【作者】 刘文兴；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 动物检疫与动物源食品安全， 2011， 博士

【摘要】 布鲁氏菌病(Brucellosis)是世界范围内发生的一种重要的人畜共患病,严重危害到流行地区畜牧业健康稳定发展,并对人的生命财产安全构成直接威胁。近年来该病在世界许多国家和地区,尤其是发展中国家,重新发生并流行。常规血清学诊断方法和常规弱毒疫苗的应用及其相应的综合防控措施,在动物布鲁氏菌病的诊断与防控史上发挥了重要作用。但是常规弱毒疫苗的残余毒力易致孕畜流产,疫苗菌株的不稳定易影响受免动物的最终免疫效果,并引起变态不良反应。常规的血清学诊断方法也不能区分自然感染与疫苗免疫引起的抗体反应。布鲁氏菌bp26基因缺失突变的新型标记疫苗,为动物布鲁氏菌病的防控与净化提供一条新途径,不仅秉承了亲本疫苗株的生物学特性和良好免疫保护性能,而且比亲本株的毒力更低,遗传稳定性更好,同时通过携带有bp26基因编码蛋白的靶标(缺失基因后的负向标记)可建立相应的鉴别诊断方法。但布鲁氏菌bp26蛋白作为诊断抗原存在假阳性现象,bp26蛋白作为诊断用抗原的抗原特异性有待提高；另外,不同宿主动物对不同毒力菌株bp26基因编码蛋白的免疫原性差异,也会影响该蛋白作为鉴别诊断用抗原的实际应用效果。试验1：布鲁氏菌bp26基因的扩增、序列测定及其生物信息学分析布鲁氏菌属各菌种间的bp26基因高度保守,该基因编码的蛋白已证明对羊、牛和人均具有良好的免疫原性,是一种有望成为该病诊断的候选抗原及作为该基因突变缺失疫苗株的分子靶标。本试验参考Rosetti et al (1996)的文章和序列(AY166766),设计一对引物,以羊种布鲁氏菌疫苗株M5-90的DNA为模板,通过PCR特异扩增,获得了预期长度为990bp的产物,经核酸电泳初步鉴定后纯化。对PCR纯化产物测序结果表明PCR扩增片段上包含了布鲁氏菌bp26基因的完整阅读框(ORF)。最后利用相关软件对该基因及其编码蛋白进行生物信息学分析,结果表明M5-90株bp26基因在布鲁氏菌属各代表种核苷酸组成(除犬种外,仅59.4%)、氨基酸组成均高度保守,同源性分别达到99.3%、97.6%以上；编码蛋白的理论分子量MW=26.569Da,等电点pI=6.62；在氨基酸第1-29位置间存在信号肽Ⅰ可能性大；该蛋白N端第29-39、64-69、109-120、171-176和204-209的五个区段较有可能为B细胞抗原的线性表位。试验2：布鲁氏菌bp26和Δbp26的基因克隆及其原核表达重组子的构建以布鲁氏菌M5-90的DNA为模板,PCR扩增bp26和截断bp26(Δbp26)基因,PCR产物经BamHⅠ和HindⅢ-L酶切后,连接到原核表达载体pQE32,重组质粒转化到感受态细胞M15[pREP4]中,在LB-Km-Amp固体培养基上筛选阳性克隆,菌落PCR鉴定,提取质粒进行单、双酶切鉴定和测序鉴定。结果表明获得了插入子序列与连接部位正确且阅读框完整的原核表达重组子(pQE32-rbp26和pQE32-r△bp26)。试验3：几种重组布鲁氏菌bp26蛋白的抗原特性比较及M5-90-26标记疫苗鉴别诊断抗原的筛选以pET30a(+), pGEX-6P-1, pQE32为载体重组构建的四种原核表达羊种布鲁氏菌M5-90株bp26基因的重组质粒pET30-rbp26,6P-1-rbp26, pQE32-rbp26和pQE32-rAbp26,转化相应的感受态细胞后,表达、纯化获得抗原,western-blot(鼠抗rbp26抗体)和iELISA试验比较分析其抗原特性(RBPT、Svanova-cELISA两种方法验证的阴性参考羊血清90份、阳性参考羊血清30份)。结果表明,特异性分别为99%、98%、99%和100%,敏感性分别为27%、30%、27%和23%,rbp26-4具有很好的抗原特异性,并命名为rAbp26,适合作为布鲁氏菌标记疫苗M5-90-26鉴别诊断用抗原。试验4：截短表达布鲁氏菌bp26基因重组融合蛋白纯化方法的建立及其抗原特性分析本研究将重组质粒pQE32-rAbp26转化E. coli M15[pREP4],经表达后,在AKTAexplorer系统上采取IMAC和SEC二步法对重组融合蛋白(rAbp26)纯化,获得了理想的纯化抗原。Western blot试验表明,rAbp26蛋白与鼠抗rbp26高免血清(抗体)、羊和牛布鲁氏菌多克隆抗体反应呈阳性结果,而鼠抗rAbp26抗体与E.coli. O:157、Salmonella group D、C1、B和Yersinia enterocolitia O:9五种革兰氏阴性菌抗原反应呈阴性结果,表明rΔbp26蛋白具有良好的抗原活性和抗原特异性。试验5：布鲁氏菌bp26抗体iELISA检测方法的建立和最适条件确定本研究将纯化的rAbp26作为抗原包被酶标板,建立了适用于新型标记疫苗M5-90-26鉴别诊断的羊种布鲁氏菌bp26抗体iELISA检测方法(△bp26-iELISA),并对该方法进行优化,从而确定该方法的最适条件：抗原最佳包被浓度为2μg/ml；血清的最佳检测浓度为1/50稀释；血清的最佳反应条件为37℃60分钟；酶标抗体的最佳反应条件为37℃60分钟；底物的最佳反应条件为37℃15分钟。试验6：布鲁氏菌bp26抗体iELISA方法的临界点确定及其在鉴别诊断上的初步应用利用△bp26-iELISA方法对来自不同背景的经过RBPT和cELISA两种方法验证过的阴性羊血清(494份)和阳性羊血清(186份)进行抗bp26蛋白抗体的检测。获得的检测数据,首先将其中92份阴性羊血清均值(X)+3倍标准差(SD)来对其临界值(Cov)进行初步确定,然后通过Medcalc 9.6.0软件进行全部数据的ROC分析,进一步优化临界点的赋值(临界值),同时对动物攻毒血清、疫苗免疫血清及现地血清进行检测。结果表明,临界值Cov=0.7816时,Dsn值为18.6%,Dsp值为96%。结合RBPT、cELISA等敏感方法,M28攻毒试验,M5-90、M5-90-26和S2免疫试验及现地检测结果,该法能鉴别羊群布鲁氏菌标记疫苗M5-90-26免疫与自然感染或常规疫苗免疫(M5-90、S2)免疫产生的抗体。试验7：布鲁氏菌bp26抗体iELISA即用型试剂盒的研制根据临床和或现地用诊断试验的要求,在我们已有研究结果的基础上进行了即用型试剂盒的研制。配制了所需的阴阳性血清、洗涤液、包被抗原的封闭液和保护液、样品稀释液、二抗工作液、显色液等试剂,并进行了敏感性与特异性试验、重复性试验(批内与批间重复试验)和保存期试验(老化试验),结果表明组装试剂盒有效期达1年以上。试验8：布鲁氏菌bp26蛋白的免疫原性研究利用建立的△bp26-iELISA方法分别对试验组1、试验组2和试验组3进行抗bp26蛋白抗体的检测,分析它们的抗体应答水平；同时通过与RBPT、cELISA试验结果的比较,分析不同诊断用抗原的免疫原性强弱。结果表明,在试验组1中M5-90疫苗在小鼠、山羊上抗体效应水平较高,表现出很好的免疫原性、而在绵羊上次之,在牛上表现较弱；通过对小鼠抗bp26蛋白抗体及其分型抗体的检测,结果表明bp26蛋白在小鼠体内能诱导较强的体液免疫反应,且以IgG1亚类抗体为主。在试验组2的山羊免疫试验中M5-90疫苗株的抗体效应水平较高,表现较好的免疫原性,S2疫苗株次之,S19疫苗株最弱。在试验组3中发现免疫状态良好的山羊对M5-90疫苗表现出较好的免疫应答水平,而免疫状态差的山羊对M5-90疫苗表现出较差的免疫应答水平。在比较试验中,发现bp26蛋白抗原的免疫原性不如含菌体蛋白与S-LPS等多种抗原成份的菌悬液和单一S-LPS抗原的免疫原性。更多还原

