【作者】 李文超；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2014， 博士

【摘要】 鸡球虫病是严重危害全世界养禽业的一类寄生虫病。目前该病的防治主要依靠化学药物，少量依靠疫苗。但化药治疗由于存在耐药性、药物残才留等不利影响，因此用疫苗免疫代表了未来鸡球虫病防治的发展方向。卡介苗具有良好的安全性和内在佐剂特性，可以诱导坚强持久的细胞免疫，是表达重组外源基因的理想活菌疫苗载体。本实验室前期研究表明表达E. tenella rhomboid与ADF基因的球虫重组卡介苗疫苗具有较好的免疫保护效果，尤其是表达rhomboid基因的重组卡介苗的ACI值达到了180以上，显示用卡介苗来构建鸡球虫疫苗具有较好的前景。巨型艾美耳球虫是集约化鸡场最常见的3种球虫之一，常引起亚临床球虫病，使鸡的饲料转化率、增重和产蛋量受到影响。本研究在克隆巨型艾美耳球虫入侵相关基因AMA1的基础上，对该基因及编码蛋白进行了详细的生物信息学分析，然后构建AMA1基因的大肠杆菌-分枝杆菌穿梭载体和整合载体，SDS-PAGE与Western blotting结果显示成功表达了AMA1基因。用构建的表达AMA1抗原的重组BCG/pMV261-AMA1和BCG/pMV361-AMA1经三种不同免疫途径接种雏鸡，同源株攻虫后显示重组BCG疫苗皮下和滴鼻途径免疫优于口服途径，而且重组卡介苗滴鼻免疫能诱导较强的细胞和体液免疫反应。安全是疫苗应具备的基本条件。鉴于目前尚无卡介苗对鸡是否安全的相关报道，本试验中，将rBCG/pMV361-AMA1以不同剂量皮下注射免疫7日龄雏鸡，通过增重、脏器系数、血液生化以及器官组织学变化等指标来评价重组卡介苗对鸡的安全性。结果显示重组卡介苗正常剂量免疫对鸡增重、脏器系数、生化指标及器官组织结构等均无显著影响（p>0.05），重组卡介苗对鸡总体上是安全的。为进一步增强重组卡介苗的免疫保护效力，本研究利用SOE-PCR将鸡IFN-γ与巨型艾美耳球虫AMA1基因通过一柔性linker串联，构建重组表达载体pMV261-IFN-γ-Linker-AMA1，电穿孔转入卡介苗中，经热诱导后，SDS-PAGE及Western blotting鉴定发现重组卡介苗在约65kDa处有一明显的特异条带，显示融合蛋白成功在卡介苗中表达。将构建的rBCG/pMV261-IFN-γ-Linker-AMA1，rBCG/pMV261-AMA1以及BCG通过滴鼻途径接种雏鸡，经同源株攻虫研究其免疫保护效力。结果显示重组卡介苗免疫对同源株攻虫具有一定的免疫保护效力，其中rBCG/pMV261-IFN-γ-AMA1免疫两次的保护力要高于rBCG/pMV261-AMA1免疫两次，表明IFN-γ具有增强AMA1免疫效果的作用。综上，本研究构建了三种重组卡介苗，免疫试验显示均对同源株攻虫有一定的保护效力，同时评价了重组卡介苗对鸡体的安全性，为重组卡介苗疫苗在鸡球虫病乃至鸡其它传染性疾病中的应用奠定了基础。更多还原

【Abstract】 E. maxima is recognized as one of the three coccidians that distributed universallyin intensive farms.The current control strategy against coccidiosis in poultry isdominated by prophylactic application of anticoccidial drugs. However, increasinggovernment regulations and bans on the use of coccidiostats, the appearance ofmulti-drug-resistant strains of Eimeria, concerns over drug residues in poultryproducts, the lack of new pipeline products and disadvantages of live vaccinesincluding the potential reversion to virulence, an early reduction in weight gain andvariable efficacy between batches suggest that new approaches are required. Althoughseveral attempts have been made to develop subunit vaccines or DNA vaccines, thereis no available vaccine against coccidiosis. A recent approach using live bacteria ascarriers to deliver and express Eimeria antigens with the long-term aim of controllingavian coccidiosis shows great potential for large-scale control of infectious diseases inthe livestock industry.Mycobacterium bovis BCG, an attenuated M. bovis strain, isparticularly attractive for the delivery of heterologous antigens based on itsremarkable safety record and intrinsic adjuvant properties. Since both M. bovis BCGand Eimeria spp. are intracellular micro-organisms, we rationalized that recombinantBCG would be appropriate for the development of a vaccine against coccidiosis.Apical membrane antigen1(AMA1), secreted by micronemes, appears to be essentialduring the invasion of host cells and is currently founded in Toxoplasma gondii,Neospora caninum,Eimeria tenella, and Eimeria maxima. AMA1in Plasmodium isone of the most promising malaria vaccine candidate and currently a hot research, hasimportant significance in the development of malaria vaccines.Based on the fact that the lack of an ideal genetically engineered vaccine againstcoccidiosis,two rBCG strains pMV261-AMA1and pMV361-AMA1expressingAMA1gene of E. maxima,were constructed and their immune protective effect wereevaluated in this study.Subsequently, safety of rBCG/pMV261-AMA1on chickenwere analyzed. For enhancement of the protective efficacy of recombinant BCG in chickens, rBCG co-expressing AMA1and IFN-γgene were constructed, and itsefficacy against homologous infection in chickens were evaluated. This report shouldprovide a new method for the future development of vaccines against coccidiosis.Cloning and bioinformatics analysis of Eimeria maxima AMA1gene:Basedon the published ORF of E. maxima AMA1gene，a pair of primers were designed