本文關(guān)鍵詞：組成型表達(dá)雞傳染性法氏囊病毒vp2基因重組乳酸菌的構(gòu)建及免疫原性分析　出處：《東北農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 傳染性法氏囊病毒(IBDV) vp2蛋白 乳酸菌 口服免疫 免疫原性

【摘要】：傳染性法氏囊病(IBD)是由傳染性法氏囊病毒(IBDV)引發(fā)的一種雞的免疫抑制性傳染病,該病給養(yǎng)禽業(yè)造成了巨大的經(jīng)濟(jì)損失。本研究中,利用乳酸菌表達(dá)載體p PG612構(gòu)建了表達(dá)IBDV免疫原性蛋白vp2的重組表達(dá)質(zhì)粒,并分別在L.plantarum,L.pentoses,和L.casei 393三株乳酸菌中進(jìn)行表達(dá)。p PG612載體是一種表面展示載體,該載體中包含HCE組成型啟動子和Pgs A錨定蛋白,同時為了提高vp2蛋白的表達(dá)量,本研究在該載體中插入了T7g10增強子。本研究對高效制備表達(dá)vp2蛋白的陽性重組乳酸菌策略進(jìn)行了改善,主要評價指標(biāo)如下:OD600值處于0.4-0.5,重組質(zhì)粒含量為2μl-6μl(100ng/μl),電壓為2.3-2.4 kv/cm。結(jié)果顯示,有兩種重組質(zhì)粒在三種乳酸菌中均表現(xiàn)出高電轉(zhuǎn)化率,即(i)p PG612-HCE-Pgs A-vp2-rrn BT1T2,(ii)p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2,之后的酶切鑒定和PCR鑒定也都均證實了該結(jié)果。該方法較之前使用的構(gòu)建重組乳酸菌的方法更加簡單且更加高效。本研究還分別對三株含有p PG612-vp2(p PG612-HCE-Pgs A-vp2-rrn BT1T2)和p PG612-T7g10-vp2(p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2)重組質(zhì)粒的重組乳酸菌作為口服疫苗的免疫原性和針對IBDV的保護(hù)率進(jìn)行了評價。實驗結(jié)果顯示,vp2蛋白成功的獲得了表達(dá)并展示在各重組乳酸菌的表面,將這些重組乳酸分別命名如下,LPL/p PG612-vp2(PL),LPL/p PG612-T7g10-vp2(TL),LPE/p PG612-vp2(PE),LPE/p PG612-T7g10-vp2(TE),LC/p PG612-vp2(PC)和LC/p PG612-T7g10-vp2(TC)。其中,含有p PG612-T7g10-vp2質(zhì)粒的重組菌的vp2蛋白表達(dá)量較高,說明T7g10增強子能夠有效提高vp2蛋白在乳酸菌中的表達(dá)水平。動物實驗結(jié)果顯示,口服免疫后,LPL/p PG612-T7g10-vp2株乳酸菌能夠刺激機(jī)體產(chǎn)生更強的免疫反應(yīng)以及更高的免疫保護(hù)率(87.5%)。通過本研究可以證明,重組乳酸菌系統(tǒng)作為口服疫苗可以刺激動物產(chǎn)生較強的抗體水平,p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2免疫組產(chǎn)生的抗體水平顯著高于p PG612-HCE-Pgs Avp2-rrn BT1T2免疫組,并且L.plantarum菌株免疫組的免疫保護(hù)率高于L.casei免疫組和L.pentoses免疫組。同時,L.casei免疫組的保護(hù)率高于L.pentoses免疫組。動物實驗中,在vp2免疫組中檢測到大量的T細(xì)胞,在IBDV攻毒組中也檢測到數(shù)量增多。與空白對照組相比,所有免疫組的CD4+和CD8+T細(xì)胞均顯著性增高,其中p PG612-HCE-T7g10-Pgs Avp2-rrn BT1T2免疫組的CD4+和CD8+T細(xì)胞比例高于p PG612-HCE-Pgs A-vp2-rrn BT1T2免疫組。TL免疫組中CD8+T細(xì)胞和CD4+T細(xì)胞的分化率分別為13.3%和21.0%,高于PL免疫組的10.4%和14.0%;TC免疫組CD8+T細(xì)胞和CD4+T細(xì)胞的分化率分別為11.4%和20.9%,高于PC免疫組的7.2%和11.5%;TE免疫組CD8+T細(xì)胞和CD4+T細(xì)胞的分化率分別為8.3%和16.7%,高于PE組的7.5%和13.5%。所有免疫組的CD8+T細(xì)胞分化率均高于CD4+T細(xì)胞。CD8+T細(xì)胞屬于細(xì)胞毒性T細(xì)胞,可以裂解感染病毒的細(xì)胞、腫瘤細(xì)胞和同種異體移植物,在免疫反應(yīng)中發(fā)揮著關(guān)鍵作用。在細(xì)胞免疫反應(yīng)的檢測結(jié)果中,重組L.plantarum菌株免疫組的CD8+T細(xì)胞和CD4+T細(xì)胞的分化率高于重組L.casei菌株組和重組L.pentoses菌株組。p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2免疫組產(chǎn)生的IFN-γ,IL-2和IL-4細(xì)胞因子水平高于p PG612-HCE-Pgs A-vp2-rrn BT1T2免疫組;其它數(shù)據(jù)顯示,IFN-γ水平高于IL-2水平,IL-2水平高于IL-4水平;重組L.plantarum菌株免疫組較其他兩個重組菌免疫組能夠刺激產(chǎn)生更高水平的Th1和Th2型細(xì)胞因子,并且重組L.casei菌株免疫組刺激產(chǎn)生Th1和Th2型細(xì)胞因子水平高于重組L.pentoses菌株免疫組。IFN-γ和IL-2等Th1型細(xì)胞因子和細(xì)胞免疫相關(guān),IL-4等Th2型細(xì)胞因子和體液免疫相關(guān)。本研究得出結(jié)論,含有p PG612-T7g10-vp2質(zhì)粒的重組L.plantarum乳酸菌免疫原性強,能夠針對IBDV提供有效的免疫保護(hù),可作為將來開發(fā)IBDV口服疫苗的候選菌株。
[Abstract]:Infectious bursal disease (IBD) is composed of infectious bursal disease virus (IBDV) immune chicken induced inhibition of infectious disease, the disease in poultry industry has caused tremendous economic losses. In this study, a recombinant expression plasmid expressing IBDV immunogenicity protein VP2 was constructed by lactic acid bacteria expression vector p PG612, and expressed in L.plantarum, L.pentoses and L.casei 393 three strains of lactic acid bacteria, respectively. P PG612 vector is a surface display vector, which contains HCE component promoter and Pgs A anchored protein. Meanwhile, in order to improve the expression level of VP2 protein, T7g10 enhancer was inserted into this vector. In this study, the strategy of efficient preparation of positive recombinant Lactobacillus expressing VP2 protein has been improved. The main evaluation indexes are as follows: the OD600 value is 0.4-0.5, the recombinant plasmid content is 2 micron L-6 L (100ng/ L), and the voltage is 2.3-2.4 kv/cm. The results showed that two recombinant plasmids exhibited high electrical conversion rate in three kinds of lactic acid bacteria, that is, (I) P PG612-HCE-Pgs A-vp2-rrn BT1T2, (II) P PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2. After that, the results of enzyme digestion and PCR identification also confirmed the results. This method is more simple and more efficient than the previous method of constructing