【Abstract】 Brucellosis is a worldwide and important bacterial zoonosis, which hits a serious threat to the healthy development of animal husbandry and human health. It outbreaks and spreads again in many countries and regions around the world, especially in developing countries in recent years.Classical serodiagnosis method, live attenuated vaccine and some corresponding measures usually taken have played important roles on preventing and controlling brucellosis. Unfortunately, they also brought about some disadvantages. For example, antibodies produced in animals vaccinated with live attenuated vaccines against Brucella spp. are indistinguishable from those produced in infected animals using current conventional serological tests. Attenuated vaccines may cause bacterial persistence within the target host, potential transmission to unintended recipients, and the possibility of reversion to virulence.A novel approach to control Brucellosis is to develop a marker vaccine through deletion the bp26 gene from these parental vaccine strains with good immunogenicity and vaccine efficacy, and to develop a novel iELISA based on BP26 protein as antigen to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains. However, serological test based on the entire sequence of recombinant bp26 protein (BP26) often lead to false-positive reactions in Brucella-free sheep, goats, or cattle. So the antigenic specificity of bp26 protein should be improved. In addition, immunogenic diversity of Brucella bp26 protein of various virulent strains of Brucella spp.exists in different host animals experimented, thus it also would impact usage of Brucella bp26 protein as antigen of differential serodiagnosis in practice.1. Amplification, sequence and bioinformation analysis of bp26 gene of Brucella melitensis M5-90 strainThe remarkable homogeneity was revealed among brucella spp. through sequence of both nucleotide in bp26 gene and amino acid in bp26 protein, and the bp26 protein of Brucella spp. has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans, so bp26 is hopefully used as a serodiagnosis antigen to develop a novel differential serological test to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains. According to the paper reported by Rosetti et al (1996), we designed a pair of primers to amplify bp26 gene by PCR with M5-90 DNA as template. The amplified fragment was about 990 bp by electrophoresis of nucleic acids, and the purified PCR product was identified to have an intact open reading frame (ORF) encoding bp26 protein of brucella spp. by sequencing on Beckman CEQTM8000 DNA Analysis System. Bioinformation analysis of bp26 protein encoded by bp26 gene showed that bp26 gene of each representative species of the genus Brucella was extremely conservative, nucleotide sequence homologies ranged more than 99.3% (except for dogs, only 59.4%) and shared deduced amino acid sequence homologies of more than 97.6%. The bp26 protein is with a calculated molecule weight of 26.569 Da and an isoelectric point of 6.62. The first 29 amino acid residues are possibly the signal peptide I. Amino acid residues of the N terminal sequences 29-39,64-69,109-120,171-176 and 204-209 are more likely to be B cell