布病分子标记、毒力缺失疫苗株免疫原性及鉴别诊断研究
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摘要
布鲁氏菌病(以下简称布病)是一种重要的人兽共患传染病,近十几年来,该病的发生和流行在世界范围内呈上升趋势。动物布病的预防主要依靠免疫预防,而目前使用的疫苗有两个缺陷,一是免疫的动物和自然感染的动物不能区别,严重地影响了布病的诊断、检疫和预防;二是所有的弱毒疫苗毒副作用比较大。因此,研制分子标记、毒力基因缺失疫苗在动物布病的免疫预防实践中具有重要的意义。本研究在我们课题组前期构建的分子标记、毒力基因缺失疫苗株(YZ-2)的基础上,对疫苗株的生物学特性、体液免疫和细胞免疫等方面进行了研究,并以亲本株S19为对照,综合评价了该疫苗株的免疫效果。结果表明,YZ-2变异为粗糙型;免疫小鼠后没有产生明显的脾脏肿大,脾脏分离的细菌数量明显降低,说明其毒力较亲本株显著减弱;YZ-2在培养基上经连续传30代,其遗传性稳定;诱发小鼠产生了与亲本株相近的抗体水平,抗体的消长规律基本一致。免疫后小鼠的淋巴细胞增殖水平、主要细胞因子含量以及脾脏T淋巴细胞亚群与亲本株没有显著差异,表明该疫苗株能引导机体产生良好的细胞免疫反应。以缺失的bp26基因表达、纯化的BP26蛋白作为检测抗原(T抗原),结合金黄色葡萄球菌A蛋白(SPA)标记胶体金技术建立了布病抗体胶体金快速鉴别诊断方法。结果表明,该方法能够有效区别S19和YZ-2免疫的动物,具有较高的敏感性、特异性和稳定性。
Brucellosis is a zoonotic disease caused by a small intracellular bacteria Brucella which is pathogenic for humans and many species of animals. This infection, which is nearly cosmopolitan in distribution, can cause allergies by Brucella invading into body, leading to severe economic loses and threatening the health of human beings and animals. At present the live attenuated vaccine is considered the best vaccine available for the brucellosis. However the live vaccine strains are of low virulence, and it induces the production of antibodies in the vaccinated animals that can interfere with diagnosis of field infection. Thus, the study on molecular marked and virulencegene deleted vaccines has become pressing needs; At the same time, we need to establish the method of identification.
Immunochromatographic technology is a new diagnostic technique which combined with colloidalgold labeling, immunoassay, chromatography analysis. Due to its advantages, such as quick, sensitive, specific, stable and easy to perform without the need of special skill, reagents or equipment, pathogens, toxin, medicine residue test. It is approved that the brucella detection can be completed quickly by this antibody diagnostic technique.
We have constructed Brucella abortus YZ-2 by homologous recombination. The novel mutant strain deleted bp26, bmp18 and added luciferase labeling has less residual virulence than strain S19 and suitable to the diagnostic test. In this study, we have evaluated the bionomics and immunogenicity of Brucella abortus YZ-2 by investigating its dyeing characteristic, toxicity,generation stability, humoral immunity and cellular immunity. The research found that the biochemistry characteristics of S19 and YZ-2 were nearly identical, and the colonies and the suspension have disisparity. The agglutination experiment and the serology experiment proved that YZ-2 had some variations in the construction process, and the brucella became rough type from smooth type. Splenomegaly was not observed in strain YZ-2-immunized mice, additionally, the result of the spleen bacteria count and the spleen index test suggested that strain YZ-2 had lost some residual virulence of the parental strain.YZ-2 has good heredity stability by stored inglycerine at 4℃for 28 months and passed on 30 generations continuously in the culture medium, and the deletedgene order in the recombinant was identical with the beginning generation.
Balb/c mice were immunized with the B. abortus YZ-2. After two weeks of immunization, the specific antibody level of mice serum was tested by indirect ELISA every week. The levels of antibodies induced by YZ-2 strain began to rise obviously at 15 days post-immunization, up to the summit at 35 days post-immunization, and thengradually declined. The result showed that the antibody level of YZ-2 is slightly higher than strain S19 in the early stage, and it hasn’t significant difference between the two groups on the whole. The lymphocyte proliferative response and the subsets of the T lymphocyte of immunized mice were also detected. Lymphocyte transformation assay and T leukomonocyte subpopulation test of mice spleen found that the S19 and YZ-2 group both could enhance ConA-induced T Lymphocyte proliferation, and the averages of the stimulation index were no significant differences. It suggests that S19 and YZ-2 have the similar immunogenicity, both induced well cellular immunity and enhanced the expression of CD4+、CD8+ on T Lymphocyte. It proved that Strain YZ-2 has the immunogenicity as well as the parent strain.
