布鲁氏菌病活疫苗S2株小鼠评价模型构建及其鉴别抗原筛选
发布时间：2019-07-04 18:19
【摘要】：布鲁氏菌病(布病)是严重危害公共卫生的人兽共患病。我国预防动物布病主要采用的是猪源分离致弱的布病活疫苗株S2,但国际上对其在牛、羊等异源动物的保护力存在争议。为此,本论文采用BALB/c小鼠为模型,系统研究了S2株残余毒力、体液和细胞免疫应答,并评价其免疫保护力。将55只小鼠皮下接种1×105CFU S2活菌,每周剖杀5只,测定脾重、脾脏含菌量、血清中IgG和细胞因子的消长情况。结果显示,小鼠脾脏含菌量随时间延长而逐步下降,4周后全部清除；小鼠免疫后2-3周IgG浓度达到最高,5周后逐步下降；IFN-y和TNF-a含量免疫后1周最高,然后迅速下降,至第3周几乎检不出。将免疫后8周小鼠,分别皮下接种3种不同种属布病强毒(猪布鲁氏菌S1330株、牛布鲁氏菌2308株和羊布鲁氏菌M28株)1×105 CFU(5只/组),30日后进行脾脏称重、脾脏分菌及病理学检测,免疫小鼠能够抵抗3种不同强毒株的攻击,证实了其良好免疫保护力。此部分工作为国际组织推荐使用S2提供模型动物实验证据。当前布病防控中的最大难题是无法区分疫苗免疫与自然感染抗体,本论文采用免疫蛋白组学方法从S2株膜蛋白中筛选鉴别诊断抗原。各用30份布病阴性健康羊、30份S2免疫羊,以及30份临床布病感染羊阳性混合血清,分别与S2株膜蛋白二维电泳胶(2D)进行Western-blot,寻找S2膜蛋白与不同布病状态(感染/免疫／阴性)绵羊血清反应差异,共寻找到113个差异蛋白点。选择免疫反应差异较大的30个蛋白点进行MALDI-TOF质谱及生物信息学分析,共鉴定出14个蛋白,这些蛋白承担13类分子功能、参与组成6类细胞组分和13类生物学过程。将3种混合血清(感染／免疫／阴性)分别与S2膜蛋白进行免疫共沉淀(IP),并对IP结合物进行Q-Extractive质谱鉴定,获得182个候选蛋白点,这些蛋白行使129类分子功能、参与组成91类细胞组分和137类生物学过程。通过2D-MALDI-TOF鉴定出的14个蛋白中有12个蛋白在IP Q-Extractive中同时被筛选出。对2种方法鉴定出的所有蛋白进行功能分析,共挑选出10个体外鉴别诊断的候选靶点(编号1#-10#)。构建10个候选靶蛋白原核表达质粒,除1#(transporter)与5# (cell division protein FtsH)外,8个蛋白成功表达。将表达蛋白分别与3种混合血清进行Western-blot和ELISA检测,显示2#(universal stress protein)、3# (glycoside hydrolase family 43)及10# (hypothetical protein)鉴别诊断效果明显。分别将3种蛋白用GST柱纯化后,作为包被抗原,通过方阵试验及单因子法优化并确定了ELISA检测参数。然后对30份感染血清及656份免疫羊场样品进行检测,2#,3#及10#蛋白建立的ELISA对感染样本检出率约60-80%。此部分工作对破解布病鉴别诊断难题,实现布病净化和根除意义重大。
[Abstract]:Brucellosis (brucellosis) is a zoonosis that seriously endangers public health. Live brucellosis vaccine strain S2, which is isolated from pigs, is mainly used to prevent brucellosis in China, but its protection in cattle, sheep and other heterogenic animals is controversial in the world. In this paper, BALB/c mice were used as a model to systematically study the residual virulence, humoral and cellular immune responses of S2 strains, and to evaluate their immune protection. 55 mice were inoculated with 1 脳 105CFU S2 live bacteria subcutaneously. five mice were killed every week. Spleen weight, spleen content, IgG and cytokine in serum were measured. The results showed that the content of bacteria in spleen of mice decreased gradually with the prolongation of time, and all of them were cleared after 4 weeks, the concentration of IgG reached the highest at 2 weeks after immunization and decreased gradually after 5 weeks, and the contents of IFN-y and TNF-a reached the highest level one week after immunization, and then decreased rapidly, and almost could not be detected at the third week. At 8 weeks after immunization, three different species of brucellosis virulent (Brucella suis S1330, Brucella bovis 2308 and Brucella sheep M28) were inoculated subcutaneously with 1 脳 105 CFU (5 mice / group). 30 days later, the spleen was weighed, the spleen was separated and pathological examination showed that the immunized mice could resist the attack of three different virulent strains, which confirmed that the mice had good immune protection. This part of the work provides evidence of model animal experiments for international organizations to recommend the use of S2. At present, the biggest problem in the prevention and control of brucellosis is that it can not distinguish vaccine immunization from natural infection antibody. In this paper, the differential diagnostic antigen of S2 strain membrane protein was screened by immunproteome method. 30 brucellosis negative healthy sheep, 30 S2 immunized sheep and 30 clinical brucellosis infected sheep positive mixed serum were used to find out the difference of S2 membrane protein reaction between S2 membrane protein and sheep with different brucellosis status (infection / immunization / negative) by Western-blot, with S2 strain membrane protein two-dimensional electrophoresis gel (2D). A total of 113 differentially protein spots were found. A total of 14 proteins were identified by MALDI-TOF mass spectrometry and bioinformatics analysis with 30 protein spots with large difference in immune response. These proteins undertake 13 kinds of molecular functions and participate in the composition of 6 types of cell components and 13 kinds of biological processes. Three kinds of mixed serum (infection / immunization / negative) were co-precipitated with S2 membrane protein by immunoprecipitation (IP), and identified by Q-Extractive mass spectrometry. 