【作者】 甄清；

【导师】 于雅琴；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2009， 博士

【摘要】 布鲁氏菌病为动物源性疾病,是由细胞内寄生的布鲁氏菌引起的传染-变态反应性人兽共患传染病,在世界范围内广泛流行,对畜牧养殖业的发展和人类健康造成严重威胁,已成为重要的公共卫生问题。布鲁氏菌病的诊断方面存在两个主要的问题:一是快速准确诊断,二是现有减毒活疫苗的鉴别诊断。PCR检测技术避免操作活菌,并能检测到标本中较低丰度的病原,适合布鲁氏菌这种高危险性病原的检测。用PCR方法对布鲁氏菌病早期、复发期以及伴随多种并发症的患者及药物治疗后患者进行诊断,具有较好的应用前景,我国目前尚无应用PCR检测方法在临床诊断上跟踪随访的研究报道。本研究针对布鲁氏菌的保守基因bp26设计了属的引物,针对插入性序列IS711设计了牛、羊、猪种引物,建立了单重和多重PCR检测方法,在临床上对免疫学诊断为患者、可疑患者和非患者人群进行检测,并结合流行病学个案数据库资料拟定了进一步跟踪随访检测的对象,为建立人布鲁氏菌病标准化PCR诊断方法提供研究依据。目前应用血清学方法不能区分布鲁氏菌减毒疫苗株的反应与自然感染,发展基因标记疫苗可解决这一问题。M5是我国广泛应用的减毒活疫苗株,本研究应用基因同源重组方法,用卡那抗性基因替换部分bp26基因,构建了bp26突变株,并根据布鲁氏菌的保守基因建立了能够区分bp26突变株与疫苗株的PCR鉴别方法。bp26突变株在胞内外的生存能力显示,bp26突变株在小鼠体内的免疫应答和免疫保护效果均低于M5疫苗株,表明bp26基因在M5疫苗株的免疫保护作用中发挥了作用。考虑到bp26基因在免疫原性和保护性方面的重要性,我们对BP26蛋白进行了表达及纯化,并致力于开发有效的布鲁氏菌亚单位疫苗和作为人布鲁氏菌病血清学的特异性诊断抗原用于临床诊断研究。更多还原

【Abstract】 Brucellosis is a kind of typical zoonosis that is caused by cytozoic Brucella, which spreads worldwide and becomes a major public health problem with the threat of the livestock industry and human being health.There are two major problems in the diagnosis of brucellosis,the one is the lack of rapid and accurate diagnostic method,the other is the lack of identifying method of diagnosis for present attenuated live vaccine.The clinical diagnostic criterion of human brucellosis is based on clinical manifestations,history exposed to Brucella and laboratory diagnosis.The diagnosis of brucellosis always requires laboratory confirmation because of its various clinical manifestations without specific clinical signs.Present laboratory testing methods is the isolation of pathogen or demonstration of high titres of specific antibodies,but all these mehthods have serious shortcomings. Although the isolation of Brucella is the best evidence of confirmation of brucellosis, it is rarely applied to clinical diagnosis because the method is a time-consuming one, and with low positive rate of isolation of patients with long-term clinical courses or with focal complications,and harm to laboratory workers’ health.As a simple method,serological testing method has been used to acquire main evidence of diagnosis of brucellosis,but it lacks specificity in spreading areas and in those persons exposed professionally to Brucella.Moreover,cross-reaction with other bacteria can also occur.Although we have developed some methods for clinical diagnosis,such as different kinds of serological methods and automated culture system,it still has some restrictions to apply.PCR assays by targeting to nucleic acids have been considered as the best way to the replacement of Brucella isolation and culture,because of its simple,rapid,sensitive,specific and no requirement of operation on live bacteria.Recent years,foreign scholars carried out studies on clinical diagnosis for brucellosis in acute stage and follow-up studies after treatment. They believed that PCR detection was useful tool in discovering patients who were in early acute stage or relapse.The standardization of detection methods,However, have not been set,the method of PCR detection is not applied to clinical diagnosis of brucellosis widely.Domestic research found results various.Most of them showed the method of PCR detection low in sensitivity.Some studies reported false-positive, all of them lack systematic and in-depth study and evaluation.As to now,there is no report concerning researching on the method of PCR detection that is used to evaluate the treatment effectiveness by follow-up patients after treatment and discover new patients who are in the early acute stage.So in our study,we are trying to do those things 1) build a standardized PCR diagnostic methods which is suitable for China’s national condition and grass roots medical staff in CDC to evaluate the treatment effectiveness by following-up patients after treatment and discover new patients who are in the early acute stage;2) apply it to isolate and culture Brucella from blood of whom are with acute febrile in clinical;3) apply the blood with bacteria to optimize the template processing method;4) evaluate its applications in clinical diagnosis and following-up patients,by combining with epidemiological information on cases,so that it could offer a tool for pharmaceutical research and the effectiveness evaluation.The prevention of Brucellosis is mainly relied on inoculating animals.Attenuated live vaccine is used to prevent Brucellosis in China now.But it is hard to distinguish difference between attenuated live vaccine and natural infection,so the efforts of inoculating animals by plan is curbed,which reveals that rebuilding marker vaccine could be the direction of future research.Objective:1.To build a standardized PCR diagnostic methods which is suitable for China’s national condition and grass roots medical staff in CDC to evaluate the treatment effectiveness by following-up patients after treatment and discover new patients who are in the early acute stage. 