本文選題：鴨坦布蘇病毒 + E蛋白��； 參考：《安徽農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鴨坦布蘇病毒病(Duck Tembusu virus disease)是由鴨坦布蘇病毒(Duck Tembusu virus,DTMUV)引起的以成年蛋鴨產(chǎn)蛋下降,生長發(fā)育遲緩并可導(dǎo)致雛鴨死亡的一種傳染病。免疫接種是預(yù)防與控制該病的關(guān)鍵性措施之一。本研究利用桿狀病毒-昆蟲細(xì)胞表達(dá)系統(tǒng)表達(dá)DTMUV的重要免疫原基因(E基因),并對(duì)表達(dá)的重組E蛋白進(jìn)行了免疫原性初步研究。首先,根據(jù)DTMUV AH-F10株的E基因全基因序列設(shè)計(jì)引物,以含有DTMUV AH-F10株E基因的pMD19-E質(zhì)粒為模板擴(kuò)增E基因,并將其插入桿狀病毒骨架載體:pFastbac載體中,再將鑒定正確的重組質(zhì)粒pFastbac-E轉(zhuǎn)入DH10Bac感受態(tài)細(xì)胞,經(jīng)過三次藍(lán)白斑篩選獲得含有重組質(zhì)粒rBacmid-E的白色單克隆菌落,提取重組質(zhì)粒并用PCR鑒定正確后,使用Cellfection轉(zhuǎn)染sf9細(xì)胞,獲得P1代重組桿狀病毒rvBac-E,經(jīng)PCR鑒定正確后,在sf9細(xì)胞中傳代至P4代,測(cè)得P4代rvBac-E的TCID50為10-5.5/0.1mL。分別用Western blot和IFA鑒定重組桿狀病毒,結(jié)果均證明,所構(gòu)建的rvBac-E可在sf9細(xì)胞中有效表達(dá)E蛋白,且表達(dá)的重組E蛋白具有良好反應(yīng)原性。將上述重組病毒在Sf9細(xì)胞中大量擴(kuò)增后,收集細(xì)胞培養(yǎng)物凍融3次,再高速離心去除細(xì)胞碎片,獲得高水平表達(dá)的重組E蛋白。然后以該重組E蛋白為抗原免疫櫻桃谷肉鴨,檢測(cè)其免疫原性。將7日齡櫻桃谷雛鴨分為4組,免疫重組E蛋白(4×106cells/只)為試驗(yàn)組;以wtBac感染sf9細(xì)胞裂解液為空Bacmid組;設(shè)置DTMUV AH-F10株全病毒油乳劑滅活苗為陽性對(duì)照組、接種等劑量PBS為陰性對(duì)照組。2周后進(jìn)行加強(qiáng)免疫。免疫前、后分別對(duì)各組雛鴨頸靜脈采血,分離血清,進(jìn)行E蛋白特異性抗體、中和抗體、IL-4、IFN-γ細(xì)胞因子檢測(cè),同時(shí)分離外周血進(jìn)行淋巴細(xì)胞增殖試驗(yàn)。二次免疫后2周使用DTMUV AH-TL15株進(jìn)行攻毒保護(hù)試驗(yàn),每只0.5mL(ELD50為10-2.38/0.1mL)。試驗(yàn)結(jié)果顯示,試驗(yàn)組櫻桃谷雛鴨首免2周后特異性抗體,血清中和抗體,IL-4、IFN-γ細(xì)胞因子水平顯著上升,與陰性對(duì)照組、空Bacmid組櫻桃谷雛鴨差異顯著(P0.05);淋巴細(xì)胞增殖試驗(yàn)發(fā)現(xiàn)試驗(yàn)組淋巴細(xì)胞SI值較陰性對(duì)照、空Bacmid組差異顯著(P0.05)。攻毒保護(hù)試驗(yàn)結(jié)果表明,試驗(yàn)組櫻桃谷雛鴨的保護(hù)率為80%,陽性對(duì)照組的保護(hù)率為100%,陰性對(duì)照組和空Bacmid組雛鴨全部發(fā)病。病理切片觀察結(jié)果顯示試驗(yàn)組和陽性對(duì)照組雛鴨均未見病變,而陰性對(duì)照組和空Bacmid組在肝臟出現(xiàn)脂肪變性,淋巴細(xì)胞浸潤;脾臟核固縮,出血;胰臟淋巴細(xì)胞浸潤;大腦血管“袖套樣”病變,有噬神經(jīng)細(xì)胞現(xiàn)象。綜上,本研究在sf9細(xì)胞中成功表達(dá)了DTMUV E蛋白,該重組E蛋白可以有效誘導(dǎo)櫻桃谷雛鴨產(chǎn)生體液免疫和細(xì)胞免疫,并能在一定程度上保護(hù)櫻桃谷雛鴨抵御病毒攻擊。提示本研究以昆蟲細(xì)胞表達(dá)的重組E蛋白具有作為研制抗DTMUV感染的新型疫苗的潛力。
[Abstract]:Duck Tembusu virus disease) is an infectious disease caused by Duck Tembusu virus (Duck Tembusu virusDTMUV) of adult laying ducks, which is slow to grow and lead to the death of ducklings. Immunization is one of the key measures to prevent and control the disease. In this study, baculovirus-insect cell expression system was used to express the important immunogen gene of DTMUV, and the immunogenicity of the expressed recombinant E protein was studied. First of all, according to the whole gene sequence of E gene of DTMUV AH-F10 strain, primers were designed. The E gene was amplified by using pMD19-E plasmid containing E gene of DTMUV AH-F10 strain as template, and inserted into the baculovirus skeleton vector: pFastbac vector. Then the identified recombinant plasmid pFastbac-E was transferred into the DH10Bac competent cells, and the white monoclonal colony containing the recombinant plasmid rBacmid-E was obtained by three times of blue and white spot screening. The recombinant plasmid was extracted and identified correctly by PCR, and the sf9 cells were transfected with Cellfection. Recombinant baculovirus rvBac-E of P1 generation was obtained. After PCR identification, the recombinant baculovirus was subcultured into P4 passage in sf9 cells. The TCID50 of rvBac-E in P4 passage was 