182 candidate protein spots were obtained. These proteins perform 129molecular functions and participate in the composition of 91 cell components and 137biological processes. 12 of the 14 proteins identified by 2D-MALDI-TOF were screened out in IP Q-Extractive at the same time. All the proteins identified by the two methods were analyzed by functional analysis, and a total of 10 candidate targets for differential diagnosis in vitro (No. 1 鈮，
本文编号：2510119
[Abstract]:Brucellosis (brucellosis) is a zoonosis that seriously endangers public health. Live brucellosis vaccine strain S2, which is isolated from pigs, is mainly used to prevent brucellosis in China, but its protection in cattle, sheep and other heterogenic animals is controversial in the world. In this paper, BALB/c mice were used as a model to systematically study the residual virulence, humoral and cellular immune responses of S2 strains, and to evaluate their immune protection. 55 mice were inoculated with 1 脳 105CFU S2 live bacteria subcutaneously. five mice were killed every week. Spleen weight, spleen content, IgG and cytokine in serum were measured. The results showed that the content of bacteria in spleen of mice decreased gradually with the prolongation of time, and all of them were cleared after 4 weeks, the concentration of IgG reached the highest at 2 weeks after immunization and decreased gradually after 5 weeks, and the contents of IFN-y and TNF-a reached the highest level one week after immunization, and then decreased rapidly, and almost could not be detected at the third week. At 8 weeks after immunization, three different species of brucellosis virulent (Brucella suis S1330, Brucella bovis 2308 and Brucella sheep M28) were inoculated subcutaneously with 1 脳 105 CFU (5 mice / group). 30 days later, the spleen was weighed, the spleen was separated and pathological examination showed that the immunized mice could resist the attack of three different virulent strains, which confirmed that the mice had good immune protection. This part of the work provides evidence of model animal experiments for international organizations to recommend the use of S2. At present, the biggest problem in the prevention and control of brucellosis is that it can not distinguish vaccine immunization from natural infection antibody. In this paper, the differential diagnostic antigen of S2 strain membrane protein was screened by immunproteome method. 30 brucellosis negative healthy sheep, 30 S2 immunized sheep and 30 clinical brucellosis infected sheep positive mixed serum were used to find out the difference of S2 membrane protein reaction between S2 membrane protein and sheep with different brucellosis status (infection / immunization / negative) by Western-blot, with S2 strain membrane protein two-dimensional electrophoresis gel (2D). A total of 113 differentially protein spots were found. A total of 14 proteins were identified by MALDI-TOF mass spectrometry and bioinformatics analysis with 30 protein spots with large difference in immune response. These proteins undertake 13 kinds of molecular functions and participate in the composition of 6 types of cell components and 13 kinds of biological processes. Three kinds of mixed serum (infection / immunization / negative) were co-precipitated with S2 membrane protein by immunoprecipitation (IP), and identified by Q-Extractive mass spectrometry. 182 candidate protein spots were obtained. These proteins perform 129molecular functions and participate in the composition of 91 cell components and 137biological processes. 12 of the 14 proteins identified by 2D-MALDI-TOF were screened out in IP Q-Extractive at the same time. All the proteins identified by the two methods were analyzed by functional analysis, and a total of 10 candidate targets for differential diagnosis in vitro (No. 1 鈮，
本文编号：2510119
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