andAMA1gene was cloned.Subsequently，the structure and functions of this gene wasanalyzed by bioinformatics method. The recults showed AMA1had an open readingframe(ORF) of1623bp encoded a secreted protein with a26th amino acid residuessignal peptide. while there were one transmembrane peptides，three N-glycosylationsites and34phosphorylation sites. AMA1had eight disulfide bond in tis extracellularregion. AMA1had the secondary structure mainly composed of helix，random coiland had the tertiary structure including ten helixs and14th-pleated sheet.Construction of pMV261-AMA1and pMV361-AMA1expression vectorsand expression in BCG:The AMA1gene segment of E. maxima was generated byPCR with primers designed based on sequences of E. maxima AMA1,pMV261andpMV361. The resulting plasmids pMV261-AMA1and pMV361-AMA1wereelectrotransfected into BCG and expressed successfully in rBCG.Western blottingindicated that the MAA1protein had a good immunogenicity.Protective efficacy of recombinant Mycobacterium bovis BCG expressingapical membrane antigen1against homologous infection:Day-old birds wereimmunized twice with rBCG/pMV261-AMA1, rBCG/pMV361-AMA1or BCG viaoral, intranasal, and subcutaneous routes, then orally challenged with homologous E.maxima sporulated oocysts. Gain of body weight, fecal oocyst output, lesion scores,serum antibody responses, numbers of splenocyte CD4+and CD8+T cells, and gutcytokine transcript levels were assessed as measures of protective immunity. Thesubcutaneous and intranasal routes were superior to the oral route based onaugmented weight gain, reduced fecal oocyst shedding, and decreased intestinallesions. Intranasal rBCG immunization induced a strong humoral and cellularresponse directed against homologous E. maxima infection. This study provides datafor the use of rBCG to develop a prophylactic vaccine against coccidiosis.Safety of rBCG/pMV361-AMA1on chickens:Chickens were immunizedsubcutaneously with rBCG/pMV361-AMA1at different dose. The safety of rBCG on chicken was evaluated based on weight gain, organ coefficient, blood biochemicalparameters and tissue structure of organs.The results showed that no significantdifference were observed in weight gain and organ coefficient between differentgroups(p>0.05). The D-BIL content of group1, group2and group3were highersignificantly compared with that of group4(p <0.05), and there were no difference inother blood biochemical parameters between different groups(p>0.05). High doses ofrecombinant BCG immunization had some impact on the chicken heart,and themyocardial cell presented a large number of lymphocytes infiltration. The liver, spleenand other organs had no changes in the different groups.Construction of pMV261-IFN-γ-Linker-AMA1and expression in BCG:Thevector of pMV261-IFN-γ-Linker-AMA1was constructed with chicken IFN-γgene,E.maxima AMA1gene by SOE-PCR, and was electrotransfected into BCG and selectedby kanamycin. After induction period at45℃, the recombinant proteins wereseparated by SDS-PAGE and Western boltting was done for immunoblot analysis. Aband with about65kDa was detected from rBCG/pMV261-IFN-γ-Linker-AMA1,indicatinging that the fusion protein IFN-γ-AMA1had expressed successfully in BCG.Protective immunity of rBCG/pMV261-IFN-γ-Linker-AMA1against Emaxima challenge:Chickens were immunized intranasally with rBCG/pMV261-IFN-γ-Linker-AMA1,rBCG/pMV261-AMA1and BCG, respectively. The efficacy ofimmunization was evaluated on the basis of oocyst output, cecal lesion scores andbody weight. The results showed that the recombinant BCG immunization had certainimmune protective efficacy against homologous challenge.The immune protectiveeffect with rBCG/pMV261-IFN-γ-Linker-AMA1were superior to that with rBCG/pMV261-AMA1,and the result indicated that IFN-γplayed a role in enhancing theimmune protection effect.更多还原