本文選題：家蠶桿狀病毒表達載體系統(tǒng) + PEDV��； 參考：《浙江理工大學》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,且效價均可達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,引起了較強的黏膜免疫反應(yīng)。脾淋巴細胞增殖實驗表明與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),對小鼠淋巴細胞的活化具有明顯的刺激作用,使小鼠產(chǎn)生了細胞免疫反應(yīng)。免疫鼠脾CD8+T淋巴細胞的流式細胞儀檢測結(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胞表現(xiàn)出較好的分化能力,具有較好的激活T細胞產(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,具有進一步開發(fā)研究的價值。
[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.
【學位授予單位】：浙江理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.651

【參考文獻】

相關(guān)期刊論文 前10條

1 桂銳;童岑;周丹娜;郭銳;劉威;田永祥;劉東宇;李坤;張輝;李家奎;;豬流行性腹瀉病毒S1蛋白表達及反應(yīng)原性分析[J];畜牧與獸醫(yī);2017年02期

2 竇磊磊;;豬流行性腹瀉的流行特點、臨床表現(xiàn)與防控措施[J];現(xiàn)代畜牧科技;2017年02期

3 趙憲義;;豬病毒性腹瀉的特點及預防措施[J];畜牧獸醫(yī)科技信息;2017年01期

4 代洪波;岳豐雄;徐宏軍;魏勝男;高鵬;胡來根;;豬流行性腹瀉病毒分離鑒定及其滅活疫苗免疫保護效果研究[J];動物醫(yī)學進展;2017年01期

5 祁正梅;;豬流行性腹瀉的防治對策[J];當代畜禽養(yǎng)殖業(yè);2017年01期

6 史春梅;高遠;;豬流行性腹瀉的流行特點及防治[J];當代畜禽養(yǎng)殖業(yè);2017年01期

7 甘紅軍;;豬流行性腹瀉的診治[J];養(yǎng)殖與飼料;2017年01期

8 張欣悅;高永翔;謝怡敏;;黏膜免疫系統(tǒng)研究進展[J];中藥與臨床;2015年05期

9 程杰;欒慧;吳家強;彭軍;;豬流行性腹瀉新型疫苗的研究進展[J];養(yǎng)豬;2015年04期

10 王悅蕓;劉琪;安欣宇;周如月;馮曉聲;賈愛卿;王貴平;;黏膜免疫系統(tǒng)及黏膜免疫研究進展[J];畜牧與獸醫(yī);2015年07期

相關(guān)會議論文 前2條

1 張志芳;;家蠶桿狀病毒生物反應(yīng)器生產(chǎn)動物基因工程疫苗[A];培育生物產(chǎn)業(yè)，，發(fā)展綠色經(jīng)濟——第五屆中國生物產(chǎn)業(yè)大會·2011基因科學與產(chǎn)業(yè)發(fā)展論壇會刊[C];2011年

2 胡桂學;刁鵬祥;陳中秋;牛偉;;PEDV COE基因重組乳酸菌仔豬和妊娠母豬免疫研究[A];中國畜牧獸醫(yī)學會家畜傳染病學分會第七屆全國會員代表大會暨第十三次學術(shù)研討會論文集（下冊）[C];2009年

相關(guān)博士學位論文 前1條

1 郎洪武;豬圓環(huán)病毒2型Cap蛋白的家蠶表達及亞單位疫苗在小鼠和仔豬的免疫效力試驗[D];中國農(nóng)業(yè)大學;2013年

相關(guān)碩士學位論文 前7條

1 史翠萍;桿狀病毒表達系統(tǒng)表達豬流行性腹瀉病毒S1蛋白及其免疫原性的研究[D];浙江理工大學;2016年

2 陳冰清;豬流行性腹瀉病毒（PEDV）基因工程疫苗載體構(gòu)建及重組病毒拯救[D];東華大學;2015年

3 李珍;家蠶桿狀病毒表達系統(tǒng)研制草魚出血病病毒和禽流感病毒疫苗[D];蘇州大學;2014年

4 張陽;載新城疫病毒F基因N-2-HACC/CMC納米粒的制備及其黏膜免疫效果評價[D];黑龍江大學;2014年

5 訾玉華;豬流行性腹瀉病毒（PEDV）基因序列分析及基因工程疫苗的研究[D];江南大學;2013年

6 王軍軍;家蠶桿狀病毒表面展示豬瘟病毒E0、E2蛋白及各自免疫原性分析[D];浙江理工大學;2012年

7 趙志剛;重組PEDV-S1抗原表位干酪乳桿菌的免疫原性分析[D];黑龍江八一農(nóng)墾大學;2010年

本文編號：2009032