本文選題：家蠶桿狀病毒表達(dá)載體系統(tǒng) + PEDV��； 參考：《浙江理工大學(xué)》2017年碩士論文

【摘要】：豬流行性腹瀉(Porcine epidemic diarrhea,PED)是由豬流行性腹瀉病毒(Porcine epidemic diarrhea virus,PEDV)的感染引起的,導(dǎo)致仔豬發(fā)病率和致死率都極高的一種豬常見的病,研制新的安全有效的疫苗十分必要。鑒于纖突蛋白S是PEDV主要的中和表位和受體結(jié)合域,與病毒抗原性和吸附入侵密切相關(guān),我們將PEDV S1(1~735 aa)區(qū)插入到GP64信號肽與特定膜錨定結(jié)構(gòu)(TMD)之間,并與eGFP基因融合,構(gòu)建到轉(zhuǎn)移載體pFastBacHTB上,利用Bac-to-Bac系統(tǒng)構(gòu)建重組家蠶桿狀病毒rvBmBacmid-SPS1-eGFP-TMD。重組病毒感染BmN細(xì)胞后,Western Blot檢測到約為150 kDa及400kDa的兩條帶,初步推斷是由于S1蛋白發(fā)生了N-糖基化以及形成了三聚體。以該重組病毒為免疫原霧化免疫BALB/c小鼠,野生病毒W(wǎng)T-BmNPV為陰性對照,TGEV/PEDV二聯(lián)滅活疫苗為陽性對照。間接ELISA檢測霧化免疫16 d后小鼠血清中PEDV特異性的抗體水平。ELISA結(jié)果表明,在重組病毒rvBmBacmid-SP-S1-eGFP-TMD和TGEV/PEDV二聯(lián)滅活苗霧化免疫16 d的小鼠血清中均檢測到較高水平的PEDV特異性的抗體,且效價(jià)均可達(dá)1:6400,說明重組病毒rvBmBacmid-SP-S1-eGFP-TMD和TGEV/PEDV二聯(lián)滅活苗霧化免疫能很好的刺激小鼠體液免疫反應(yīng)。針對分泌型免疫球蛋白A(sIgA)的酶聯(lián)免疫分析結(jié)果顯示,不管是血清還是肺泡灌洗液,rvBmBacmidSP-S1-eGFP-TMD免疫組產(chǎn)生的sIgA濃度均顯著高于TGEV/PEDV二聯(lián)滅活苗免疫組,說明rvBmBacmid-SP-S1-eGFP-TMD免疫小鼠后能刺激小鼠產(chǎn)生高水平的sIgA,引起了較強(qiáng)的黏膜免疫反應(yīng)。脾淋巴細(xì)胞增殖實(shí)驗(yàn)表明與WT組相比,rvBmBacmid-SP-S1-eGFP-TMD組和TGEV/PEDV二聯(lián)滅活苗組均發(fā)生明顯增殖(P0.05),說明rvBmBacmid-SP-S1-eGFP-TMD和TGEV/PEDV二聯(lián)滅活苗免疫組小鼠淋巴細(xì)胞對抗原的重新接觸表現(xiàn)出了增殖效應(yīng),對小鼠淋巴細(xì)胞的活化具有明顯的刺激作用,使小鼠產(chǎn)生了細(xì)胞免疫反應(yīng)。免疫鼠脾CD8+T淋巴細(xì)胞的流式細(xì)胞儀檢測結(jié)果顯示rvBmBacmidSP-S1-eGFP-TMD組和TGEV/PEDV二聯(lián)滅活苗組CD8+T細(xì)胞比例均高于WT組,說明rvBmBacmid-SP-S1-eGFP-TMD、TGEV/PEDV二聯(lián)滅活苗免疫小鼠可以使小鼠淋巴細(xì)胞表現(xiàn)出較好的分化能力,具有較好的激活T細(xì)胞產(chǎn)生免疫應(yīng)答的能力。綜上所述,我們制備的重組家蠶桿狀病毒霧化免疫小鼠取得了初步的效果,說明該重組病毒可以作為PEDV黏膜疫苗的候選疫苗,具有進(jìn)一步開發(fā)研究的價(jià)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.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.651

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