2.To isolate Brucella from patients blood and to identify its phenotype so as to offer bacteriological evidence for PCR diagnosis research.3.To establish two database which contain epidemiological information on cases and sample information separately.4.To build safe and effective vaccine strains with genetic markers so that infection could be distinguished between the attenuated live vaccine and wild strains.Methods:1.To establish database which contains epidemiological information on cases collecting at CDC Brucellosis outpatient service,such as personal information, clinical manifestation,laboratory diagnosis.At the same time,the blood and serum samples of out-patients would be collected and preserved at -20℃which are used to extract genome and test PCR.2.To establish a standardized PCR diagnostic methods.Primers were designed separately to optimize single-PCR and multi-PCR detection and reaction conditions, which were specific to BP26 encoding gene and IS711 gene sequence,so that they could be applied to identifying Brucella spp.3.To establish a boiling method in order to extract Brucella genome from patients blood.M5 strains and BP26 primers were used to amplify Brucella DNA by extracting lysate,simulate and experimental animals at different time points,so as to determine a method fit for clinical application.4.To isolate and culture Brucella from patients’ blood,and BP26 encoding gene and primers were used to identify bacterial strains phenotype,so as to verify specificity of amplified target sequence.5.Preparations of templates from blood isolated Brucella.spp.then tested with different primers by PCR detection,so as to find most sensitive method for PCR detection applying to patients,no-patients and suspect patients who immunology diagnosed.Furthermore,we would use it to track target people in three,six and twelve months separately combining with epidemiological information of cases and to analyze and evaluate the reliability of PCR diagnosis and evaluation of clinical application.6.In the method of homologous recombination,a genetic marked vaccine strain was rebuilt by replacing attenuated live Brucella vaccine strain of the bp26 gene M5 with kanamycin resistance gene.Two pairs of primers which were towards deleted sequences of bp26 gene and various types of highly conserved sequence were designed to rebuild PCR detection so as to identify vaccine and mutant strains.Wild Brucella strains were used to attack mice immune system so as to explore mice immune protection from marked vaccine strain.Results:1.Database of Brucellosis epidemiological information on cases was built in Songyuan CDC.Information on demography,clinical manifestation,history exposed to Brucella spp.,results of laboratory diagnosis,treatment protocols,samples of whole blood and serum blood were collected.2.Methods of single-PCR and multi-PCR detection were established.Primers for diagnosing Brucella BP26 encoding gene were designed.And primers used to diagnose Brucella insert sequences IS711 and to identify B.mellitensis,B.abortus, and B.suis were designed too.The specificity was verified among genomes of standard Brucella spp.and vaccine strains.3.Ways of Brucella DNA extraction from blood were established.With the PCR detection of mice blood among lystae,simulate and experimental animals at different time points,results showed that the sensitivity of pyrolysis method was better than inhibitor stimulation,and the sensitivity of PCR assay was better than isolated culture.4.Ten strains of Brucella were identified as B.melitensis which were isolated cultured from patients’ blood samples.In the positive isolates and other isolates in PCR identification,BP26,primers for B.melitensis were amplified the target bands in the ten bacteria strains,and other isolates were not amplified the target bands.5.In the templates of blood which were positive isolated and cultured Brucella, the sensitivity of B4/B5 was the highest in accordance with the results of isolated culture.The