家蚕杆状病毒表达的猪流行性腹泻病毒S1蛋白及其黏膜免疫原性研究

发布时间：2018-06-12 08:25

本文选题：家蚕杆状病毒表达载体系统 + PEDV　；参考：《浙江理工大学》2017年硕士论文

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的感染引起的,导致仔猪发病率和致死率都极高的一种猪常见的病,研制新的安全有效的疫苗十分必要。鉴于纤突蛋白S是PEDV主要的中和表位和受体结合域,与病毒抗原性和吸附入侵密切相关,我们将PEDV S1(1~735 aa)区插入到GP64信号肽与特定膜锚定结构(TMD)之间,并与eGFP基因融合,构建到转移载体pFastBacHTB上,利用Bac-to-Bac系统构建重组家蚕杆状病毒rvBmBacmid-SPS1-eGFP-TMD。重组病毒感染BmN细胞后,Western Blot检测到约为150 kDa及400kDa的两条带,初步推断是由于S1蛋白发生了N-糖基化以及形成了三聚体。以该重组病毒为免疫原雾化免疫BALB/c小鼠,野生病毒WT-BmNPV为阴性对照,TGEV/PEDV二联灭活疫苗为阳性对照。间接ELISA检测雾化免疫16 d后小鼠血清中PEDV特异性的抗体水平。ELISA结果表明,在重组病毒rvBmBacmid-SP-S1-eGFP-TMD和TGEV/PEDV二联灭活苗雾化免疫16 d的小鼠血清中均检测到较高水平的PEDV特异性的抗体,且效价均可达1:6400,说明重组病毒rvBmBacmid-SP-S1-eGFP-TMD和TGEV/PEDV二联灭活苗雾化免疫能很好的刺激小鼠体液免疫反应。针对分泌型免疫球蛋白A(sIgA)的酶联免疫分析结果显示,不管是血清还是肺泡灌洗液,rvBmBacmidSP-S1-eGFP-TMD免疫组产生的sIgA浓度均显著高于TGEV/PEDV二联灭活苗免疫组,说明rvBmBacmid-SP-S1-eGFP-TMD免疫小鼠后能刺激小鼠产生高水平的sIgA,引起了较强的黏膜免疫反应。脾淋巴细胞增殖实验表明与WT组相比,rvBmBacmid-SP-S1-eGFP-TMD组和TGEV/PEDV二联灭活苗组均发生明显增殖(P0.05),说明rvBmBacmid-SP-S1-eGFP-TMD和TGEV/PEDV二联灭活苗免疫组小鼠淋巴细胞对抗原的重新接触表现出了增殖效应,对小鼠淋巴细胞的活化具有明显的刺激作用,使小鼠产生了细胞免疫反应。免疫鼠脾CD8+T淋巴细胞的流式细胞仪检测结果显示rvBmBacmidSP-S1-eGFP-TMD组和TGEV/PEDV二联灭活苗组CD8+T细胞比例均高于WT组,说明rvBmBacmid-SP-S1-eGFP-TMD、TGEV/PEDV二联灭活苗免疫小鼠可以使小鼠淋巴细胞表现出较好的分化能力,具有较好的激活T细胞产生免疫应答的能力。综上所述,我们制备的重组家蚕杆状病毒雾化免疫小鼠取得了初步的效果,说明该重组病毒可以作为PEDV黏膜疫苗的候选疫苗,具有进一步开发研究的价值。
[Abstract]:Porcine epidemic diarrhea (PED) is caused by porcine epidemic diarrhea virus (PEDVV), which is a common disease in piglets with high morbidity and mortality. Therefore, it is necessary to develop a new safe and effective vaccine. Since fibrin S is the main neutralizing epitope and receptor binding domain of PEDV and is closely related to the antigenicity and adsorption invasion of PEDV, we inserted the PEDV S _ (1) ~ (1) ~ (1) ~ (1) ~ 1 ~ 735aa) region between GP64 signal peptide and a specific membrane anchoring structure (TMDD), and fused it with eGFP gene. The recombinant Bombyx mori baculovirus rvBmBacmid-SPS1-eGFP-TMDS was constructed by using Bac-to-Bac system on the transfer vector pFastBacHTB. Two bands of 150 kDa and 400 kDa were detected by Western blot after the recombinant virus was infected with BmN cells. It was inferred that S1 protein had N-glycosylation and trimer formation. The recombinant virus was used as immunogen to immunize BALB / c mice, and the wild virus WT-BmNPV was used as negative control and TGEV / PEDV dual inactivated vaccine as positive control. Indirect Elisa was used to detect the specific antibody level of PEDV in mouse serum after 16 days of atomization immunization. The results of Elisa showed that a higher level of PEDV specific antibody was detected in the sera of mice immunized with recombinant virus rvBmBacmid-SP-S1-eGFP-TMD and TGEV / PEDV combined inactivated vaccine for 16 days. The titer of recombinant virus rvBmBacmid-SP-S1-eGFP-TMD and TGEV / PEDV inactivated vaccine was 1: 6400, which indicated that the nebulization of recombinant virus rvBmBacmid-SP-S1-eGFP-TMD and TGEV / PEDV inactivated vaccine could stimulate humoral immune response in mice. The results of enzyme-linked immunosorbent assay (Elisa) for secretory immunoglobulin A (IgA) showed that the serum and alveolar lavage fluid rvBmBacmidSP-S1-eGFP-TMD immunized group were significantly higher than that of TGEV / PEDV combined inactivated vaccine immunization group. The results showed that rvBmBacmid-SP-S1-eGFP-TMD could stimulate mice to produce a high level of Siga and induce a strong mucosal immune response after immunization with rvBmBacmid-SP-S1-eGFP-TMD. Compared with WT group, the proliferation of spleen lymphocytes in rvBmBacmid-SP-S1-eGFP-TMD group and TGEV / PEDV combined inactivated vaccine group was significantly increased (P 0.05), which indicated that the lymphocytes of RvBm Bacmid-S1-eGFP-TMD group and TGEV / PEDV double inactivated vaccine group showed proliferative effect on the recontact of antigen. It can stimulate the activation of lymphocytes in mice and induce cellular immune response in mice. The results of flow cytometry showed that the percentage of CD8 T cells in rvBmBacmidSP-S1-eGFP-TMD group and TGEV / PEDV combined inactivated vaccine group was higher than that in WT group, which indicated that rvBmBacmid-SP-S1-eGFP-TMD-TGEV / PEDV biphasic inactivated vaccine could induce the differentiation of lymphocytes in mice. It has better ability to activate T cells to produce immune response. In conclusion, we prepared recombinant Bombyx mori baculovirus nebulized mice and achieved preliminary results, indicating that the recombinant virus can be used as a candidate vaccine for PEDV mucosal vaccine, which has the value of further development and research.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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