【作者】 王秋悦；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2009， 博士

【摘要】 鸡球虫病(Coccidiosis)是由艾美耳属(Eimeria)的各种球虫寄生在鸡的肠道引起的寄生性原虫病,所有日龄和品种的鸡都易感,严重影响了养殖业的发展。卡介苗(BCG)集佐剂与抗原于一身,是表达外源基因的理想活菌疫苗载体。为探讨重组卡介苗(rBCG)对鸡球虫感染的免疫保护作用,本研究首先构建了E.tenella rhomboid基因穿梭表达载体pMV261-Rho和整合表达载体pMV361-Rho,并在BCG内进行了rhomboid基因的诱导表达和免疫原性分析;然后将两种rBCG疫苗通过不同途径免疫雏鸡,观察其对动物免疫保护效果;在此基础上构建EGFP标记的rBCG,对Rhomboid蛋白在鸡体内各组织器官中的分布及稳定性和消长规律进行了研究;此外还将rhomboid基因与鸡IL-2基因(ChIL-2)联合构建rBCG,观察抗球虫效果。结果表明在鸡肝、脾、肺、肾、盲肠各组织器官均检测到rhomboid基因表达,雏鸡免疫14天后基因表达量达到最高,随后开始下降,至28天后消失;动物免疫保护性实验表明rBCG疫苗对E.tenella卵囊的攻击具有一定的免疫保护作用,诱导产生细胞免疫和体液免疫反应,且以滴鼻方式免疫效果最佳,抗球虫指数为174.6;将rhomboid基因与ChIL-2基因联合在卡介苗中表达,结果显示IL-2具有明显增强Rhomboid蛋白的免疫保护作用,其中pMV261-Rho-IL-2滴鼻免疫组保护效果最为明显,抗球虫指数ACI达到180以上,已达到临床使用的要求。本研究为球虫病的预防奠定了基础。更多还原

【Abstract】 Coccidiosis, caused by intestinal protozoan parasites of the genus Eimeria, is a ubiquitous disease in poultry. E.tenella is one of the most important and serious species causing coccidiosis in chickens. Currently the control strategies of coccidiosis rely on chemotherapy and live parasite vaccines. However, due to the continual emergence of drug resistant strains, higher costs for new medicine development and the risks of reversion to highly virulent strains, novel vaccines are urgently needed to prevent further economic loss from the E.tenella infection. Rhomboid protein is the signal-generating component of epidermal growth factor (EGF) receptor signaling during development. Apicomplexan Rhomboid protein, having a potential role in microneme protein cleavage during host cell invasion, is a candidate antigen for development of a coccidiosis vaccine. BCG, the attenuated strain of Mycobacterium bovis, has been considered a promising candidate for the delivery of foreign antigens to the immune system. Several features have encouraged its use as a live carrier for recombinant antigens, including its known adjuvant properties, its ability to elicit humoral or cellular immune responses toward heterologous antigens, its thermostability, its low frequency of side effects and it is inexpensive to produce. A wide range of recombinant BCG vaccine candidates containing foreign viral, bacterial, parasitic or immunomodulatory genetic materials have been developed and evaluated for stimulation of immune response to the foreign antigen, but there is no report has been published on rBCG used in avian species against coccidiosis.Based on our aim to combine the two, two rBCG strains pMV261-Rho and pMV361-Rho expressing rhomboid gene of E.tenella were constructed in this study. Animal experiments via intranasal, oral and subcutaneous route in chickens were carried out to evaluate the immune efficacy of the vaccines. The protective effects of immunization were evaluated by mortality, cecal lesion scores, oocysts output and growth performance after a challenge with E.tenella oocysts. We also constructed M. bovis BCG recombinants expressing enhance green fluorescent protein (EGFP) as a fusion protein with Rhomboid antigen and evaluate the stability and the localization of the rBCG in tissues of chickens. For enhancement of the protective efficacy of E. tenella infection in chickens, recombinant BCG (rBCG) co-expressing rhomboid and ChIL-2 gene were constructed, and its efficacy against coccidiosis in chickens were evaluated. This report should provide a new method for the future development of vaccines against coccidiosis.Construction of pMV261-Rho and pMV361-Rho expression vectors and expression in BCGThe rhomboid gene segment (GenBank accession No. DQ323509) of E.tenella F2 strains was generated by PCR with primers, and was subcloned into the PstI/ClaI sites of pMV261 and PvuII/ClaI sites of pMV361, respectively. The resulting plasmids pMV261-Rho and pMV361-Rho were electrotransfected into BCG and selected by kanamycin. The results showed that the two vectors pMV261-Rho and pMV361-Rho containing rhombid gene were successful constructed, and Western blotting indicated that the Rhomboid protein was successfully expressed in rBCG. This study has established foundation for further study of