铜绿假单胞菌LasR基因反义核酸原核表达载体的构建及对该菌毒力的影响

PCR扩增LasR基因,用反向克隆法构建LasR基因反义核酸原核表达载体pUCP18/lasRantisense并转化铜绿假单胞菌,经酶切、PCR及测序鉴定重组载体.RT-PCR检测LasB基因和LasI基因mRNA的表达,NAD法测定外毒素A的活性,紫外分光光度计测定绿脓菌素的产生水平.用转化pUCP18/lasRantisense质粒菌株感染大鼠呼吸道并进行病理组织切片检查.PCR扩增出约720bp片段,与GenBank(No:NC_002516)报道的基因片段大小一致,构建了LasR基因反义核酸原核表达载体pUCP18/lasRantisense,并成功转化铜绿假单胞菌,且有效表达,使转化菌的毒力因子弹性蛋白酶、外毒素A和绿脓菌素表达水平均降低.与标准株感染的大鼠比较,转化pUCP18/lasRantisense质粒菌株感染的大鼠支气管炎症明显减轻.上述结果表明,LasR基因反义核酸原核表达载体pUCP18/lasRantisense可以降低铜绿假单胞菌的毒力.

Construction of Prokaryotic Expression Vectors of Antisense Nucleic Acid of LasR Gene and Its Effect on The Virulence of Pseudomonas aeruginosus

The LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and recombined with plasmid pUCP18 reversely. The recombinant pUCP18/lasRantisense was verified with restriction analysis, PCR and sequence and was transformed in Pseudomonas aeruginosus. The biological effect of pUCP18/lasRantisense was detected by RT-PCR, NAD method and the assay of pyocyanin. The air tubes of rats were infected by pUCP18/lasRantisense strain and then carried on histopathologic slide check. Expected full length LasR fragment (721bp) can be extended from Pseudomonas aeruginosus gene with PCR technology. And it is consistent with LasR gene of Pseudomonas aeruginosa covered in GenBank (NO. NC_002516). The recombinant plasmid was constructed and transformed into Pseudomonas aeruginosus sucessfully. Compared with the rats which were infected by standard strain, the bronchitis of the rats which were infected by pUCP18/lasRantisense strain was obviously eased. It can be concluded that the antisensenucleic acid of LasR gene can depress the virulence of Pseudomonas aeruginosus and reveal a new target site for treatment.