【作者】 徐贤坤；

【导师】 曾宪垠； 刘棋；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 博士

【摘要】 牛结核病（tuberculosis,TB）是由牛结核分枝杆菌引起的慢性消耗性疾病,呈世界性分布。牛结核病的诊断方法主要有病原鉴定和皮内变态反应。细菌学检查需要5～8周时间,检出率低,且常有假阴性结果出现而不适合于流行病学调查和日常监测;结核菌素（PPD）皮内变态反应（TST）是国际贸易指定试验,该法技术简单,价格低廉,但其非特异性强,不是理想的牛结核病诊断方法;而且该检测方法只是针对牛属结核病检测设定,不适用于水牛属、羊等家畜及其他的野生动物结核病诊断。抗原特异性γ-干扰素试验是一种新型体外T细胞免疫检测试验,由于新的结核杆菌特异性抗原CFP10,ESAT6等的发现,提高了检测的敏感性和特异性,其应用于牛结核病的诊断受到广大研究者的关注。然而,目前该法的研究对象主要集中在牛属动物,而未见水牛属结核病检测方法的报道。针对近年来我国南方水牛规模不断扩大,养殖密度不断增加,水牛结核病感染率和发病率急剧攀升却无适合的方法开展检疫的现状,本研究拟利用抗原特异性γ-干扰素原理,建立一种适用于水牛结核病检测的方法,为水牛结核病诊断提供一种新的手段,为检疫和控制水牛结核病提供技术支持。主要的研究工作和成果如下:1、提取经刀豆素（ConA）诱导培养的水牛外周血淋巴细胞培养物总RNA,通过RT-PCR扩增出水牛IFN-γ（WBIFN-γ）前体的eDNA序列,然后将其定向克隆入PMD18-T质粒中,构建重组质粒PMD18-T-WBIFN-γ,并进行测序。序列分析结果表明,克隆获得的IFN-γ基因全长为544个碱基,水牛IFN-γ基因开放阅读框（ORF）为501个碱基,编码166个氨基酸,基因登录号为EF567075。与GenBank上已发表的水牛、牛的IFN-γ基因进行同源性比较,其核苷酸同源性均高于97%,推导氨基酸同源性高于96%。通过亚克隆从重组质粒PMD18-T-WBIFN-γ中扩增出WBIFN-γ的成熟肽段序列,并将其定向插入原核表达载体pGEX-6P-1和PET-32a,经DNA测序确认后,分别转化至BL21（DE3）重组菌,经IPTG诱导表达后,pGEX-WBIFN-γ/BL21与PET-WBIFN-γ/BL21重组菌各自表达出相对分子量分别为43Ku和37.4Ku左右的融合蛋白rGST-WBIFN-γ和rHIS-WBIFN-γ,融合蛋白的相对表达量最高可分别达到菌体总蛋白的约30%和45%,并分别用GST FF蛋白纯化柱和HIS FF蛋白纯化柱对融合蛋白rGST-WBIFN-γ和rHIS-WBIFN-γ进行纯化。将重组菌pGEX-WBIFN-γ/BL21与PET-WBIFN-γ/BL21及其纯化蛋白进行Western-blotting和ELISA试验,检测结果表明两种重组蛋白均能与牛IFN-γ单抗发生特异性反应,均具有良好的免疫活性。2、将pGEX-WBIFN-γ/BL21重组菌经诱导表达后取菌体进行SDS-PAGE电泳,切取rGST-WBIFN-γ目的条带及可溶性纯化蛋白分别免疫Balb/c小鼠,均能使免疫小鼠产生高效价的多克隆抗体。以可溶性重组蛋白rGST-WBIFN-γ和rHIS-WBIFN-γ作为双重筛选抗原建立间接ELISA方法,应用淋巴细胞杂交瘤技术,选取血清效价高的小鼠脾细胞与骨髓瘤细胞融合,经过3轮亚克隆筛选出2株能稳定分泌抗rWBIFN-γ单克隆抗体的杂交瘤细胞株,分别命名为1E4,4A11;免疫球蛋白亚类鉴定其重链均为IgG1型,轻链为κ链,间接ELISA检测杂交瘤细胞培养上清效价均为1:3200,腹水效价均为1:819200;Western-blotting和Dot-ELISA分析显示,2株单抗均能特异性结合rGST-WBIFN-γ和rHIS-WBIFN-γ,而不与BL21（DE3）菌体裂解上清及其他重组蛋白反应,表明2株单抗均为针对水牛IFN-γ的特异性单抗。3、以罗氏培养法培养牛结核分枝杆菌标准株AN5并提取DNA模板,采用重组PCR方法从牛结核分枝杆菌AN5基因组中扩增CFP10-Linker-ESAT6融合基因,并将其定向克隆至原核表达载体PET-32a,构建原核表达质粒PET-CFP10/ESAT6。重组子经酶切及测序鉴定后转化BL21（DE3）大肠杆菌,IPTG诱导后经SDS-PAGE电泳鉴定,其在相对分子量为42Ku表达带有6×HIS蛋白标签的rHIS-CFP10/ESAT6融合蛋白,表达量约占菌体总蛋白的40%,并用HIS FF蛋白纯化柱纯化该蛋白。Western blotting分析显示:该融合蛋白能与牛结核阳性血清发生特异性反应。将重组的该融合蛋白用于刺激单次皮内变态反应阳性牛全血,可以产生高水平的IFN-γ。用Bovigam^TM牛型结核ELISA试剂盒检测结果与皮内变态反应反应对比显示,PPD抗原敏感性为85.71%,特异性为69.23%,符合率为75%:rHIS-CFP10/ESAT6抗原的敏感性为77.78%,特异性为72.27%,符合率为75%。研究结果显示,应用牛和禽PPD比较反应检测牛结核阳性牛IFN-γ释放反应的准确度较低,混合感染牛可能被误判为结核阴性牛,而基于rHIS-CFP10/ESAT6的IFN-γ释放反应不受环境分枝杆菌的影响,检测具有良好的特异性。4、利用筛选得到的两株抗水牛γ-干扰素的单克隆抗体,对其中的一株纯化单抗进行HRP标记作为检测抗体,另一株纯化单抗作为捕获抗体,建立检测水牛结核病的γ-干扰素双单克隆抗体夹心ELISA。通过对封闭液、稀释液等条件进行优化,经过方阵滴定实验确定捕获抗体的最佳浓度为625ng/mL,检测抗体的工作效价为1:100,确定样本经rHIS-CFP10/ESAT6抗原刺激血浆OD值减去阴性OD值大于0.3判定为阳性,检测灵敏度为25ng/mL,批内变异系数为0.52%～6.86%,批间变异系数为5.6%～7.07%,稳定性良好。采集60头单次皮内变态反应水牛肝素抗凝全血,利用牛结核分枝杆菌特异性抗原rHIS-CFP10/ESAT6体外刺激水牛外周血淋巴细胞释放IFN-｜。用所建立的夹心ELISA检测所有样本,与皮内变态反应试验比较结果显示,自制夹心ELISA方法检测水牛结核病的敏感性为81.81%,特异性为71.05%,符合率为75%。该法的初步建立可作为皮内变态反应的辅助方法应用于水牛结核病的检测,为将来摸清广西水牛结核病流行情况,制定相应的防控措施提供技术支持。更多还原