E.tenella rBCG.Protective immunity of rBCG pMV261-Rho and pMV361-Rho against E.tenella challengeChickens were immunized with rBCG pMV261-Rho and pMV361-Rho via intranasal, oral and subcutaneous route, with BCG immunized as control. After 1 and 2 week intervals, each sample was boosted. Chickens were then challenged with E.tenella sporulated oocysts and the efficacy of immunization was evaluated on the basis of oocyst output, cecal lesion scores, ACI, response immunity of cellular and antibody. All the rBCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+ and CD8+ cells compared to the control(P<0.01). Vaccination via intranasal and oral produced better protective effect, The anticoccidiosis index(ACI) of intranasal immunized rBCG pMV261-Rho-IL-2 groups was 174.6. Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge.Construction of EGFP-tagged rBCG of E.tenella and distribution in chickensThe fragment of EGFP and rhomboid were subcloned into the pMV361 integrative expression vector, with correct structures was then transformed into BCG by electroporation. Chickens were immunized with this rBCG pMV361-Rho-EGFP, and one week after the immunization, various tissues containing liver, spleen, lung, kindey and caecum from rBCG pMV361-Rho-EGFP immunized chickens were removed and processed to frozen section. Expression of Rhomboid protein in liver, spleen, lung, kindey and caecum was observed by laser confocal microscopy. At the 7th, 14th, 21st, 28th day after the immunization, RNA from liver, spleen, lung, kindey, caecum tissues was extracted. The results showed that EGFP fluorescence expression of various tissues containing liver, spleen, lung, kindey and caecum from rBCG pMV361-Rho-EGFP immunized chickens was observed by laser confocal microscopy one week after the immunization. Real-time quantitative RT-PCR revealed that a rise of rhomboid expression level at 7th day and a peak at 14th day and a disappearance at the 28th day after immunization.Construction of pMV261-Rho-IL-2 and pMV361-Rho-IL-2 and expression in BCGChIL-2 is an important cytokine with the ability of enhancement of the immune responses. ChIL-2 gene segment was ligated together with rhomboid fragment and subcloned into the pMV261 and pMV361 expression vector, separately. The recombinant plasmids pMV261-Rho-IL-2 and pMV361-Rho-IL-2 was electrotransfected into BCG and selected by kanamycin. After induction period at 45℃, the recombinant proteins were separated by SDS-PAGE and Western boltting was done for immunoblot analysis. A band with the molecular masses of about 40 kDa was detected from rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2, suggesting that the fusion protein Rho-IL-2 had expressed successfully in rBCG Protective immunity of rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2 against Eimeria tenella challengeChickens were immunized with rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2 via intranasal, oral and subcutaneous route, and boosted after 1 and 2 week intervals. Chickens were then challenged with E.tenella sporulated oocysts and the efficacy of immunization was evaluated on the basis of oocyst output, cecal lesion scores, ACI, cellular immunity response and antibody immunity. All the rBCG immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+and CD8+cells compared to the control(P<0.01). Challenge experiments demonstrated that the two rBCG strains could provide significant protection against E. tenella challenge. The level of CD4+,CD8+ lymphocyte and the ratio of CD4+/CD8+ of immunized chicken were increased significantly compared with controls(P<0.01). Chickens immunized rBCG revealed a significant decrease in oocyst output, especially in the group of intranasal immunized rBCG pMV261-Rho-IL-2. The ACI of rBCG pMV261-Rho-IL-2 and pMV361-Rho-IL-2 immunized groups were above 160. The highest ACI is 184.0 in the group of intranasal immunized rBCG pMV261-Rho-IL-2. The protective effect in pMV261-Rho-IL-2 and pMV361-Rho-IL-2 immunized groups are better than the groups received rBCG pMV261-Rho and pMV361-Rho.更多还原