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

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

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。

We constructed pertactin （PRN） deletion mutant strain of rabbit Bordetella bronchisep- tica （Bb） to study the function of pathogenesis of Bb, and to provide a theoretical basis for re- searching live attenuated vaccine against Bb. The 550 bp fragment of PRNl（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 re- combinant 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 theprinciple 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 differ- ence 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.