【Abstract】 Bovine tuberculosis(TB) is a chronic bacterial disease of animals and humans caused by Mycobacterium bovis.The disease has a worldwide distribution.In many countries bovine TB is still a major infectious disease among domestic animals and the wildlife population,and transmission to humans constitutes a public health problem.Microbiological diagnosis of M.bovis is an extremely slow procedure which may take as long as 5 to 8 weeks,the sensitivity of culture is not 100%,and false-negative culture results may occur.The tuberculin skin test(TST) is the official test used in the eradication campaign for the detection of infected animals in the field,but the PPD can’t distinguish between M.bovis infection and BCG vaccination.,it isn’t a good BTB diagnosis method for water buffalo,goat and other wildlife.,The interferon gamma(IFN-g) assay is a recently developed whole-blood in vitro assay.Although early versions of IFN-γassays used purified protein derivative(PPD) as the stimulating antigen,PPD contains many mycobacterial antigens,some of which are shared among pathogenic mycobacteria belonging to the M.tuberculosis complex,environmental nontuberculous mycobacteria (NTM),and the vaccine substrain M.bovis BCG.Therefore,specific antigens expressed by M.bovis but absent from BCG constitute prime candidates for differential diagnostic reagents.Recently,two such antigens,ESAT-6 and CFP-10,have been reported to be promising candidates as diagnostic reagents for the detection of M.bovis infection in cattle.Using this peptide cocktail in T-cell assays,M.bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond.This study was aimed to develop McAbs against recombinant Water Buffalo IFN-γand established a sandwich indirect ELISA to detect water buffalo IFN-γ.The results of research are summarized as following:1.Total RNA was isolated from water buffalo peripheral blood lymphocytes stimulated with ConA.The water buffalo interferon-γ(WBIFN-γ) cDNA was amplified by RT-PCR,and then cloned and sequenced.The ORF of the cloned water buffalo IFN-γgene was 501bp in length,encoding a polypeptide with 166 amino acid.Compared with other IFN-γ,the nucleotide homologies between the water buffalo IFN-γgene and the cattle IFN-γgenes were higher than 97%,the amino acid homologies were higher than 96%.The Water Buffalo mature peptide IFN-γwas subcloned and constructed into the expression plasmid of pGEX-WBIFN-γand PET-WBIFN-γ,and then transformed into E.coli BL21(DE3),the rGST-WBIFN-γprotein and rHIS-WBIFN-γprotein was highly expressed under IPTG.The SDS-PAGE results showed that the predicted fusion protein rGST-WBIFN-γwas 43Ku in molecular weight with 30%of bacterial whole proteins,the fusion protein rHIS-WBIFN-γwas 37.4 Ku in molecular weight with 45%of bacterial