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【目的】由于现有的减毒活疫苗仍存在较强的毒力,因抗原与毒株的差异不大而很难区分疫苗免疫和自然感染等缺点,限制了现有布鲁氏菌减毒活疫苗的广泛应用。本文拟对布鲁氏菌的减毒活疫苗株M5进行遗传改造,克服这些缺点。【方法】本研究利用同源重组的方法,用卡那抗性基因替换了布鲁氏菌减毒疫苗株M5的BP26基因,得到了新的标记疫苗株M5ΔBP26。分别用标记疫苗株和野生株侵染巨噬细胞和感染小鼠,比较分析标记株在细胞内和小鼠体内的存活能力。根据种特异性保守基因dnaK和缺失的BP26基因设计引物,建立双重PCR,用于区分标记株与野生株。【结果】成功构建了BP26基因标记疫苗株,细胞实验和动物实验结果表明,标记株仍能在胞内和小鼠内存活,具备作为减毒活疫苗的特性。小鼠实验结果显示,感染后两周野生株的细菌数为102.9,而突变株为101.1(P<0.01),至第3周野生株的细菌数为102.2,而突变株未能检出,表明与原疫苗株相比,标记株的感染力进一步减弱。根据DNA序列的差异,建立了能够区分标记疫苗株与野生株的双重PCR方法,标记株因只能扩增出一条带而能与野生株和毒株相区分,从而可以区分自然感染和疫苗免疫。【结论】基因标记疫苗株的构建及鉴别PCR方法的建立,为布鲁氏菌疫苗的进一步研发奠定了基础。
【OBJECTIVE】 Because the existing live attenuated vaccines still have strong virulence, the difference between antigen and strain is very small and it is difficult to distinguish the shortcomings of vaccine immunity and natural infection, which limits the existing Brucella attenuated Widely used live vaccine. This article intends to Brucella attenuated live vaccine strain M5 genetically modified to overcome these shortcomings. 【Method】 In this study, homologous recombination method was used to replace the BP26 gene of Brucella attenuated vaccine strain M5 with kanamycin resistance gene, and a new marker vaccine strain M5ΔBP26 was obtained. Macrophages and infected mice were respectively infected with the labeled vaccine strain and the wild strain, and the viability of the marker strains in the cells and in mice was comparatively analyzed. Primers were designed based on the species-specific conserved DNA (dnaK) and the deleted BP26 gene to establish a dual-PCR for distinguishing marker strains from wild-type strains. 【Result】 The BP26 gene-tagged vaccine strain was successfully constructed. The results of cell and animal experiments showed that the marker strain still survived both intracellular and mouse, and possessed the characteristics of live attenuated vaccine. The results of the mouse experiments showed that the number of bacteria in the wild-type strain was 102.9 and the mutant strain was 101.1 (P <0.01) two weeks after infection. The number of bacteria in the wild-type strain was 102.2 by the third week while the mutant strain failed to detect Compared with the original vaccine strain, the infectivity of the marker strain further weakened. According to the difference of DNA sequences, a dual-PCR method was established to distinguish the vaccine strains from the wild-type strains. The marker strains can distinguish between natural infection and vaccine immunity because only one band can be amplified and differentiated from the wild strains and strains . 【Conclusion】 The construction of gene-tagged vaccine strains and the establishment of differential PCR methods laid the foundation for the further development of Brucella vaccine.