布鲁氏菌BP26基因标记疫苗株的构建及鉴别PCR方法的建立

Construction of BP26 Tagged Vaccine Strain and Development of Discriminating PCR for Brucella

摘要:

摘要：【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)，至第三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。

Abstract:

Abstract: [Objective] The wide application of live attenuated vaccine strains is limited because of drawbacks of residual virulence, similar antigenenicity to virulent strain and the difficulty to differentiate vaccination and natural infection. In this study, we modified the vaccine strain to prevent the drawbacks. [Methods] By using homologous recombination, we replaced the BP26 gene by the kanamycin gene in a live attenuated vaccine strain M5. The new tagged vaccine strain, M5ΔBP26, was generated. The wild type strain and M5ΔBP26 were used to infect macrophage and mice to compare their intracellular survival capability. According to the conservative sequence of dnaK and the deleted region of BP26, primers were designed to develop a duplex PCR for discriminating the wild type strain and M5ΔBP26. [Results] A new tagged strain, M5ΔBP26, was successfully constructed. The tagged strain could survive in both macrophage and mice, indicating the feasibility as live attenuated vaccine strain. Results from mice infection showed that, at 2 weeks p.i., 102.9 CFU of Brucella were isolated from M5 infected mice, whereas only 101.1 CFU of Brucella were isolated from M5ΔBP26 infected mice (P<0.01). At 3 weeks p.i., 102.2 CFU of Brucella whereas no M5ΔBP26 were isolated. These results indicated that infection capability of M5ΔBP26 was decreased. Based on the sequence differences between M5ΔBP26 and M5, a new discriminating duplex PCR was developed. With the duplex PCR, only one product was amplified from M5ΔBP26, by which it can be differentiated from wild type and virulent strains. [Conclusion] The construction of tagged strain and the development of discriminating PCR provide a new candidate for further vaccine development.