【作者】 姜勇；

【导师】 郭爱珍；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2012， 硕士

【摘要】 结核病是一种人兽共患的慢性消耗性传染病,是目前致死亡人数最多的传染病,由结核分枝杆菌复合群(Mycobacterium tuberculosis complex, MTC)成员引起。猕猴(Macaca mulatta, Mm)为我国Ⅱ级重点保护野生动物,也是医学研究中具有很高价值的实验动物,在我国分布广泛。非人灵长类是结核病的敏感物种,其中猕猴最为易感。猴群发生结核病,会导致猴群的损失,同时实验猴发生结核分枝杆菌感染会对实验项目产生严重干扰,并可能传染人。目前,尚无有效疫苗预防猕猴结核病,控制猕猴结核病的主要措施是“检疫—扑杀”。因此,一种敏感性和特异性俱佳的猕猴结核病检测方法是实施该措施的前提条件。结核菌素皮肤试验(tuberculin skin test, TST)是目前最常用的结核病检测方法。此方法操作繁琐,主观性强,假阳性率高。干扰素是一种具有多种生物活性的低分子量糖蛋白,结核分枝杆菌感染能刺激体内免疫细胞产生IFN-γ。利用这一原理建立的结核病IFN-γ体外释放检测法在发达国家已被广泛用于牛结核和人结核的感染检测。国外也有猴结核IFN-γ检测试剂盒。但这些产品均价格高,且货源难得。本研究旨在自主建立猕猴结核IFN-y检测法,为我国猕猴结核的诊断与防控提供技术手段。主要研究内容包括：克隆猕猴IFN-γ(MmIFN-y)基因,进行了原核和真核表达,进一步纯化了IFN-γ重组蛋白,制备和筛选了抗MmIFN-y的单克隆抗体；制备了抗MmIFN-y的多克隆抗体。在此基础上建立了检测MmIFN-y的间接夹心ELISA方法,通过临床样本检测验证了该方法的可行性。1.猕猴IFN-γ的克隆与原核和酵母表达根据本实验室保存的猕猴IFN-γ cDNA设计一对引物并进行扩增,将MmIFN-y基因克隆至原核表达载体pET-30a,转化大肠杆菌BL21,经IPTG诱导,结果成功表达出大小为24kDa左右的His-tag-MmIFN-y重组融合蛋白。同时,根据本实验室保存的猕猴IFN-γ cDNA设计另一对引物并进行扩增,进一步将MmIFN-y基因克隆至酵母表达载体pPICZaA,电转化酵母GS115感受态细胞,经甲醇诱导,结果表达出17kDa的MmIFN-γ和约22kDa的糖基化重组MmIFN-y蛋白。2.猕猴IFN-γ单克隆抗体、多克隆抗体的制备以及鉴定利用原核表达的MmIFN-y免疫Balb/C小鼠,用原核表达的MmIFN-y建立间接ELISA筛选杂交瘤细胞株,获得10株单克隆抗体,分别命名为2A1、2C3、2F6、2H8、3A1、3B2、3E5、3F6、3G7、3H9。单抗腹水效价在3.2×103(3G7)-6.5×106(2A1)之间,单抗相对亲和力在104-107之间。间接ELISA表明10株单抗都能和酵母表达MmIFN-y进行反应。利用原核表达的MmIFN-y免疫日本大耳白兔,获得高免血清,经纯化获得兔抗MmIFN-y多克隆抗体,ELISA抗体效价为5.12×104。3.间接双夹心ELISA方法的建立和应用用2F6株单抗、多抗及HRP标记的羊抗兔IgG,建立检测MmIFN-y的双夹心ELISA万法。此ELISA方法检测原核表达的MmIFN-y灵敏度达到125pg/mL,同时能检测猕猴天然IFN-y。用该方法检测了7份结核菌素皮试阴性猴的外周血细胞经牛PPD刺激后的培养液,结果全为阴性。检测阴性猴外周血细胞ConA刺激后的培养液,结果为阳性。检测灵敏度为125pg/mL时,其对应OD630值为0.18,相当于7份阴性样本的OD630平均值+3.5SD。一般阳性样本判断值标准为OD630值+2-3SD,因此,推测本试剂盒能够用作猕猴结核病的检测。此外,据报道,结核感染猕猴的全血经PPD刺激后释放的IFN-γ量都在200pg／mL以上。因此,虽然本本研究尚未应用于结核感染阳性猕猴的检测,但所建立的ELISA方法为猕猴结核病的检测和防治奠定了良好基础。更多还原

