【作者】 张月美；

【导师】 步志高；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2009， 硕士

【摘要】 干扰素(Interferon,IFN)是在特定的诱生剂作用下,由细胞产生的一种具有高度生物学活性的糖蛋白,在同种动物细胞上具有广谱的抗病毒活性,其活性的发挥又受细胞基因组的调节和控制。IFN-γ的免疫学检测(IFN-γ实验)是在特异性抗IFN-γMcAb基础上建立的用于样本中IFN-γ定性定量分析的实验方法。国外学者已经将牛γ干扰素(Bovine Interferonγ,BoIFN-γ)的单克隆抗体用于多种牛疫病的研究。最近几年来,国内这方面的报道也逐渐增多,但检测方法建立的报道比较少。本研究克隆并表达了BoIFN-γ,制备了两株针对不同表位的高亲和力BoIFN-γ单克隆抗体,并在此基础上建立了BoIFN-γ的抗原捕获ELISA检测方法。本研究可分为两个部分:第一部分:本研究以原核表达并纯化的BoIFN-γ免疫6周龄雌性BALB/c小鼠,加强免疫后取脾细胞与SP2/0细胞融合,用重组杆状病毒表达的BoIFN-γ作为包被抗原采用ELISA方法筛选阳性克隆,经3-5次亚克隆,最后获得2株抗体效价较高的抗BoIFN-γ特异性单克隆抗体,分别命名为2G6和5F9,其重链分别为IgG1、IgG2b,轻链均为Kappa链。经间接ELISA、Western blot和抗病毒活性阻断实验表明两株单抗具有高度特异性。对2G6和5F9单克隆抗体进行相加ELISA实验,证明两株单抗针对BoIFN-γ不同的抗原表位。第二部分:抗原捕获ELISA检测方法的建立。2G6和5F9腹水经Protein G纯化后,将其中效价高的5F9进行辣根过氧化物酶标记作为检测抗体,并利用2G6作为包被抗体建立了抗原捕获ELISA方法,用于检测BoIFN-γ。对ELISA各个反应条件进行优化后,得到的最佳工作条件为:包被抗体浓度0.127μg/孔,标准蛋白1:20稀释,酶标抗体浓度0.130μg/孔,包被抗体4℃过夜,标准蛋白反应时间1.0h,酶标抗体作用时间1.0h,包被液为20mM pH8.5的Tris-Cl,封闭液为3%鱼皮胶。重复性实验表明,同一样品的板内和板间的变异系数均小于10%。特异性试验表明本试验制备的单抗与牛α、β干扰素不反应。本试验所建立的ELISA方法可检测到pET-BoIFN-γ表达的BoIFN-γ的敏感性为98.43ng/mL,可分别检测到2IU/100μL的rBac-BoIFN-γ表达的BoIFN-γ、10IU/100μL的r-NDV-lasota-BoIFN-γ表达的BoIFN-γ。包被抗体的酶标板经老化试验和未做此试验的板同时保存于30℃、4℃和-20℃,定期进行ELISA检测,结果表明:未做老化试验的板在30℃、4℃保存时极不稳定,而做老化试验的板在30℃、4℃、-20℃可稳定保存4个月,未作老化试验的板在-20℃可稳定保存3个月。该方法的建立为抗原捕获ELISA定量检测BoIFN-γ试剂盒的研制奠定了基础。该研究建立了高效的免疫学检测BoIFN-γ方法,在牛疫病防治和检测上具有十分重要的意义。更多还原

【Abstract】 Interferon (IFN) is a kind of inducing glycoprotein which possesses high bioactivity and broad-spectrum antiviral activity on the homogeneity animal cells, its function is controlled and regulated by cell genome. IFN-γtest is established on the base of IFN-γMcAb which can detect the qualitation and quantity of interferon gamma. IFN-γand its monoclonal antibody also have a broad application in veterinary medicine. Some foreign scientists have reported that monoclonal antibody has been used for the research of bovine diseases. Some researches have been reported in China in recent years, but a few researches for detection. This study cloned and expressed recombinant BoIFN-γ(bovine interferon gamma), produced monoclonal antibodies against BoIFN-γand established a sandwich direct ELISA to detect BoIFN-γ.Our study includes two parts:Part one: The BoIFN-γgene fragment was cloned into prokaryotic expression plasmid. The recombinant protein was purified by affinity chromatography and used to immunize BALB/c mice. After three to five sub-clone,two hybridomas 2G6 and 5F9 secreting monoclonal antibodies to BoIFN-γwere established by ELISA . The two McAbs recognized BoIFN-γspecifically by western blot and indirect ELISA. The antiviral activity of BoIFN-γexpressed by recombinant baculovirus could be blocked by the two McAb ascites.The additivity ELISA demonstrated that the two McAbs recognized different antigenic regions.Part two: A sandwich ELISA for detection of BoIFN-γwas developed utilizing 2G6 McAb and HRP-labled 5F9 McAb which could detect BoIFN-γspecifically.The optimal working condition was listed as below. The dilution of capture McAb was 0.127μg/well, the standard protein was diluted to 1:20 and the dilution of HRP-labeled 5F9 was 0.130μg/well , the reaction time for coating antibody and HRP-labled 5F9 were all 1.0h in the assay,the coating buffer was 20mM Tris-cl pH8.5,the blocking solution was 3% gelatin from cold water fish skin, CV of intra-plate and inter-plate was lower than 10%. All the specific test in this paper demonstrated the two McAbs had good specificity .The result showed that the ELISA had a detection limit of 98.43ng/mL BoIFN-γexpressed by pET-30a(+)-BoIFN-γin BL21, 2IU/100μL BoIFN-γexpressed by rBac-BoIFN-γ, 10IU/100μL BoIFN-γexpressed by r-NDV-lasota-BoIFN-γ.After accelerated temperature studies for protein activity of 2G6 coating microplate the result showed that it could endure 30℃for 4 months.更多还原