IBDV抗原表位与HBcAg嵌合体基因的构建及其表达产物免疫功能分析

发布时间：2018-05-25 01:26

本文选题：传染性法氏囊病 + 乙肝病毒核心抗原　；参考：《南京农业大学》2010年硕士论文

【摘要】：传染性法氏囊病(Infectious Bursal Disease, IBD)是由传染性法氏囊病病毒(Infectious Bursal Disease Virus, IBDV)引起的以侵害雏鸡淋巴组织,特别是中枢免疫器官—法氏囊为主要特征的传染病。该病不仅引起患病动物死亡,而且还导致机体免疫抑制,使机体的免疫防御能力降低和疫苗免疫接种失败,对易感鸡群实施疫苗接种是预防该病最有效的方法,但由于各地流行的IBDV毒株的毒力与抗原性的差异,给疫苗毒株的选择造成较大的困难,每年由于IBD免疫失败或免疫抑制造成的经济损失巨大。为了抵抗超强毒的攻击以及母源抗体的干扰,目前广泛应用中等毒力的IBD活疫苗,给IBD的防控带来极大的隐患,灭活疫苗存在着抗原制备的困难。鉴于此,进一步分析当前IBDV的分子流行病学、研制新型的基因工程疫苗,仍是当前禽病防控工作中亟待解决的重要课题。研究内容包括两个部分：试验1：IBDV抗原表位与HBcAg嵌合体基因的构建及其表达产物分析为了提高模拟IBDV多表位基因5epis的免疫效力,运用重叠延伸PCR技术,在乙型肝炎病毒核心抗原(Hepatitis B core antigen, HBcAg)基因的231位～232位核苷酸(77位～78位氨基酸残基)之间插入BamH I和EcoR I酶切位点,构建基于HBcAg修饰基因(Modified HBc, mHBc)的抗原表位嵌合体基因分子载体pET-mHBc。将模拟IBDV多表位基因5epis定向插入到pET-mHBc的mHBc基因中,获得嵌合体基因mHBc-5epis表达质粒pET-mHBc-5epis,在大肠杆菌中表达。用SDS-PAGE分析,重组嵌合体蛋白的分子量约为29ku；在Western-blotting试验中,重组嵌合体蛋白与IBDV抗体反应形成1条特异性蛋白带。将重组菌超声破碎后,用电镜观察,可见重组嵌合体蛋白呈病毒样颗粒(VLP),直径约100nm～120nm；用IBDV抗体夹心ELISA检测,抗原效价达到1：200。本试验成功构建了5epis与HBcAg嵌合体基因,在大肠杆菌中高效表达具有IBDV抗原反应性的重组病毒样颗粒嵌合体蛋白,为表位型IBD基因工程疫苗的研究提供了一条新途径。试验2：重组mHBc-5epis嵌合体蛋白的免疫功能分析将重组菌pET-mHBc-5epis诱导表达,用SDS-PAGE胶回收方法纯化重组mHBc-5epis嵌合体蛋白,免疫1月龄SPF鸡,双抗体夹心ELISA法检测免疫鸡血清中的IFN-y、 IL-2、IL-4、IL-6动态变化,在免疫后的第2天IL-2、IL-4表达量都有明显上升;用间接ELISA去检测免疫鸡血清中IBDV抗体,在免疫后的第14天IBDV抗体效价达到1：100。试验表明,嵌合体基因HBcAg分子载体能够增强鸡产生特异性的体液免疫及细胞免疫应答。本试验首次证明了HBcAg在鸡体内能增强抗原表位的免疫保护效力,为新型IBD颗粒化多表位疫苗的研究奠定了基础。
[Abstract]:Infectious bursal disease (Bursal Disease, IBD) is an infectious disease caused by infectious bursal disease virus (IBDV) Infectious Bursal Disease Virus, IBDV), which is characterized by invasion of chicken lymphoid tissue, especially central immune organ-bursa of Fabricius. The disease not only causes the death of diseased animals, but also leads to immune suppression, which reduces the immune defense ability of the body and fails to vaccinate the susceptible chickens. The most effective way to prevent the disease is to vaccinate susceptible chickens. However, because of the difference of virulence and antigenicity of IBDV strains in different places, it is difficult to select vaccine strains. Every year, the economic loss caused by the failure of IBD immunization or immunosuppression is enormous. In order to resist the attack of super virulence and the interference of maternal antibody, the medium virulence IBD live vaccine is widely used, which brings great hidden trouble to the prevention and control of IBD, and the inactivated vaccine has the difficulty of antigen preparation. In view of this, further analysis of molecular epidemiology of IBDV and development of new genetic engineering vaccine are still an important task to be solved in poultry disease prevention and control. The study consists of two parts: Construction of 1:IBDV epitope and HBcAg chimera gene and analysis of its expression products In order to improve the immune potency of 5epis, a mimic IBDV polyepitope gene, the overlapping extension PCR technique was used. To construct the chimeric gene vector pET-mHBcbased on modified HBcAg modified gene, the restriction sites of BamH I and EcoR I were inserted between the amino acid residues at the 77th and 78th position of nucleotide at the 231st and 232nd nucleotides of hepatitis B virus core antigen (B core antigen, HBcAg), and the molecular vector pET-mHBcwas constructed based on the modified HBcAg gene (modified HBc, mHBcc). The chimeric gene mHBc-5epis expression plasmid pET-mHBc-5epis was obtained by inserting the mimic IBDV polyepitope gene 5epis into the mHBc gene of pET-mHBc and expressed in Escherichia coli. By SDS-PAGE analysis, the molecular weight of recombinant chimeric protein was about 29ku. in Western-blotting test, the recombinant chimeric protein reacted with IBDV antibody to form a specific protein band. The recombinant chimeric protein was observed by electron microscope after ultrasonic fragmentation. The recombinant chimeric protein was shown to be a virus like particle with a diameter of about 100 nm and 120 nm in diameter. The titer of the recombinant chimeric protein was 1: 200 detected by IBDV antibody sandwich ELISA. In this study, 5epis and HBcAg chimeric genes were successfully constructed and highly expressed in Escherichia coli with IBDV antigen-responsive recombinant virus-like granular chimeric proteins, which provided a new approach for the study of epitope IBD gene engineering vaccine. Test 2: analysis of immune function of Recombinant mHBc-5epis Chimeric protein The recombinant mHBc-5epis chimeric protein was purified by SDS-PAGE gel recovery method. The recombinant mHBc-5epis chimeric protein was immunized with 1-month-old SPF chicken. The dynamic changes of IFN-y, IL-2, IL-4 and IL-6 in serum of immunized chicken were detected by double antibody sandwich ELISA method. On the second day after immunization, the expression of IL-4 increased obviously, and the titer of IBDV antibody reached 1: 100 on the 14th day after immunization by indirect ELISA to detect the IBDV antibody in the serum of immunized chicken. The results showed that the chimeric gene HBcAg molecular vector could enhance the specific humoral and cellular immune responses of chickens. It was proved for the first time that HBcAg could enhance the immuno-protective effect of antigen epitopes in chicken and lay a foundation for the study of novel IBD granulated multiepitope vaccine.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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