本文選題：豬繁殖與呼吸障礙綜合征病毒(PRRSV)　切入點(diǎn)：nsp10　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：豬繁殖與呼吸障礙綜合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)屬于動(dòng)脈炎病毒科動(dòng)脈炎病毒屬成員,基因組為單股正鏈RNA,大小約為15kb,含有9個(gè)開放閱讀框。主要引起妊娠母豬繁殖障礙和仔豬呼吸道疾病。豬繁殖與呼吸障礙綜合征是一種病毒性傳染病,已在全世界流行,給養(yǎng)豬業(yè)帶來嚴(yán)重?fù)p失。目前為止,該病沒有特異性治療方法且疫苗防治效果不理想。只有進(jìn)一步研究病毒復(fù)制機(jī)理及其相關(guān)蛋白功能,尋找針對(duì)PRRSV特異性預(yù)防與治療策略,對(duì)于控制該病發(fā)生,減少養(yǎng)豬業(yè)的經(jīng)濟(jì)損失具有重要意義。PRRSV nsp10蛋白為一種多功能蛋白,由N端的鋅結(jié)合結(jié)構(gòu)域(Zinc-binding domain,ZBD)和C端的超家族1解螺旋酶域組成,具有結(jié)合活性和解旋酶活性。ZBD由一個(gè)a-螺旋和兩個(gè)反平行的β-折疊共三個(gè)肽段組成,a-螺旋能鑲嵌于DNA的大溝中,從而結(jié)合DNA或者RNA。本實(shí)驗(yàn)主要對(duì)PRRSV nsp10蛋白解旋活性與結(jié)合活性進(jìn)行了研究,在細(xì)胞水平上進(jìn)行抗病毒藥物的篩選,為該病毒的致病機(jī)理研究及藥物預(yù)防與治療提供參考。主要工作如下:1.Nsp10蛋白解旋方向及其底物突出端最小長(zhǎng)度的檢測(cè)利用非變性聚丙烯酰胺凝膠實(shí)驗(yàn)方法驗(yàn)證nsp10的解旋活性,測(cè)定蛋白的解旋方向以及底物突出端所需的最小長(zhǎng)度。實(shí)驗(yàn)證明nsp10具有5’端到3’端解旋活性,底物5’突出端的長(zhǎng)度應(yīng)大于5bp,才能保證解旋反應(yīng)的正常進(jìn)行。2.Nsp10蛋白的ss DNA及ds DNA結(jié)合活性檢測(cè)Nsp10蛋白N-端存在鋅結(jié)合結(jié)構(gòu)域,可以與DNA相結(jié)合,因此利用凝膠阻滯實(shí)驗(yàn)檢測(cè)了蛋白核酸結(jié)合活性,結(jié)果表明,nsp10具有非特異性的ss DNA與ds DNA結(jié)合活性,且不需要ATP的參與。3.鋅結(jié)合結(jié)構(gòu)域突變體蛋白的表達(dá)純化及活性檢測(cè)通過對(duì)動(dòng)脈炎病毒科動(dòng)脈炎病毒屬四種(EAV、LDV、SHFV、PRRSV)病毒ZBD區(qū)域的對(duì)比,以及根據(jù)EAV晶體結(jié)構(gòu)的分析,選擇6個(gè)氨基酸進(jìn)行突變,結(jié)果顯示C10、C25、H28、H32可溶性表達(dá)發(fā)生變化,嘗試不同表達(dá)載體p ET-28a-SUMO、p GEX6p-1進(jìn)行摸索,以獲得可溶性表達(dá)。突變體蛋白在相同濃度下,檢測(cè)其解旋活性以及結(jié)合活性,C25、H32位活性消失,為ZBD的關(guān)鍵位點(diǎn)。4.化合物庫的高通量篩選在細(xì)胞水平上建立了抗PRRSV高通量篩選方法,最終確定Marc-145細(xì)胞最適的接板密度5000 cells/孔、檢測(cè)時(shí)間120h以及0.01個(gè)MOIs病毒的感染劑量。并且在滿足S/B5、Z’≥0.5和CV10%條件下對(duì)實(shí)驗(yàn)室的LOPAC1280庫進(jìn)行篩選,得到了抑制率大于60%的6種化合物,分析復(fù)篩結(jié)果,考慮到臨床實(shí)際應(yīng)用,選擇其中兩種A1895、D8555進(jìn)行后續(xù)實(shí)驗(yàn)。5.抗PRRSV化合物的檢測(cè)A1895、D8555具有價(jià)格便宜,抑制率高的優(yōu)點(diǎn),通過Western blot和間接免疫熒光實(shí)驗(yàn)檢測(cè)兩種化合物的抑制效果,結(jié)果顯示,兩種藥物的抗病毒效果良好。A1895為DNA拓?fù)洚悩?gòu)酶ⅱ抑制劑,D8555為高親和力配體神經(jīng)膠質(zhì)線粒體苯二氮受體,是一種抗焦慮藥。
[Abstract]:Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) belongs to the Arteriviridae arterivirus member genome is a single strand RNA, about 15KB in size, containing 9 open reading frames. The main cause reproductive failure in pregnant sows and piglets respiratory diseases. Porcine reproductive and respiratory disorders in general syndrome is a viral infectious disease, which has been popular in the world, caused serious losses to the pig industry. So far, the disease has no specific treatment and vaccine prevention effect is not ideal. Only further research on the function mechanism of virus replication and related protein, in order to find PRRSV specific prevention and treatment strategies for the control of the disease occurrence, reduce economic losses in pig industry has important significance of.PRRSV Nsp10 protein is a multifunctional protein, from the N side of the zinc binding domain (Zinc-binding domain, ZB D) and C terminal superfamily 1 helicase domain, the binding activity and helicase activity of.ZBD