【Abstract】 Tuberculosis (TB) is a zoonotic, chronic and consumptive infectious disease with the highest death in infectious diseases of human. It was caused by Mycobacterium tuberculosis complex (MTC). Macaque (Macaca mulatta, Mm) is widely distributed in China and put in the list of Grade II protectivewild animal species in our country. Furthermoreand it is of a high value as an experimental animal species in medical research. Macaques are the most susceptible species to TB among the non-human primates. The tuberculosis will lead to loss of monkeys and interfere experiments. In addition, monkey TB may transmit to human. Since there is not an effective vaccine for Macaque TB prevention, the only method to control macaque TB is "detection-slaughtering". Therefore, a diagnostic method for TB which has a good sensitivity and specificity is a prerequisite for the implementation of this policy.The most commonly diagnostic method for TB is the tuberculin skin test (TST). This method is cumbersome and subjectivity, often gives false positivedue to the cross-reactivity to the non-tuberculosis mycobacteria. Interferon-y(IFN-y) is a multifunctional glycoproteins. MTC infection can stimulate IFN-y production. Therefore, IFN-y in vitro release assays based on the IFN-y production by pheriperal blood cells in vitro after stimulation of Mycobacterium tuberculosis specific proteins are widely used in cattle TB and human TB detection in developed countries. Monkey IFN-y in vitro release assay is available in international market, but expensive and unavailable in Chinese market.Thus this paper was aimed to establish a monkey IFN-y in vitro release assay and provide an effective tool for monkey TB detection. The main contents include cloning and expressing the Macaque IFN-y (MmIFN-y) gene, making the purified recombinant MmIFN-y, preparing monoclonal antibodies and polyclonal antibodies of MmIFN-y, and finally establishing a indirect sandwich ELISA for detection of MmIFN-y.1. Cloning and expression of MmIFN-y in Escherichia coli and Pichia pastoris YeastBasing on the sequence of MmIFN-y cDNA saved in our lab, one pair of primers was designed and the MmIFN-y cDNA was cloned into the prokaryotic expression vector pET-30a. Then the recombinant vector was transformed it into E. coli BL21and the expression was induced by IPTG. The soluble protein was expressed with molecular mass of about24kDa. It is a fusion protein of his-tag and MmIFN-y. Basing on the sequence of MmIFN-y cDNA saved in our lab, another pair of primers was designed and MmIFN-y cDNA was cloned into yeast expression plasmid pPICZaA. The resulting recombinant vector was transformed by electroporation into yeast competent cells GS115. The expression of MmIFN-y was then induced by methanol and the resultant proteins were recombinant MmIFN-y of about17kDa and glycosylated MmIFN-y of22kDa.2. Preparation and characterization of MmIFN-y monoclonal antibodies and polyclonal antibodies.In this research, we used the recombinant MmIFN-y expressed in E. coli to immunize Balb/C mice. The protein was used to screen hybridoma cells which were able to produce monoclonal antibodies to MmIFN-y with an indirect ELISA. Finally10hybridoma cells secreting monoclonal antibodies were obtained, and designated as2A1,2C3,2F6,2H8,3A1,3B2,3E5,3F6,3G7and3H9respectively. The titers of the monoclonal antibodies ascites were between3.2×103(3G7) and6.5×106(2A1). The relative affinity of the monoclonal antibodies ranged between104and107. By using the indirect sandwich, it was showed that these10monoclonal antibodies can react to MmIFN-y expressed in yeast.At the same time, we used the MmIFN-y expressed in E. coli to immunize rabbits, then the rabbit hyperimmune antisera were produced. After purification, the polyclonal antibody IgG against MmIFN-y with the titer of5.12×104was obtained.3. Establishment and application of indirect sandwich ELISABy using McAb2F6as the captive antibody and PcAb as the detection antibody, an indirect sandwich ELISA was established. The sensitivity of this assay to detect MmIFN-y was125pg/mL. Meanwhile, this ELISA is able to detect the natural IFN-y produced by pheriperal blood cells after stimulated with ConA. Seven blood samples from TB negative monkeys were detected to be negative with this ELISA after stimulated with bovine PPD, while positive after stimulated with ConA. Furthermore, the detectable limit125pg/mL was corresponding to0.18of OD630. This was equal to value of Mean+3.5SD for the negative samples. Since the cutoff value is generally defined as Mean+2-3SD, this sensitivity of the ELISA can meet the requirement of clinical detection. In addition, it was reported that the pheriperal blood cells from infected macaques usually release IFN-y more than500pg/mL. Therefore, this home-made macaque IFN-y in vitro release assay can be used in the detection of macaque TB And Thereby this research provides a promising tool for macaque TB detection and thereby TB control.更多还原