title: [Construction of BP26 tagged vaccine strain and development of discriminating PCR for Brucella].
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 with the kanamycin gene in a live attenuated vaccine strain M5. The new tagged vaccine strain, M5DeltaBP26, was generated. The wild type strain and M5DeltaBP26 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 M5DeltaBP26.
  
  
  RESULTS
  A new tagged strain, M5DeltaBP26, 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., 10(2.9) CFU of Brucella were isolated from M5 infected mice, whereas only 10(1.1) CFU of Brucella were isolated from M5DeltaBP26 infected mice (P < 0.01). At 3 weeks p. i., 10(2.2) CFU of Brucella whereas no M5DeltaBP26 were isolated. These results indicated that infection capability of M5DeltaBP26 was decreased. Based on the sequence differences between M5DeltaBP26 and M5, a new discriminating duplex PCR was developed. With the duplex PCR, only one product was amplified from M5DeltaBP26, by which it can be differentiated from wild type and virulent strains.
  
  
  CONCLUSIONS
  The construction of tagged strain and the development of discriminating PCR provide a new candidate for further vaccine development.
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Science, Beijing 100071, China. grace-zhuji@163.com
authors.forename: Zhoujia
authors.initials: Z
authors.lastname: Wang
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhen
authors.forename: Feng
authors.initials: F
authors.lastname: Qiao
authors.forename: Yufei
authors.initials: Y
authors.lastname: Wang
authors.forename: Xinying
authors.initials: X
authors.lastname: Du
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Zhong
authors.forename: Jin
authors.initials: J
authors.lastname: Zhao
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Yu
authors.forename: Liuyu
authors.initials: L
authors.lastname: Huang
authors.forename: Yansong
authors.initials: Y
authors.lastname: Sun
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000818:Animals; D002002:Brucella / Q000235:genetics* / Q000254:growth & development / Q000276:immunology* / Q000302:isolation & purification; D002004:Brucella Vaccine / Q000235:genetics* / Q000276:immunology*; D002460:Cell Line; D005260:Female; D007612:Kanamycin / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011897:Random Allocation
chemical_list: C106040:BP26 protein, Brucella; D002004:Brucella Vaccine; D008565:Membrane Proteins; D007612:Kanamycin