consists of a a- helix and two antiparallel beta sheet total of three peptides, a- can be embedded in the major groove of spiral DNA, which combined with DNA or RNA. in the experiment of PRRSV Nsp10 protein Jiexuan activity and binding activity were studied. The screening of antiviral drugs at the cellular level, and provide a reference for the study of pathogenic mechanism and drug prevention and treatment of the virus. The main work is as follows: the activity of 1.Nsp10 protein solution solution spin spin direction and the minimum length of substrate protruding end detection using non denaturing polyacrylamide Nsp10 verification the gel test method, determination of the minimum length of protein unwinding direction and substrate protruding end required. Experiments show that Nsp10 is the 5 'end to the 3' end of helicase activity, substrate 5 'overhang length should be greater than 5bp, in order to ensure Unwinding reaction of normal SS DNA and DS DNA protein.2.Nsp10 binding activity of Nsp10 protein was detected at N- zinc binding domain, can be combined with DNA, so using gel retardation assay of protein nucleic acid binding activity, the results show that the binding activity of Nsp10 with nonspecific SS DNA and DS DNA, and do not need to ATP.3. in the zinc binding domain mutant expression purification and activity detection of proteins by Arteriviridae arteriviruses genera and four species (EAV, LDV, SHFV, PRRSV) of viral ZBD region, and according to the analysis of the crystal structure of EAV, 6 amino acid mutations, the results showed that C10, C25, H28. The expression changes of soluble H32, try a different expression vector of P ET-28a-SUMO and P GEX6p-1 were explored to obtain soluble expression of mutant protein. At the same concentration, detect the unwinding activity and binding activity, C25, H32 An activity disappeared for high-throughput key sites of.4. ZBD compound library screening at the cellular level established for anti PRRSV high-throughput screening methods, and ultimately determine the optimal Marc-145 cell plate density 5000 cells/ hole infection dose detection time 120h and 0.01 MOIs virus. And in the LOPAC1280 library to meet S/B5. Laboratory Z '= 0.5 and CV10% under the condition of 6 kinds of compounds were screened, the inhibition rate was higher than 60% were obtained. The analysis of screening results, considering the clinical application, select one of two kinds of A1895, A1895 D8555 detection for subsequent experiments of.5. anti PRRSV compounds, D8555 has the advantages of low price, restrain the advantage of high efficiency. The inhibitory effect of Western, blot and indirect immunofluorescence assay showed that two compounds, two kinds of drugs for good antiviral effect of DNA topoisomerase inhibitors.A1895, D8555 for high affinity ligands The neuroglia mitochondrial two nitrogen receptor is an anti anxiety drug.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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