本文選題：豬繁殖與呼吸綜合征病毒 + GP5　； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：豬繁殖與呼吸綜合征(Porcine reproductive and respiratory syndrome,PRRS)是由豬繁殖與呼吸綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的豬的繁殖障礙和呼吸系統(tǒng)的傳染病,是目前嚴(yán)重危害養(yǎng)豬業(yè)的重要病毒性傳染病之一。PRRSV為有囊膜,單股正鏈RNA病毒,分類屬于尼多病毒目,動脈炎病毒科成員。PRRSV基因是由一條15kb的單股正鏈RNA構(gòu)成,包括9個開放閱讀框(ORF),分別編碼2個非結(jié)構(gòu)蛋白(Nsp1和Nsp2),和7個結(jié)構(gòu)蛋白(GP2a、E、GP3、GP4、GP5、M和N)。GP5作為主要結(jié)構(gòu)蛋白含有病毒重要的抗原中和表位,外源性免疫GP5蛋白或利用GP5 DNA疫苗內(nèi)源性的表達GP5蛋白可以有效抑制PRRSV對豬體的感染。然而,在實際PRRSV感染豬只過程中,GP5表達誘導(dǎo)中和抗體的產(chǎn)生卻發(fā)生了延遲或是被抑制。這表明,在病毒感染過程中GP5的功能受到了其它PRRSV的蛋白或宿主蛋白的修飾或干擾。為了排除其它PRRSV蛋白的干擾作用,明確GP5蛋白表達在病毒感染過程中的作用,本課題研究的內(nèi)容和結(jié)果如下:1.通過RT-PCR克隆PRRSV ORF5基因片段,利用overlap PCR擴增獲得帶有Flag標(biāo)簽的ORF5基因片段。構(gòu)建重組慢病毒表達載體pTRIP-GP5-Flag,將重組質(zhì)粒和包裝載體共轉(zhuǎn)染293T細胞獲得重組慢病毒。利用重組慢病毒轉(zhuǎn)導(dǎo)Marc-145細胞,通過嘌呤霉素和單克隆篩選,獲得到穩(wěn)定表達GP5Flag的細胞系。IFA和Western blot檢測結(jié)果證明GP5Flag在Marc-145細胞內(nèi)穩(wěn)定表達。2.利用CCK-8 Kit和Annexin V-FITC Apoptosis Detection Kit分別對篩選獲得細胞系進行細胞增殖和細胞凋亡檢測,分析GP5蛋白表達對Marc-145細胞活性的影響。檢測結(jié)果顯示,與對照組細胞相比,GP5蛋白在Marc-145細胞內(nèi)表達不影響Marc-145細胞的增殖,同時也未誘導(dǎo)細胞凋亡。3.為了分析GP5表達對PRRSV感染的影響,我們選擇三種不同的PRRSV毒株SD16,VR-2332和JXA1分別感染表達GP5的Marc-145細胞,結(jié)果顯示GP5的表達顯著抑制三種病毒在Marc-145細胞上的增殖,但并不影響PRRSV對Marc-145的吸附。4.I型干擾素基因水平檢測結(jié)果顯示,GP5蛋白的表達顯著提高了細胞IFN-β的產(chǎn)生和顯著增強了細胞內(nèi)IFN-β的啟動子活性。為了進一步證明GP5蛋白表達誘導(dǎo)IFN-β的產(chǎn)生從而抑制PRRSV的感染,設(shè)計合成針對IFN-β的siRNA,通過敲低細胞內(nèi)IFN-β的表達進而檢測PRRSV的感染變化,實驗結(jié)果顯示,當(dāng)GP5表達細胞IFN-βmRNA水平被敲低后,PRRSV對Marc-145細胞的感染可以得到顯著性恢復(fù)。綜上所述,為了探究GP5的表達對PRRSV感染的影響,本研究構(gòu)建了GP5穩(wěn)定表達的Marc-145細胞系,細胞活性測定和病毒感染實驗表明GP5蛋白的表達沒有影響細胞的活性但卻顯著抑制了病毒的感染,并且這種對病毒的抑制作用是由GP5蛋白誘導(dǎo)IFN-β產(chǎn)生導(dǎo)致的。該研究通過對PRRSV GP5蛋白表達對宿主的調(diào)控分析,明確了GP5蛋白的預(yù)表達不利于PRRSV的后期感染,為更加深入了解PRRSV的感染與致病機制提供新的思路。
[Abstract]:Porcine reproductive and respiratory syndrome (PRRSs) is a reproductive disorder and respiratory disease caused by porcine reproductive and respiratory syndrome virus. PRRSv is one of the most important viral infectious diseases which seriously endangers the pig industry at present. PRRSv is a envelope, single-stranded RNA virus, which belongs to the order Nidovirus. The member of arteritis virus family. PRRSv gene is composed of a single strand positive strand RNA of 15kb. Nine open reading frames (ORF) encoding two nonstructural proteins, Nsp1 and Nsp2, respectively, and seven structural proteins, GP2aEGP3, GP4, GP5Mand N).GP5, were used as major structural proteins to contain important antigen-neutralizing epitopes of the virus. Exogenous immunizing GP5 protein or endogenous expression of GP5 protein using GP5 DNA vaccine can effectively inhibit the infection of PRRSV to pig body. However, the production of neutralizing antibodies induced by GP5 expression was delayed or inhibited during actual PRRSV infection in pigs. This suggests that the function of GP5 is modified or interfered