10 -5.5% 0.1 mL. The recombinant baculovirus was identified by Western blot and IFA, respectively. The results showed that the constructed rvBac-E could effectively express E protein in sf9 cells, and the expressed recombinant E protein had good reactivity. After the recombinant virus was amplified in Sf9 cells, the cell culture was frozen and thawed for 3 times, then the fragment was removed by high speed centrifugation, and the recombinant E protein expressed at high level was obtained. Then the recombinant E protein was used as antigen to immunize Cherry Valley Meat Duck and its immunogenicity was tested. The 7-day-old Cherry Valley ducklings were divided into four groups, the experimental group was immunized with recombinant E protein 4 脳 106cells/, the wtBac infected sf9 cell lysate was used as the empty Bacmid group, and the DTMUV AH-F10 virus oil emulsion inactivated vaccine was used as the positive control group. Inoculation with equal dose of PBS as negative control group was carried out after 2. 2 weeks. Before and after immunization, blood samples were collected from the jugular vein of each group of ducklings, serum was isolated, E protein specific antibody, neutralizing antibody IL-4 and IFN- 緯 cytokines were detected, and peripheral blood lymphocytes proliferation test was carried out at the same time. Two weeks after the second immunization, DTMUV AH-TL15 strain was used to protect the virus, and the 0.5mL(ELD50 was 10 ~ (-2.38) / 0.1 mL 路L ~ (-1) 路L ~ (-1) 路min ~ (-1) per mouse. The results showed that the levels of specific antibodies, serum neutralizing antibody IL-4 and IFN- 緯 cytokines of Cherry Valley ducklings in the experimental group were significantly higher than those in the negative control group. The results of lymphocyte proliferation test showed that the SI value of lymphocytes in the test group was higher than that in the negative control group, and the difference in the empty Bacmid group was significant (P 0.05). The results showed that the protective rate of Cherry Valley ducklings in the test group was 80%, the protective rate of the positive control group was 100%, and that of the negative control group and the empty Bacmid group was 100%. Pathological observation showed that there was no pathological changes in both the experimental group and the positive control group, while the fatty degeneration and lymphocyte infiltration occurred in the liver of the negative control group and the empty Bacmid group, the spleen nucleus was pyknosis, hemorrhage, pancreatic lymphocyte infiltration. Cerebral vascular "cuff like" lesions, the phenomenon of phagocytosis. In conclusion, DTMUV E protein was successfully expressed in sf9 cells. The recombinant E protein could effectively induce humoral and cellular immunity of Cherry Valley ducklings and protect Cherry Valley ducklings from virus attack to some extent. These results suggest that the recombinant E protein expressed by insect cells has the potential to be a novel vaccine against DTMUV infection.
【學(xué)位授予單位】：安徽農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.65

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本文編號(hào)：1805765