antigen linear epitopes.2. Cloning of intact or truncated bp26 gene of Brucella melitensis M5-90 strain, and construction of recombinant plasmid pQE32-rbp26 and pQE32-rAbp26A specific fragment of the bp26 gene or truncated bp26 gene of Brucella melitensis M5-90 was amplified through PCR, and the recombinant plasmid was constructed through ligating the purified PCR product and pQE32 vector digested with both BamH I and Hind III respectively, then was transformed into competent M15 [pREP4] cells. The competent cells grew on solid LB media containing kanamycin and ampicillin. The positive colonies were selected and then identified by colony PCR, digestion of one or double enzymes, and sequencing. We have gained the recombinant plasmid, named as pQE32-rbp26 or pQE32-rAbp26, and their integrity and the orientation of the amplicons were verified by sequencing the inserted DNA fragments with a Genetic Analysis system, so.3. Selection of antigenic specificity and antigenic comparison of four recombinant bp26 proteins of Brucella melitensis M5-90 strainFour recombinant plasmids, pET30-rbp26,6P-1-rbp26, pQE32-rbp26 and pQE32-rAbp26, were used to transform corresponding E. coli host cells. They expressed the four recombinant bp26 proteins of Brucella melitensis M5-90 strain as a poly-His or GST tagged fusion, named as rbp26-1, rbp26-2, rbp26-3 and rbp26-4. Subsequently, antigenicities of the purified recombinant proteins were analyzed by western blot and iELISA using mouse anti-bp26 protein antibody and reference sera from sheep and goats (90 Brucella-negative sera and 30 Brucella-positive sera verified by RBPT and cELISA) as Abl. The results showed the specificities of the four reconstructed antigens were 99%,98%, 99% and 100%, and their sensitivities were 27%,30%,27% and 23%, respectively. Among them the rbp26-4 showed the best antigenic reactivity and antigenic specificity, named as rAbp26, was suitable as diagnosis antigen for brucellosis marked vaccine (M5-90-26)4. Antigenic analysis, purification of truncated recombinant bp26 protein of Brucella melitensis M5-90 strainRecombinant plasmid pQE32-rAbp26 was transformed into Escherichia coli M15 (pREP4) host cells to express the rAbp26 protein. The rAbp26 protein was then purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC) on an AKTA explorer system. Its antigenicity was detected by western blot using anti-rAbp26 hyperimmune serum (HIS), Brucella-positive serum from goat or bovid as Ab1 respectively, and analysis of cross-reactions between the rAbp26 protein and other antigens, E.coli. O:157, Salmonella group D, C1, B, and Yersinia enterocolitia 0:9, was also performed using anti-rAbp26 HIS as Ab1. The results showed the purified protein could react with either mouse anti-rAbp26 protein antibody or positive serum from cattle and goats, and only the rAbp26 protein reacted with mouse anti-rAbp26 HIS, and the other antigens of Gram-negative bacteria did not react with HIS. So the rAbp26 protein showed fine antigenic reactivity and antigenic specificity again.5. Development and optimization of reaction conditions of△bp26-iELISA test to detect Brucella bp26 antibodyAn iELISA test to detect Brucella bp26 antidody wae developed through coating the purified rAbp26 