组成型表达鸡传染性法氏囊病毒vp2基因重组乳酸菌的构建及免疫原性分析

发布时间：2017-12-28 07:24

本文关键词：组成型表达鸡传染性法氏囊病毒vp2基因重组乳酸菌的构建及免疫原性分析　出处：《东北农业大学》2016年博士论文　论文类型：学位论文

【摘要】：传染性法氏囊病(IBD)是由传染性法氏囊病毒(IBDV)引发的一种鸡的免疫抑制性传染病,该病给养禽业造成了巨大的经济损失。本研究中,利用乳酸菌表达载体p PG612构建了表达IBDV免疫原性蛋白vp2的重组表达质粒,并分别在L.plantarum,L.pentoses,和L.casei 393三株乳酸菌中进行表达。p PG612载体是一种表面展示载体,该载体中包含HCE组成型启动子和Pgs A锚定蛋白,同时为了提高vp2蛋白的表达量,本研究在该载体中插入了T7g10增强子。本研究对高效制备表达vp2蛋白的阳性重组乳酸菌策略进行了改善,主要评价指标如下:OD600值处于0.4-0.5,重组质粒含量为2μl-6μl(100ng/μl),电压为2.3-2.4 kv/cm。结果显示,有两种重组质粒在三种乳酸菌中均表现出高电转化率,即(i)p PG612-HCE-Pgs A-vp2-rrn BT1T2,(ii)p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2,之后的酶切鉴定和PCR鉴定也都均证实了该结果。该方法较之前使用的构建重组乳酸菌的方法更加简单且更加高效。本研究还分别对三株含有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)重组质粒的重组乳酸菌作为口服疫苗的免疫原性和针对IBDV的保护率进行了评价。实验结果显示,vp2蛋白成功的获得了表达并展示在各重组乳酸菌的表面,将这些重组乳酸分别命名如下,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质粒的重组菌的vp2蛋白表达量较高,说明T7g10增强子能够有效提高vp2蛋白在乳酸菌中的表达水平。动物实验结果显示,口服免疫后,LPL/p PG612-T7g10-vp2株乳酸菌能够刺激机体产生更强的免疫反应以及更高的免疫保护率(87.5%)。通过本研究可以证明,重组乳酸菌系统作为口服疫苗可以刺激动物产生较强的抗体水平,p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2免疫组产生的抗体水平显著高于p PG612-HCE-Pgs Avp2-rrn BT1T2免疫组,并且L.plantarum菌株免疫组的免疫保护率高于L.casei免疫组和L.pentoses免疫组。同时,L.casei免疫组的保护率高于L.pentoses免疫组。动物实验中,在vp2免疫组中检测到大量的T细胞,在IBDV攻毒组中也检测到数量增多。与空白对照组相比,所有免疫组的CD4+和CD8+T细胞均显著性增高,其中p PG612-HCE-T7g10-Pgs Avp2-rrn BT1T2免疫组的CD4+和CD8+T细胞比例高于p PG612-HCE-Pgs A-vp2-rrn BT1T2免疫组。TL免疫组中CD8+T细胞和CD4+T细胞的分化率分别为13.3%和21.0%,高于PL免疫组的10.4%和14.0%;TC免疫组CD8+T细胞和CD4+T细胞的分化率分别为11.4%和20.9%,高于PC免疫组的7.2%和11.5%;TE免疫组CD8+T细胞和CD4+T细胞的分化率分别为8.3%和16.7%,高于PE组的7.5%和13.5%。所有免疫组的CD8+T细胞分化率均高于CD4+T细胞。CD8+T细胞属于细胞毒性T细胞,可以裂解感染病毒的细胞、肿瘤细胞和同种异体移植物,在免疫反应中发挥着关键作用。在细胞免疫反应的检测结果中,重组L.plantarum菌株免疫组的CD8+T细胞和CD4+T细胞的分化率高于重组L.casei菌株组和重组L.pentoses菌株组。p PG612-HCE-T7g10-Pgs A-vp2-rrn BT1T2免疫组产生的IFN-γ,IL-2和IL-4细胞因子水平高于p PG612-HCE-Pgs A-vp2-rrn BT1T2免疫组;其它数据显示,IFN-γ水平高于IL-2水平,IL-2水平高于IL-4水平;重组L.plantarum菌株免疫组较其他两个重组菌免疫组能够刺激产生更高水平的Th1和Th2型细胞因子,并且重组L.casei菌株免疫组刺激产生Th1和Th2型细胞因子水平高于重组L.pentoses菌株免疫组。IFN-γ和IL-2等Th1型细胞因子和细胞免疫相关,IL-4等Th2型细胞因子和体液免疫相关。本研究得出结论,含有p PG612-T7g10-vp2质粒的重组L.plantarum乳酸菌免疫原性强,能够针对IBDV提供有效的免疫保护,可作为将来开发IBDV口服疫苗的候选菌株。
[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.
【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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