【作者】 许金俊；

【导师】 秦爱建；

【作者基本信息】 扬州大学 ， 预防兽医学， 2004， 博士

【摘要】 γ-干扰素(IFN-γ)是在病毒等特定诱生剂的诱导下，由机体的T细胞和NK细胞所分泌产生的一种具有抗病毒、抗肿瘤和免疫调节功能的细胞因子，一直是现代医学和生物学领域研究的热点，利用基因工程技术生产的商品化重组γ-干扰素已经成功应用于人类疾病的预防和控制。与人IFN-γ相比，动物IFN-γ的研究普遍滞后，自Cerretti 1986年首次报道牛IFN-γ(BoIFN-γ)基因后，国外学者先后利用多种表达系统表达出了重组奶牛IFN-γ(rBoIFN-γ)，并对应用rBoIFN-γ控制奶牛乳房炎进行了一些研究探讨，国内这方面的研究尚处于空白。为了开拓利用rBoIFN-γ控制奶牛乳房炎的新途径，克服传统的依靠药物和疫苗手段控制乳房炎所存在的效果差、细菌耐药性增加、药物残留和免效失败等诸多方面的缺陷，本研究在克隆出BoIFN-γ基因的基础上，利用多种表达系统对BoIFN-γ基因进行了表达和表达产物活性比较分析，选择出具有较高生物学活性的rBoIFN-γ，并在奶牛乳房炎治疗中进行了小规模田间应用试验，旨在为临床上奶牛乳房炎的防治提供新方法和新手段。 一、奶牛γ-干扰素基因成熟肽片段在大肠杆菌中的高效可溶性表达及其特异性抗体的制备 根据已发表的奶牛BoIFN-γ基因的cDNA序列设计—对引物，应用一步法逆转录—聚合酶链式反应(RT-PCR)技术从经PHA刺激诱导的奶牛脾脏淋巴细胞的总RNA中扩增出BoIFN-γ基因不含信号肽的成熟肽特异性cDNA片段。将RT-PCR产物克隆到pUCm-T载体，经过限制性酶切分析、测序证实扩增得到的BoIFN-γ基因序列完全正确。用限制性内切酶将该基因片段从pUCm-T载体切下后按正确的阅读框架亚克隆入大肠杆菌表达载体pGEX-6P-1谷胱甘肽-S-转移酶(GST)基因的下游，经IPTG诱导和SDS-PAGE比较分析证实表达出了与预期大扬州大学博士论文小42 kDa相符的GST一BOIFN·丫融合蛋白。超声波裂解分析发现融合蛋白主要以可溶性形式得到表达，经过薄层扫描测定可溶性融合蛋白表达量约占菌体可溶性总蛋白的25%。通过VSV一MDBK细胞病变抑制试验证实，融合蛋白具有一定的生物学活性，在MDBK细胞上抑制vsv病毒的活性可达到1 .6384x104U/mg。 通过SDS一PAGE电泳分离融合蛋白，切取含目的条带凝胶，液氮冻干碾碎后作为免疫原腹腔免疫BALB/C小鼠，免疫4一5次后采集血清，经过间接EUSA试验证实所制备的抗体特异性的针对GST一BOIFN一丫融合蛋白，为下一步制备抗rBOIFN一Y特异性单抗和rBofFN一丫的表达鉴定提供了有力的保障。二、抗重组奶牛Y一干扰素特异性单克隆抗体的研制及鉴定 利用大肠杆菌表达的融合蛋白GST-BOIFN一丫经SDS一PAGE电泳后，切取融合蛋白条带作为抗原腹腔免疫BALB/C小鼠，取脾脏细胞与SP刀O细胞进行细胞融合，以表达GST-BOIFN一丫的大肠杆菌超声波裂解物和表达GST的大肠杆菌的超声波裂解物分别作为包被抗原，通过间接EUSA方法比较筛选到2株效价高、性质稳定的只与表达GsT一BOIFN一的大肠杆菌超声波裂解物反应而不与表达GST的大肠杆菌的超声波裂解物反应的针对rBOIFN一丫的特异性单克隆抗体，分别命名为4A3和4G5。Westem一blotting印迹试验证实，本研究所获得的单克隆抗体 4A3和4G5均能与IPTG诱导过的表达GST一BofFN一Y融合蛋白的重组菌反应，识别分子量为42kDa特异性蛋白质条带，与IPTG诱导的表达GST的非重组菌不反应，进一步确认得到的单抗特异性识别rBOIFN一Y。间接ELSIA试验检测证实，4A3单抗腹水的效价可以达到1:1.3x107，细胞上清效价可达到卜25600，4G5单抗腹水和细胞上清的效价均比4A3略低。应用捕获ELisA方法进行亚类鉴定结果表明单抗4A3为IgGZb亚型，单抗4G5抗体亚型为191迈。中和试验结果表明，单抗4A3对大肠杆菌表达的rBofFN一丫具有一定的中和作用。本研究所获得的两株单抗为下一步利用peDNA和杆状病毒真核表达系统表达rBofFN一Y时表达产物的检测鉴定和表达产物的亲和层析纯化提供了可靠的手段和保障。三、奶牛补干扰素基因的克隆及其在COs一1细胞中的暂态表达 由于原核表达系统的诸多缺陷，为了开发真核表达的安全、高效、廉价的rB01FN一丫用于生产实践，同时鉴于许多基因在真核表达系统中表达产量极低或不表达，我们对刀口刀刁叭Y基因在COS一1细胞中进行了暂态表达试验.应用两步法RT·PCR技术从经PHA刺激诱导的奶牛脾脏淋巴细胞的总RNA中扩增出含信号肤许金俊:奶牛丫一干扰素基因的表达及其在乳房炎防治中的初步应用的刀协刀刁叭Y基因cDNA片段.将扩增出的刀。刀刁瞬r基因cDNA克隆到pMD 18一T载体中，经过限制性酶切分析筛选正向插入的克隆并测序.将该基因片段从pMD18一载体中用角”dm和刀。功Hl双酶切后克隆到真核表达载体peDNA3.l龙eo。(+)中，构建真核表达载体peDNA一BOIFN一丫，通过脂质体转染COS一1细胞，用抗rBOIFN一Y单抗和FrrC标记的羊抗鼠IgG分别作为一抗和二抗进行间接免疫荧光实验(IEA)检测，结果表明，在COS一1细胞的细胞膜和胞浆内均有rBOIFN一Y的表达，通过VSV一MDBK细胞病变抑制试验证实，转染后细胞培养上清液具有一定的干扰素活性，转染后72h时细胞培养上清液在MDBK细胞上抑更多还原