protein in 96 microplate as antigen, and its reaction conditions of Abp26-iELISA was optimized. The optimal concentration of the coated r△bp26 protein was 2μg/ml, the optimal dilution of detected sera (Ab1) was 1:50, the optimal reacting time of Ab1 with the r△bp26 protein(Ag) was 60min at 37℃, the optimal reacting time of Ab2 with the Abl-Ag complex was 60min at 37℃, and the optimal time of TMB coloration was 15min at 37℃.6. Determination of the cut-off value of△bp26-iELISA test, and initial application for differential serodiagnosis of brucellosis in sheep and goats△bp26-iELISA test was used to detect 494 reference negative sera and 186 reference positive sera verified by RBPT & Svanova-cELISA tests of sheep and goats from different sites. Among the two groups of data,92 reference negative sera was used to initial determinate the threshold value (Cov) of Abp26-iELISA, and its value was calculated as the mean specific OD450 of control (negative) sera plus threefold standard deviation (SD), and then the two groups of data were analyzed by ROC with Medcalc 9.6.0 software in order to optimize the threshold value of Abp26-iELISA. Sera from sheep and goats inoculated with M28 virulent strain, sera from sheep and goats immunized with vaccine strains, and field sera were detected by Abp26-iELISA test in order to optimize correlating Dsn and /or Dsp with Dsn+Dsp. Results showed the Dsn and Dsp value was equal to 18.6% and 96% when the threshold value (Cov) was 0.7816. The method can distinguish antibodies produced in animals vaccinated with brucellosis marked vaccine (M5-90-26) from those with live attenuated vaccines or infected animals, combined with the results of RBPT, cELISA, attacking test of M28, immunization test of M5-90, M5-90-26 and S2.7. Preparament of ready-to-use△bp26-iELISA kit for detecting Brucella bp26 antibodyNegative and positive reference serum,wash solution, block and protection solution, sample dilution solution, working solution of HRP-Ab2, and one competent TMB solution were respectively prepared and assemblized into a ready-to-use Abp26-iELISA kit for detecting Brucella bp26 antibody. Then sensitivity test, specificity test, repetition test (inter-bulks, and exter-bulks) and storage life test of Abp26-iELISA were accomplished. The results showed the kit’s shelf life could reach up to more than one year.8. Immunogenic analysis of Brucella bp26 proteinAccording to this test’s design, samples of sera from test 1, test 2 and test 3 were detected by Abp26-iELISA, RBPT, and Svanova-cELISA. In the test 1, the immunogenicity of M5-90 in goats and mice was the strongest, the second place is in sheep, that in calfhoods is the worst according to levels of anti-bp26 protein antibodies. In the test 2, the immunogenicity of M5-90 in goats was the strongest among them, and its immunogenicity of S2 in goats was stronger than that of S19 in goats. In the test 3, the immunogenicity of M5-90 in goats under good immunity condition was better than that under poor immunity condition. According to levels and durations of anti-bp26 protein antibodies, anti-’bacteria suspensions’antibodies, and anti-sLPS antibodies, its immunogenicity of S-LPS or tropina (eg,OMPs) was stronger than that of bp26 protein.更多还原