畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (2): 299-305.doi:

• 预防兽医 • 上一篇    下一篇

兔支气管败血波氏杆菌PRN基因缺失突变株的构建及特性研究

李洪广1,王芳1*,姜平2,范志宇1 ,胡波1   

  1. 1. 江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,南京 210014; 2. 南京农业大学动物医学院,南京 210095
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-02-15 发布日期:2012-02-15
  • 通讯作者: 王芳

Construction of Defined Mutations of PRN of Rabbit Bordetella bronchiseptica and Property Research

LI Hongguang1, WANG Fang1*, JIANG Ping2, FAN Zhiyu1, HU Bo1   

  1. 1. National Center for Engineering Research of Veterinary Bioproducts, Key Laboratory of Veterinary Biologicals engineering and technology of Ministry of Agriculture, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. College of Veterinary Medicine, Nanjing Agriculture University, Nanjing 210095, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2012-02-15 Published:2012-02-15
  • Contact: WANG Fang

摘要: 利用自杀性质粒构建兔支气管败血波氏杆菌百日咳黏附素(PRN)缺失突变株以研究PRN在支气管败血波氏杆菌(Bb)致病机理中的作用,同时为支气管败血波氏杆菌病减毒活疫苗的研究提供理论依据。PCR扩增出PRN1(PRN上游基因)和PRN2(PRN下游基因)2个目的基因片段,运用基因重组技术将庆大霉素抗性基因(GM)连接到PRN1和PRN2之间,将连接好的基因片段克隆到pMEG375自杀性载体中,构建自杀性载体pMEG375PRN1GMPRN2,将其转化到宿主菌SM10中,通过宿主菌SM10与受体菌Bb固相滤膜交配,自杀性载体转移到受体菌,根据同源重组原理,抗性筛选得到基因缺失突变株,命名为Bb(△PRN)。对突变株Bb(△PRN)与野生株WT进行了遗传稳定性、生长特性、溶血特性、细胞黏附特性、毒力、免疫保护性等比较研究。结果表明:Bb(△PRN)具有遗传稳定性;与野生株相比,突变株生长速度较慢,毒力有所下降,溶血活性及对Hep2细胞的黏附能力没有明显变化;小鼠免疫原性试验结果显示,突变株免疫小鼠后可以产生强有力的免疫力,能够抵抗野生株的攻击。Bb(△PRN)突变株构建成功并具有良好的免疫原性,为支气管败血波氏杆菌病减毒活疫苗的研究奠定了基础。

Abstract: We constructed pertactin (PRN) deletion mutant strain of rabbit Bordetella bronchiseptica (Bb) to study the function of pathogenesis of Bb, and to provide a theoretical basis for researching live attenuated vaccine against Bb. The 550 bp fragment of PRN1(upstream of PRN)and 440 bp fragment of the PRN2(downstream of PRN)were amplified by PCR respectively. The two fragments together with Gentamycin (GM) gene were linked and inserted into suicide vector, and the recombinant suicide vector was named as pMEG375PRN1GMPRN2. The recombinant suicide vector was transformed into host strain SM10. The transformed SM10 was mated with recipient strain Bb on the solid phase membrane, and the recombinant suicide vector was transformed into the recipient bacteria. According to homologous recombination and the principle of resistance screening, mutant strain was generated and was named as Bb△PRN. We compared Bb△PRN with wild type(WT)strain on genetic stability, growth characteristics, hemolytic activity, cell adhesion properties, immunogenicity. The results showed that the mutant strain stabilized genetically and grew slower than the wild type. There was no significant difference between them on hemolytic activity and adhesion on Hep2 cells. Comparing with parent strain, the virulence of mutant stain was attenuated about two fold. The assay of immunogenicity showed that Bb△PRN could produce a strong immune capacity. All of results indicated that mutant strain had good immunogenicity, and it could provide a theoretical basis for researching live attenuated vaccine against Bb.