sensitivity of BP26,primers of B.melitensis and 26A/26B were lower, with the coincidence rate of 67%.6.Serological diagnosis was performed to test patients’ blood samples.The results showed that the sensitivity of BP26 primer and primer of B.melitensis were lower than that tested by serological detection methods.The sensitivity of B4/B5 primer was high with high rate of coincidence.Furthermore,positive amplification was shown among suspected cases and non-patients,but was not shown among healthy people.7.Analysis of clinical data showed that there were seven cases who suffered Brucellosis(SAT＞1:100),two cases who were confirmed as suspected cases (PAT0.04),one case confirmed as non-patient among the ten Brucella sufferers who were proved as existing Brucella in the blood samples.Positive predictive value of PCR detection was 67%～100%.8.Positive rate of PCR detection were 82.9%,80.0%and 60.0%separately among those who were diagnosed as patients(34 cases),non-patients(25 cases) and suspected cases(16 cases) by immunology.9.By constructing mutative box,which means the recombinant plasmid of pUC19K-NC,and under the principle of gene homologous recombination,part of bp26 gene in the M5 vaccine strain was deleted,M5△bp26 tagged by molecular was constructed.Two pairs of primers were designed specific to deletion sequences of bp26 gene and every highly conserved sequences,respectively.Double-PCR detection methods were established and optimized which could be used to identify vaccine strains and mutant strains.10.Bacterial strains marked by M5△bp26 could live in macrophages and mice body,but the viability of it was lower than mild strain.Marked strains and vaccine strains M5 could both induce specific antibody in the body of Balb/c mice,but humoral immune response induced by marked strains was weaker and had shorter duration than that induced by M5.Immune protective effect of marked strains was dissatisfactory when it was attacked by B.mellitensis 16M. Conclusions:1.Primers could be used to identify the strains of Brucella,which were designed to target to BP26 coding gene of Brucella diagnostic antigen and parenthetical sequences of IS711 designed to identify B.melitensis,B.abortus and B.suis separately.The sensitivity of boiling method was better than inhibitor stimulation,so it could be performed on clinical research of PCR detection.2.When being used to detect blood infected by Brucella or came from Brucellosis patients,it showed that the sensitivities of PCR detection using BP26 primers or primers of B.melitensis were low,but specificities were high;the sensitivities of PCR detection using B4 primers and B5 primers were high,and high coincidence rate with indicators of isolated culture and indicators of immunology. Positive amplification was shown among suspected cases and non-patients,but was not shown among healthy people.The method of PCR detection could be used to follow up Brucellosis patients after treatment.3.Database of Brucellosis epidemiological information on cases which were collected at Songyuan CDC was built.4.Molecular marked strains M5△bp26 which deleted part of bp26 gene were built,and dual-PCR detection methods were established to identify vaccine strains and mutant strains.Bacterial strains marked by M5△bp26 could live in macrophages and mice body,but the its viability was lower than mild strain.Marked strains and vaccine strains M5 could both induce specific antibody in the body of Balb/c mice,but humoral immune response induced by marked strains was weaker and had shorter duration than that induced by M5.5.Objectives remaining to study are:(1)To follow up those people whose blood are proved positive when being cultured isolated or detected positive with PCR assay or who are proved to be Brucellosis patients diagnosed by immunological detection after their treatment in three,six,twelve months separately.To evaluate status and future of PCR techniques for the diagnosis and follow-up of human brucellosis used to discover relapses patients.(2) Following up those people who are diagnosed as Brucella positive by PCR detection but non-patients when diagnosed by immunology detection,so as to make sense of the PCR detection method when it is used to discover missed diagnosis.(3) Immune protective effect of marked strains was dissatisfactory,which were deleted of bp26 gene.But BP26 antigen had been expressed and purified,which could be used as subunit vaccine to be continued to research on.更多还原