【作者】 窦永喜；

【导师】 才学鹏；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2008， 博士

【摘要】 IFN-γ是由机体内活化的T淋巴细胞和NK细胞产生的一种糖蛋白,丝裂原以及抗原特异性刺激的T细胞克隆也可产生和分泌。IFN-γ除具有抗病毒、抗胞内寄生菌、抗胞内寄生原虫、抗细胞增殖的作用外,还具有独特而强大的免疫调节功能,可促进MHCⅡ类抗原的表达,增强APC与T细胞的相互作用,增强Th细胞和CTL转化的能力等。IFN-γ有较为严格的种属特异性,不同物种之间通用使其活性降低或失去作用。IFN-γ活性形式为两条单体链结合形成的同型二聚体形式,单体没有生物学活性。IFN-γ作为机体免疫应答的产物,一方面是机体发挥免疫功能,清除胞内寄生病原体不可缺少的成分,与疾病的发生、发展有着密切的关系;另一方面,机体内IFN-γ分泌过量,可引起病理性反应。由于IFN-γ具有上述诸多重要的生物学活性,故其在疾病的诊断、治疗和预防等方面具有广阔的应用前景。但是,IFN-γ在临床应用时可引起发热、寒战、恶心等一系列的副作用,而且有些不良反应是不可逆的损害,这限制了其在临床中的应用。因此,开展新型、低毒副作用的pIFN-γ研究意义重大。此外,IFN-γ必须与受体相结合,通过受体的介导才能发挥其生物学效应,对其受体的研究将有利于明确IFN-γ的作用机制,进而发现疾病治疗和预防的新途径。本论文在对克隆的pIFN-γ基因进行适当的改造,去除其信号肽序列,将该基因与pGEX-4T-1载体上的谷胱甘肽S-转移酶(GST)拼接,然后在原核表达系统中进行表达,并对其诱导表达条件、蛋白纯化方法进行系统研究,最终获得了纯度高达95%的GST-pIFN-γ融合蛋白,产量为0.4~0.5g/L培养液。体外试验证实该重组蛋白具有较高的生物活性,可有效抑制PRV(DNA病毒)和FMDV(RNA病毒)的复制,并且具有稳定、不宜降解和低毒副作用的优点。开展了重组pIFN-γ体外抗弓形虫研究,结果表明pIFN-γ可诱导猪腹腔巨噬细胞产生抗虫作用,且随pIFN-γ量的增加抗弓形虫作用越明显,但任何浓度的重组pIFN-γ对弓形虫入侵PK15细胞无明显影响。对pIFN-γ诱导永生化细胞(PK15细胞)凋亡的作用及其对细胞端粒酶的影响进行了研究,结果发现在高浓度pIFN-γ的长时间诱导下,PK15细胞端粒酶显著下降,因此PK15细胞(肿瘤样细胞)发生凋亡,具体表现为细胞变圆脱落,DNA出现典型的“梯状带”,荧光染色可发现细胞核固缩,有凋亡小体出现;但如果用低浓度pIFN-γ进行诱导作用,PK15细胞端粒酶明显升高;该研究结果为IFN-γ临床治疗肿瘤提供了理论依据和指导。以表达纯化的GST-pIFN-γ作为抗原免疫BALB/C小鼠,取其脾细胞和SP2/0细胞融合,再经纯化的GST-pIFN-γ和的带组氨酸标签pIFN-γ作为包被抗原进行筛选,最终获得2株分泌pIFN-γ单抗的杂交瘤细胞株,经鉴定这两株单抗抗体亚类均属于IgG2a,轻链为κ型,对杂交瘤细胞株的染色体组型、单抗的稳定性及其识别的抗原位点等进行分析,结果证明所获2株单抗均表现稳定,可满足常规实验的要求。在对不同动物IFNGR两条链基因序列比对基础上,选择保守区段作为引物;然后分离猪外周血淋巴细胞,经刺激后提取细胞总RNA,应用RT-PCR方法克隆了猪IFNGR两条链基因,序列测定表明猪IFNGR1基因ORF为1413bp,共编码470个氨基酸;猪IFNGR2基因ORF为1110bp,共编码369个氨基酸。对不同物种IFNGR序列比较发现,不同物种间的差异比较大,据此推测这可能是IFN-γ种属特异性的重要原因之一。然后利用分子生物学软件对两条链进行结构预测,设计引物扩增猪IFNGR胞外区片段并对其进行原核表达,经鉴定该基因在原核中得到正确表达;开展了两条受体链胞外区对pIFN-γ抗病毒活性的影响研究,发现猪IFNGR1胞外区可降低pIFN-γ抗病毒活性,而IFNGR2胞外区可增强pIFN-γ抗病毒活性,由此可知猪IFNGR两条链均可结合pIFN-γ;此外,推测猪IFNGR1具有信号转导功能且是IFN-γ信号转导的主要执行者,而IFNGR2则在IFN-γ信号转导中起次要作用或没有信号转导的能力。更多还原