【Abstract】 Interferon-gamma is a pleiotropic cytokine characteristically prouduced by natural killer cells and T helper 1 type cells in response to antigenic or mitogenic stimulus. Apart from its capacity to directly exert anti-viral activity and anti-tumor activity, it plays many crtitical roles in the modulation of immune reponses. Due to its important biological activity, IFN- Y now is a focus among the medical and biological fields, and recombinant IFN- Y produced by recombinant technology has been successfully implemented for the therapy and prevention of some human diseases. In comparison with human IFN- Y , animal IFN- Y have only recently been investigated. Since it first reported by Cerretti et al in 1986, bovine interferon-gamma (BoIFN- r) gene has been expressed in some prokaryotic and eukaryotic expression systems, and recombinant bovine interferon-gamma (rBoIFN- Y ) has been applied for the control of bovine mastitis at abroad, there is few report in china. Traditional means of mastitis control involve antibiotic therapy and vaccination, which often company with some defects such as antibiotic residues, drug resitance and immune failure, therefore, new and innovative approaches for mastitis control are needed. In this paper, We described the selection of highly active rBoIFN- Y expressed in three different expreesion systems and its preliminary application in the treatment of bovine mastitis.1. Expression of bovine interferon-gamma gene in soluble form in Esherichia Coli and preparation of specific antisera to recombinant bovine interferon-gammaBovine interferon-gamma gene without signal peptide encoding region was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes in response to Phytohemagglutinin (PHA). The products of RT-PCR were cloned into pUCm-T vector. Postive recombinant clone named BoIFN-Y -Tl was identified by restriction enzyme digestion and sequencing. The fragment of BoIFN- Kin the pUCm-T vector was subcloned into pGEX-6P-1. Partly soluble 42 kDaGST-BoIFN- Y fusion protein was expressed after induced by EPTG, which was confirmed by SDS-PAGE and accounted for 25 % of total soluble bacterial proteins detected by Tic-scanning. Recombinant fusion protein showed some anti-viral activity in the cytopathic effect inhibition assay with VSV. The interferon titer in the supernatant of bacterial sonicates was up to 1.6384x104U per milligram.Fusion protein GST-BoIFN- Y separated by SDS-PAGE was extracted from the gel and injected into BALB/C mice once a week for five times. The antisera was collected from the immunized mice, and its specificity to GST-BoIFN- Y was identified by indirect ELISA. The above results suggested that the specific antisera can be used as a useful tool in preparing a specific monoclonal antibody to rBoIFN- Y and identifying the expression of rBoIFN- Y in the next eukaryotic expression system.2. Development and identification of the monoclonal antibody against recombinant bovine interferon-gammaGST-BoIFN- Y fusion protein in the gel was used to immunize the BALB/C mice. Two hydromas secreting specific monoclonal antibodies against rBoIFN- Y were developed by cell fusion between SP2/0 cells and spleen cells from the BALB/C mice immunized with GST-BoIFN- Y fusion protein extracted from the gel. In direct ELISA, these two monoclonal antibodies named as 4A3 and 4G5, respectively, only reacted with sonic supernatant of E. coli expressing GST-BoEFN- Y, and not reacted with sonic supernatant of E. coli expressing GST protein, the tilers of the cultured supernatant and ascites of 4A3 can achieve 1:25600 and 1:1.3x107 respectively. Subclasses of these two McAbs were identified by capture ELISA as IgG2b and IgM respectively. McAb 4A3 showed some potent neutralizing activity against E. coli derived rBoIFN- Y. The hereby reported two McAbs will provided useful tools for detection and purification of rBoIFN- Y expressed in eukaryotic expression systems.3. Expression of bovine interferon-gamma gene in COS-1 cellsBoIFN- rg更多还原