In our study, we developed colloidalgold test strip by using BP26 protein, which we had expressed and purified in initial stage, as detection antigen (T line) for rapid detection of specific antibody of Brucella. BP26 protein was purified by affinity chromatography, and the gold-labeled pond was prepared by SPA labeling technique. Our experiment has confirmed that the strips had no cross-reaction in detection of the sera of certain strains which had nearlygenetic relationship with Brucella, and it could discriminate the mice which had inoculated with with S19 or YZ-2. The assembled test strips were also used for detecting the known sera, the coincidence with Elisa was above 90%; The stability test of the strips suggested that the test strip could test correctly by stored at 4℃for 180 days. The results indicated that the colloidalgold test strip has the highly specificity, sensitivity, and it's suitable for brucellosis detection.
Development of the test strip can be potentially used to differentiate of Yz-2-vaccinated animals from animals infected with field strains.
The challenge test and proteomics research is in progress.
Brucellosis is a zoonotic disease caused by a small intracellular bacteria Brucella which is pathogenic for humans and many species of animals. This infection, which is nearly cosmopolitan in distribution, can cause allergies by Brucella invading into body, leading to severe economic loses and threatening the health of human beings and animals. At present the live attenuated vaccine is considered the best vaccine available for the brucellosis. However the live vaccine strains are of low virulence, and it induces the production of antibodies in the vaccinated animals that can interfere with diagnosis of field infection. Thus, the study on molecular marked and virulencegene deleted vaccines has become pressing needs; At the same time, we need to establish the method of identification.
Immunochromatographic technology is a new diagnostic technique which combined with colloidalgold labeling, immunoassay, chromatography analysis. Due to its advantages, such as quick, sensitive, specific, stable and easy to perform without the need of special skill, reagents or equipment, pathogens, toxin, medicine residue test. It is approved that the brucella detection can be completed quickly by this antibody diagnostic technique.
We have constructed Brucella abortus YZ-2 by homologous recombination. The novel mutant strain deleted bp26, bmp18 and added luciferase labeling has less residual virulence than strain S19 and suitable to the diagnostic test. In this study, we have evaluated the bionomics and immunogenicity of Brucella abortus YZ-2 by investigating its dyeing characteristic, toxicity,generation stability, humoral immunity and cellular immunity. The research found that the biochemistry characteristics of S19 and YZ-2 were nearly identical, and the colonies and the suspension have disisparity. The agglutination experiment and the serology experiment proved that YZ-2 had some variations in the construction process, and the brucella became rough type from smooth type. Splenomegaly was not observed in strain YZ-2-immunized mice, additionally, the result of the spleen bacteria count and the spleen index test suggested that strain YZ-2 had lost some residual virulence of the parental strain.YZ-2 has good heredity stability by stored inglycerine at 4℃for 28 months and passed on 30 generations continuously in the culture medium, and the deletedgene order in the recombinant was identical with the beginning generation.
Balb/c mice were immunized with the B. abortus YZ-2. After two weeks of immunization, the specific antibody level of mice serum was tested by indirect ELISA every week. The levels of antibodies induced by YZ-2 strain began to rise obviously at 15 days post-immunization, up to the summit at 35 days post-immunization, and thengradually declined. The result showed that the antibody level of YZ-2 is slightly higher than strain S19 in the early stage, and it hasn’t significant difference between the two groups on the whole. The lymphocyte proliferative response and the subsets of the T lymphocyte of immunized mice were also detected. Lymphocyte transformation assay and T leukomonocyte subpopulation test of mice spleen found that the S19 and YZ-2 group both could enhance ConA-induced T Lymphocyte proliferation, and the averages of the stimulation index were no significant differences. It suggests that S19 and YZ-2 have the similar immunogenicity, both induced well cellular immunity and enhanced the expression of CD4+、CD8+ on T Lymphocyte. It proved that Strain YZ-2 has the immunogenicity as well as the parent strain.
In our study, we developed colloidalgold test strip by using BP26 protein, which we had expressed and purified in initial stage, as detection antigen (T line) for rapid detection of specific antibody of Brucella. BP26 protein was purified by affinity chromatography, and the gold-labeled pond was prepared by SPA labeling technique. Our experiment has confirmed that the strips had no cross-reaction in detection of the sera of certain strains which had nearlygenetic relationship with Brucella, and it could discriminate the mice which had inoculated with with S19 or YZ-2. The assembled test strips were also used for detecting the known sera, the coincidence with Elisa was above 90%; The stability test of the strips suggested that the test strip could test correctly by stored at 4℃for 180 days. The results indicated that the colloidalgold test strip has the highly specificity, sensitivity, and it's suitable for brucellosis detection.
Development of the test strip can be potentially used to differentiate of Yz-2-vaccinated animals from animals infected with field strains.
The challenge test and proteomics research is in progress.
引文
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