signal can be detected in the PAM infected by PCV2, indicating that the ORF5 gene coding virus protein, that is, the ORF5 protein, is found in the PCV2 infection. The expression of white at the level of translation needs further verification. After all, Western blot failed to detect the specific ORF5 protein bands. The results of the study have preliminarily verified the transcription and translation of the PCV2 ORF5 gene from the gene and protein levels, and laid the foundation for the study of the properties and functions of the protein. (2) the study of the function of the PCV2 ORF5 protein. The function of the PCV2 ORF5 protein was analyzed, and the PCV2 infected clone (PCV2 delta) missing ORF5 protein was constructed. After the analysis of its replication kinetics, it was found that ORF5 protein was not the necessary viral protein for PCV2 replication. Using the saved ORF5 protein deletion somatic virus PCV2 Delta, the recombinant virus rpcv2 and the wild type virus wpcv2 infected PAM. The expression curves of ORF1, ORF2, ORF3, ORF4 and orf5mrna were analyzed by real time fluorescence quantitative PCR method (RT-qPCR). The results showed that the ORF5 gene expressed the highest 48h expression after the virus infected cells, and the mutation of the starting codon did not affect the transcription of the ORF5 gene, suggesting that the starting site of the ORF5 gene transcription may be located in a more upstream position. Besides, PCV. The expression of ORF1 and orf2mrna after 2 delta infection was significantly lower than that of wpcv2 and rpcv2, indicating that the deletion of ORF5 protein may affect the transcriptional process of the ORF1 and ORF2 genes. In order to further analyze the role of ORF5 protein in the PCV2 infection process, the eukaryotic expression carrier of the ORF5 protein was constructed, and then the PAM cells were detected. The effect of the protein on the physiological function of PAM cells shows that the GFP labeled ORF5 protein can be degraded through the proteasome pathway, which can inhibit the proliferation of PAM cells, prolong the S phase of the PAM cell cycle, locate the endoplasmic reticulum and induce the endoplasmic reticulum stress, activate the nf- kappa B and regulate the expression of the downstream genes. However, due to the gfp-orf5 protein. The function may not fully represent the function of ORF5 protein itself, so the description of the function of ORF5 protein needs further confirmation. (3) screening of intercellular proteins of pcv2orf4 and ORF5 proteins. In order to screen the interacting proteins of the two new viral proteins of pcv2orf4 and ORF5 in the host cells, the porcine alveolar macrophages were constructed. The cDNA library, using pcv2orf4 and ORF5 protein as bait protein, screened 4 proteins interacting with pcv2orf4 protein (FHC, SNRPN, cox8a and laminc) and the 5 proteins interacting with pcv2orf5 protein (gpnmb, CYP1A1, ywhab, laminc and pcv2orf5). (4) study on the mechanism of apoptosis in antagonistic cells. 4 protein has been presumed to be an anti apoptotic protein, but so far the specific mechanism about the anti apoptosis of the protein is not clear. In order to clarify this problem, we choose the heavy chain ferritin (FHC) in the results of the yeast two hybrid results as the starting point. First, gstpull-down and co-Ip are used to verify that the ORF4 protein can interact with FHC in the fine cell and in the cells respectively. In addition, the co localization of ORF4 protein and FHC protein in HEK293T cells was demonstrated by laser confocal technique at the cell level, indicating the interaction between the pcv2orf4 protein and the host FHC protein. Considering the anti apoptosis effect of FHC and the dose dependent effect, the first use of expression (lv-fhc) and gene interference (sh-fhc) The method confirmed that FHC was involved in PCV2 induced apoptosis, and then using the method of overexpression (gfp-orf4) and gene deletion (infectious clone PCV2 delta), it was proved that ORF4 protein could not affect the FHC gene transcription of the host cell, but it could reduce the concentration of the bioactive FHC protein in the cell by some kind of protein modification, suggesting pcv2orf4. Proteins are likely to play an anti apoptotic role by stabilizing the FHC protein level in the host cells. To sum up, this study has preliminarily confirmed that the pcv2orf5 protein is a non essential protein for viral replication, but the GFP labeled ORF5 protein can inhibit cell proliferation by prolonging the S phase of the cell cycle and can be located in the endoplasmic reticulum and can be located in the endoplasmic reticulum. And it can induce endoplasmic reticulum stress to activate nf- kappa B and up-regulate its downstream genes; ORF5 protein can interact with gpnmb, CYP1A1, ywhab, znf511 and srsf3 in cells to interact with FHC, SNRPN, cox8a, and srsf3 in cells. It can play a role in antagonizing cell apoptosis and provide help for early replication of the virus.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.65
，
本文編號(hào)：2134359
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