【摘要】：布魯氏菌病(布病)是嚴重危害公共衛(wèi)生的人獸共患病。我國預防動物布病主要采用的是豬源分離致弱的布病活疫苗株S2,但國際上對其在牛、羊等異源動物的保護力存在爭議。為此,本論文采用BALB/c小鼠為模型,系統(tǒng)研究了S2株殘余毒力、體液和細胞免疫應答,并評價其免疫保護力。將55只小鼠皮下接種1×105CFU S2活菌,每周剖殺5只,測定脾重、脾臟含菌量、血清中IgG和細胞因子的消長情況。結果顯示,小鼠脾臟含菌量隨時間延長而逐步下降,4周后全部清除；小鼠免疫后2-3周IgG濃度達到最高,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周幾乎檢不出。將免疫后8周小鼠,分別皮下接種3種不同種屬布病強毒(豬布魯氏菌S1330株、牛布魯氏菌2308株和羊布魯氏菌M28株)1×105 CFU(5只/組),30日后進行脾臟稱重、脾臟分菌及病理學檢測,免疫小鼠能夠抵抗3種不同強毒株的攻擊,證實了其良好免疫保護力。此部分工作為國際組織推薦使用S2提供模型動物實驗證據。當前布病防控中的最大難題是無法區(qū)分疫苗免疫與自然感染抗體,本論文采用免疫蛋白組學方法從S2株膜蛋白中篩選鑒別診斷抗原。各用30份布病陰性健康羊、30份S2免疫羊,以及30份臨床布病感染羊陽性混合血清,分別與S2株膜蛋白二維電泳膠(2D)進行Western-blot,尋找S2膜蛋白與不同布病狀態(tài)(感染/免疫／陰性)綿羊血清反應差異,共尋找到113個差異蛋白點。選擇免疫反應差異較大的30個蛋白點進行MALDI-TOF質譜及生物信息學分析,共鑒定出14個蛋白,這些蛋白承擔13類分子功能、參與組成6類細胞組分和13類生物學過程。將3種混合血清(感染／免疫／陰性)分別與S2膜蛋白進行免疫共沉淀(IP),并對IP結合物進行Q-Extractive質譜鑒定,獲得182個候選蛋白點,這些蛋白行使129類分子功能、參與組成91類細胞組分和137類生物學過程。通過2D-MALDI-TOF鑒定出的14個蛋白中有12個蛋白在IP Q-Extractive中同時被篩選出。對2種方法鑒定出的所有蛋白進行功能分析,共挑選出10個體外鑒別診斷的候選靶點(編號1#-10#)。構建10個候選靶蛋白原核表達質粒,除1#(transporter)與5# (cell division protein FtsH)外,8個蛋白成功表達。將表達蛋白分別與3種混合血清進行Western-blot和ELISA檢測,顯示2#(universal stress protein)、3# (glycoside hydrolase family 43)及10# (hypothetical protein)鑒別診斷效果明顯。分別將3種蛋白用GST柱純化后,作為包被抗原,通過方陣試驗及單因子法優(yōu)化并確定了ELISA檢測參數。然后對30份感染血清及656份免疫羊場樣品進行檢測,2#,3#及10#蛋白建立的ELISA對感染樣本檢出率約60-80%。此部分工作對破解布病鑒別診斷難題,實現布病凈化和根除意義重大。
[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，

本文編號：2510119