血清7型胸膜肺炎放线杆菌apxⅡC-/apxⅠA+基因缺失重组菌株的构建与鉴定

通过接合转移和SacB负向筛选方法,成功构建了一株apxⅡC缺失的血清7型胸膜肺炎放线杆菌重组菌株。首先构建重组转移质粒pEHA1。将pEHA1转化供体菌大肠杆菌(E.coliβ2155),并将其与野生型APP血清7型亲本菌混合培养约5h,然后涂到含氯霉素抗性的培养基培养,挑取阳性克隆,接种到无抗性液体培养基,培养后涂于含有蔗糖的的固体培养基,培养一定时间后挑取蔗糖抗性的克隆,即可得到目的突变株。通过PCR、遗传稳定性、外毒素分泌、重组位点序列分析证明重组菌构建成功。通过对重组菌生物学特性进行初步研究,表明突变株生长能力未受影响,对小鼠毒力显著降低。该突变株构建体系的建立为猪传染性胸膜肺炎减毒活疫苗的开发及对胸膜肺炎放线杆菌新基因的功能研究奠定了良好基础。

Construction and characteristics of a recombinant strain apx II C- /apx I A+ of Actinobacillus pleuropneumoniae serotype 7]

An Actinobacillus pleuropneumoniae apx II C mutant was constructed by transconjugation and counterselection method. Briefly, a transconjugation plasmid pEHA1 was constructed, and transformed into donor strain Escherichia coli 32155. After mixed the donor cells with A . pleuropneumoniae acceptor cells, the mixture was cultivated for about 5 hours and plated on solid medium containing chloromycetin. Then the Cm(R) positive clones were picked and inoculated into liquid medium in the absence of any antibiotic. Cultures were pelleted, plated on sucrose plates and incubated overnight. Finally, Sucrose-resistant colonies (Suc(R)) were selected and considered as mutant. The mutant was verified by PCR, heredity stability, exotoxin secretion and sequence analysis, suggested that the construction of the mutant was sucessful. The biological characteristics of this mutant strain was further investigated. Compared with parental strain, the results indicated that the mutant hold the same growth rate in vitro and reduced virulence on mice. Altogether, this mutation system will facilitate development of live attenuated vaccines and research on functions of novel genes of A. pleuropneumoniae.