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

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.