鸭坦布苏病毒E蛋白在昆虫细胞中的表达及其免疫原性的初步研究

发布时间：2018-04-26 11:08

本文选题：鸭坦布苏病毒 + E蛋白　；参考：《安徽农业大学》2017年硕士论文

【摘要】：鸭坦布苏病毒病(Duck Tembusu virus disease)是由鸭坦布苏病毒(Duck Tembusu virus,DTMUV)引起的以成年蛋鸭产蛋下降,生长发育迟缓并可导致雏鸭死亡的一种传染病。免疫接种是预防与控制该病的关键性措施之一。本研究利用杆状病毒-昆虫细胞表达系统表达DTMUV的重要免疫原基因(E基因),并对表达的重组E蛋白进行了免疫原性初步研究。首先,根据DTMUV AH-F10株的E基因全基因序列设计引物,以含有DTMUV AH-F10株E基因的pMD19-E质粒为模板扩增E基因,并将其插入杆状病毒骨架载体:pFastbac载体中,再将鉴定正确的重组质粒pFastbac-E转入DH10Bac感受态细胞,经过三次蓝白斑筛选获得含有重组质粒rBacmid-E的白色单克隆菌落,提取重组质粒并用PCR鉴定正确后,使用Cellfection转染sf9细胞,获得P1代重组杆状病毒rvBac-E,经PCR鉴定正确后,在sf9细胞中传代至P4代,测得P4代rvBac-E的TCID50为10-5.5/0.1mL。分别用Western blot和IFA鉴定重组杆状病毒,结果均证明,所构建的rvBac-E可在sf9细胞中有效表达E蛋白,且表达的重组E蛋白具有良好反应原性。将上述重组病毒在Sf9细胞中大量扩增后,收集细胞培养物冻融3次,再高速离心去除细胞碎片,获得高水平表达的重组E蛋白。然后以该重组E蛋白为抗原免疫樱桃谷肉鸭,检测其免疫原性。将7日龄樱桃谷雏鸭分为4组,免疫重组E蛋白(4×106cells/只)为试验组;以wtBac感染sf9细胞裂解液为空Bacmid组;设置DTMUV AH-F10株全病毒油乳剂灭活苗为阳性对照组、接种等剂量PBS为阴性对照组。2周后进行加强免疫。免疫前、后分别对各组雏鸭颈静脉采血,分离血清,进行E蛋白特异性抗体、中和抗体、IL-4、IFN-γ细胞因子检测,同时分离外周血进行淋巴细胞增殖试验。二次免疫后2周使用DTMUV AH-TL15株进行攻毒保护试验,每只0.5mL(ELD50为10-2.38/0.1mL)。试验结果显示,试验组樱桃谷雏鸭首免2周后特异性抗体,血清中和抗体,IL-4、IFN-γ细胞因子水平显著上升,与阴性对照组、空Bacmid组樱桃谷雏鸭差异显著(P0.05);淋巴细胞增殖试验发现试验组淋巴细胞SI值较阴性对照、空Bacmid组差异显著(P0.05)。攻毒保护试验结果表明,试验组樱桃谷雏鸭的保护率为80%,阳性对照组的保护率为100%,阴性对照组和空Bacmid组雏鸭全部发病。病理切片观察结果显示试验组和阳性对照组雏鸭均未见病变,而阴性对照组和空Bacmid组在肝脏出现脂肪变性,淋巴细胞浸润;脾脏核固缩,出血;胰脏淋巴细胞浸润;大脑血管“袖套样”病变,有噬神经细胞现象。综上,本研究在sf9细胞中成功表达了DTMUV E蛋白,该重组E蛋白可以有效诱导樱桃谷雏鸭产生体液免疫和细胞免疫,并能在一定程度上保护樱桃谷雏鸭抵御病毒攻击。提示本研究以昆虫细胞表达的重组E蛋白具有作为研制抗DTMUV感染的新型疫苗的潜力。
[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- 纬 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- 纬 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.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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