with by other PRRSV proteins or host proteins during viral infection. In order to eliminate the interference of other PRRSV proteins and clarify the role of GP5 protein expression in the process of virus infection, the contents and results of this study are as follows: 1. The PRRSV ORF5 gene fragment was cloned by RT-PCR, and the ORF5 gene fragment with Flag tag was obtained by overlap PCR amplification. The recombinant lentivirus expression vector pTRIP-GP5-Flag. was constructed and co-transfected into 293T cells to obtain the recombinant lentivirus. The recombinant lentivirus was used to transduction Marc-145 cells. By purine mycin and monoclonal screening, the stable expression of GP5Flag in Marc-145 cell lines. IFA and Western blot were obtained. The results showed that GP5Flag expressed stably in Marc-145 cells. CCK-8 Kit and Annexin V-FITC Apoptosis Detection Kit were used to detect the proliferation and apoptosis of the selected cell lines, and the effect of GP5 protein expression on the activity of Marc-145 cells was analyzed. The results showed that the expression of GP5 protein in Marc-145 cells did not affect the proliferation of Marc-145 cells, nor did it induce apoptosis. In order to analyze the effect of GP5 expression on PRRSV infection, we selected three different PRRSV strains SD16VR-2332 and JXA1 to infect Marc-145 cells expressing GP5, respectively. The results showed that the expression of GP5 significantly inhibited the proliferation of the three viruses on Marc-145 cells. The results showed that the expression of GP5 protein significantly increased the production of IFN- 尾 and the promoter activity of IFN- 尾. In order to further prove that the expression of GP5 protein induces the production of IFN- 尾 and thus inhibits the infection of PRRSV, we designed and synthesized siRNAs against IFN- 尾 and detected the changes of PRRSV infection by knocking down the expression of IFN- 尾 in cells. When the IFN- 尾 mRNA level of GP5 expression cells was knocked down, the infection of Marc-145 cells could recover significantly. In conclusion, in order to investigate the effect of GP5 expression on PRRSV infection, we constructed a Marc-145 cell line with stable expression of GP5. Cell activity test and virus infection test showed that the expression of GP5 protein did not affect the cell activity, but significantly inhibited the virus infection, and the inhibitory effect on the virus was caused by the production of IFN- 尾 induced by GP5 protein. Through the analysis of the regulation of PRRSV GP5 protein expression on the host, it is clear that the pre-expression of GP5 protein is not conducive to the late infection of PRRSV, which provides a new idea for further understanding the infection and pathogenesis of PRRSV.
【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】：S858.28

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