whole proteins,the two existed in soluble portion and inclusion bodies.The soluble fusion protein was purified using GST and HIS affinity columns respectively.Western-blotting and ELISA analysis were employed to determine the expression,purification and activity identification of the expected protein and indicated that the two recombinant protein possess good immunological activity.2.Balb/c mice(Mus musculus) were immunized four times byrecombinant buffalo interferon-gamma(rGST-WBIFN-γ) expressed in E.coli BL21(DE3) at a dose of 100μg per mice.Murine myeloma sp2/0 cells were fused with splenocytes from the immunized mice. An indirect ELISA using recombinant protein rHIS-WBIFN-γand rGST-WBIFN-γas coating antigens were used to screen antibody-producing hybridomas.Two hybridomas cell stains could produce monoclonal antibody(McAbs) steadily after 3 cycles of subcloning.The titer of the two rWBIFN-γMcAbs in the culture Supernatant are 1:3200, the titer of the two rWBIFN-γMcAbs in the ascite are 1:819200.Both McAbs blonging to IgG1 isotype andκchain,showed positive reaction to rHIS-WBIFN-γin western blotting.3.The gene encoding protein CFP10-ESAT-6 was amplified from mycobacterium tuberculosis AN5 chromosomal DNA by PCR.The PCR product was cloned into the expression plasmid PET-32a to generate the recombinant plasmid PET-CFP10/ESAT-6.The recombinant expression plasmid was transformed into E.coli BL21(DE3).The fused protein CFP10/ESAT-6 with His-tag was expressed after inducing with IPTG.The SDS-PAGE results showed that the predicted fusion protein rHIS-CFP10/ESAT-6 was 42Ku in molecular weight with 40%of bacterial whole proteins.The soluble protein was purified with HIS affinity chromatography.Western-blotting analysis was employed to determine the activity identification of the expected protein and indicated that the recombinant protein possess good immunological activity.The protein was used to stimulate the whole blood to generate high level IFN-γ,the result showed that the IFN-γresponses to rHIS-CFP10/ESAT-6 is better than PPDa and PPDb.4.Double antibody Sandwich ELISA for IFN-γin water buffalo was developed.Using McAbs 1E4 as a capture antibody and HRP-labeled McAbs 4A11 as a detection antibody. The optimal reaction conditions for the assay were determined.The result indicated that the optimal concentration of capture antibody is 625ng/mL,the working concentration of HRP-labeled 4A11 was 1:100,the threshold for serum sample was 0.3,the detection limit of the sandwich ELISA for IFN-γwas 25ng/mL.The diagnosis specificity,sensitivity and coincidence test of DAS-ELISA compared with TST is 81.81%,71.05%,75%,respectivity. Therefore,we propose this DAS-ELISA assay will be a useful tool in the bovine tuberculosis diagnosis in water buffalo.更多还原