recombinant lactic acid bacteria. In this study, three strains of recombinant Lactobacillus containing P PG612-vp2 (P PG612-HCE-Pgs A-vp2-rrn BT1T2) and P PG612-T7g10-vp2 (P PG612-HCE-T7g10-Pgs A-vp2-rrn) recombinant plasmid were evaluated for their immunogenicity and protection rate. The experimental results show that the VP2 protein was successfully expressed and displayed on the surface of the recombinant lactic acid bacteria, the recombinant lactic acid were named as follows, LPL/p PG612-vp2 (PL), LPL/p PG612-T7g10-vp2 (TL), LPE/p PG612-vp2 (PE), LPE/p PG612-T7g10-vp2 (TE), LC/p PG612-vp2 (PC) and LC/p PG612-T7g10-vp2 (TC). Among them, the expression of VP2 protein containing P PG612-T7g10-vp2 plasmid was higher, indicating that T7g10 enhancer can effectively improve the expression level of VP2 protein in Lactobacillus. Animal experiments showed that after oral immunization, LPL/p PG612-T7g10-vp2 strain Lactobacillus could stimulate the body to produce stronger immune response and higher immune protection rate (87.5%). This research proved that recombinant lactic acid bacteria system as an oral vaccine can stimulate the animal to produce the antibody level of strong, P PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2 antibody level of immunized group was significantly higher than that of P PG612-HCE-Pgs Avp2-rrn BT1T2 immune group, and immune protection strain L.plantarum immune group was higher than that of L.casei group and L.pentoses group of immune immunity. At the same time, the protection rate of L.casei immune group was higher than that of L.pentoses immunization group. In animal experiments, a large number of T cells were detected in the VP2 immunization group, and the number of IBDV in the attack group was also increased. Compared with the blank control group, the CD4+ and CD8+T cells in all the immunological groups were significantly increased, and the proportion of CD4+ and CD8+T cells in P PG612-HCE-T7g10-Pgs Avp2-rrn BT1T2 group was higher than that in P PG612-HCE-Pgs A-vp2-rrn, and the immunization group. The TL group in CD8+T and CD4+T cells differentiation rates were 13.3% and 21%, higher than the 10.4% and 14% PL group; TC group of immune CD8+T cells and CD4+T cells differentiation rates were 11.4% and 20.9%, higher than the 7.2% and 11.5% PC group; TE group of immune CD8+T cells and CD4+T cell differentiation rates were 8.3% and 16.7%, 7.5% and 13.5% higher than that of PE group. The differentiation rate of CD8+T cells in all immune groups was higher than that of CD4+T cells. CD8+T cells belong to cytotoxic T cells, which can cleavage infected virus cells, tumor cells and allogeneic grafts, and play a key role in immune response. In the detection results of cellular immune response, the differentiation rate of CD8+T cells and CD4+T cells in recombinant L.plantarum strain was higher than that in recombinant L.casei strain group and recombinant L.pentoses strain group. IFN- P PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2 gamma immunohistochemistry, IL-2 and IL-4 cytokine levels higher than the P PG612-HCE-Pgs A-vp2-rrn BT1T2 immunohistochemistry; other data showed that IFN- levels higher than the IL-2 level, IL-2 level higher than the level of IL-4; recombinant strain L.plantarum immune group compared to the other two recombinant immune group can stimulate the production of Th1 and Th2 cytokines a higher level, and the recombinant strain L.casei immune group stimulates the production of Th1 and Th2 type cytokine levels higher than the group immunized with recombinant L.pentoses strains. Th1 type cytokines such as IFN- gamma and IL-2 are related to cellular immunity, and Th2 type cytokines such as IL-4 are related to humoral immunity. This study concluded that the recombinant L.plantarum Lactobacillus containing P PG612-T7g10-vp2 plasmid has strong immunogenicity and can provide effective immune protection for IBDV, and it can be used as a candidate vaccine for the development of IBDV oral vaccine in the future.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.65

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本文編號：1345125