【Abstract】 Interferon-gamma (IFN-γ) is a kind of glycoprotein generated by activated T cells and NK cells and could also be produced and secreted by T cells which were stimulated by mitogen or special antigen. Besides the function of antivirus, anti-intracellular bacteria and parasitic protozoa, anti-tumor, IFN-γhas also the distinct immune regulation function. It can promote expression of MHCⅡantigens, enhance the interaction between antigen present cells (APC) and T cells, accelerate differentiation of Th cells and cytotoxicity T lymphocyte (CTL), and so on. IFN-γposses strict species-specificity and the activity would be depressed or missed if it were in common use in different species. IFN-γneeds to form homodimer through combination of two monomer chains to have the bioactivity since the monomer chain do not have the bioactivity. As a product of host immune response, IFN-γis a necessary component to eliminate intracellular pathogen and is closely related to the nosogenesis and development of disease, on the other hand, if IFN-γwere over secreted, it would cause pathological reaction. Accordingly, IFN-γhas an extensive application in the aspects of diagnosis, therapy and disease prevention, however, IFN-γhas some side effect including fever, shakes, nausea, and so on. Furthermore, some side effect could cause nonreversible damage. All of these limited the clinical application of IFN-γand it is important to develop a new type IFN-γwith lower side effect. IFN-γexerts the biological activities through binding to its receptors which will transform the signal into intracellular. So the receptor play a critical role and the study on IFN-γreceptor (IFNGR) would be benefit to demonstrate the mechanism of IFN-γand find the new methods for treatment and prevention of clinical disease.In this paper, the cloned pIFN-γgene was reconstructed to cut off the signal sequence and fused with GST presented in the pGEX-4T-1 vector. The recombinant gene was expressed in prokaryotic expression system. A series of study was carried out such as induction conditions, recombinant protein purified methods and eventually the recombinant protein was obtained with the purity of up to 95% and the yield of 0.4~0.5 g per litre culture medium. In vitro experiment indicated that the purified protein had a higher bioactivity and could efficiently inhibit the replication of PRV and FMDV. In addition, the recombinant protein had a good stability and lower side effect. The effect of GST-IFN-γanti-toxoplasma gondii was studied and the results showed that pIFN-γcould induce the anti-toxoplasma gondii effect of the porcine abdominal macrophage cell, and the function of anti-toxoplasma goddii presented obviously with the increase of pIFN-γdose. But it could not induce PK15 cell to generate anti- toxoplasma goddii effec. pIFN-γhas the ability of inducing immortalized cells (PK15 cell) apoptosis. At the high concentration of pIFN-γ, the level of PK15 cell telomerase significantly decreased and apoptosis appeared, such as cell rounding and falling off and the“ladder pattern”of the DNA, pyknotic nucleus and apoptotic body appeared by fluorescein stain. However, at the low concentration of pIFN-γ, the level of PK15 cell telomerase significantly increased. This provided the direction of clinical treatment of the tumor with pIFN-γ. The BALB/C mice were immunized with purified GST-pIFN-γand then the spleen cell and SP2/0 were fused. The fusion cell was selected by GST-pIFN-γand pIFN-γwith His tag as antigens. Finally 2 hybridoma cell strains which secreted anti-pIFN-γmonoclonal antibody were obtained. The identification of the monoclonal antibody showed all of them belong to IgG2a subclass withκtype light chain. Analysis of the caryotype, antibody stability and antigen site showed that antibody was stable. After clustal alignment of IFNGR gene sequence from many species, conservative region was selected as primer to amplify porcine IFNGR gene. Porcine peripheral blood lymphocyte was isolated and stimulated in vitro, then total RNA was extracted. Porcine IFNGR two chain genes were cloned by RT-PCR method. Sequence analysis showed that porcine IFNGR1 ORF had 1413 bp, encoding 470 amino acid and porcine IFNGR2 ORF had 1110 bp encoding 369 amino acid. Sequence comparison showed that they were obviously different from those of other species and that is the possible reason that IFN-γpossesed species-specificity. The primers used to amplify extracellular region of IFNGR was designed based on the protein structure prediction. The amplified fragment was expressed in E.coli system. The study of relationship between IFNGR extracellular region and character of pIFN-γantivirus was carried out. The results showed IFNGR1 extracellular region decreased the antivirus activity of pIFN-γ, but IFNGR2 extracellular region increased the antivirus activity of pIFN-γ. These results proved that 2 chains of IFNGR has the function of binding pIFN-γ. In addition, according to the results, it can draw a conclusion that porcine IFNGR1 occupied mainly position in signal transmit, but IFNGR2 had occupied secondary position in signal transmit or not function of signal transmit.更多还原