类鼻疽伯克霍尔德菌BPSL1549基因敲除株的构建及鉴定  

Construction and identification of Burkholderia pseudomallei BPSL1549 gene knockout mutant strain

作　　者：胡治强[1] 方瑶[1] 朱攀[1] 任春艳[1] 胡艺[1] 马腾飞[1] 毛旭虎[1] 

机构地区：[1]第三军医大学西南医院医学检验系临床微生物及免疫学教研室,重庆400038

出　　处：《第三军医大学学报》2016年第11期1210-1214,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81471914)~~

摘　　要：目的构建类鼻疽菌BPSL1549基因敲除株[BP(△BPSL1549)],建立有效的类鼻疽菌毒力基因敲除平台。方法设计引物扩增类鼻疽菌BPSL1549基因上下游同源臂,连接至p K18mob Sac B自杀质粒上,通过大肠杆菌S17-1λpir以接合方式将其转入类鼻疽菌中。利用同源重组原理替换野生株中的BPSL1549基因,经过蔗糖筛选靶标基因敲除株,并采用PCR、Western blot检测方法鉴定敲除株,利用动物模型评价敲除株表型变化。结果 BP(△BPSL1549)菌株与野生株的BPSL1549基因两侧同源臂片段PCR产物相比缺少600 bp,Western blot检测敲除株不表达BPSL1549基因编码蛋白,成功构建类鼻疽菌BPSL1549基因敲除株。动物实验证实敲除株相比野生株毒力显著降低(P<0.05)。结论利用同源重组原理成功构建类鼻疽菌BPSL1549基因敲除株,完善了类鼻疽菌敲除平台和评价体系。Objective To construct Burkholderia pseudomallei（ B. pseudomallei） BPSL1549 gene knockout mutant strain,and establish efficient B. pseudomallei virulence gene knockout platform. Methods Primers were designed to amplify upstream and downstream homologous arms of BPSL1549 gene,and then the arms were linked to p K18 mob Sac B suicide plasmid. The recombinant suicide plasmid was introduced into B. pseudomallei BPC006 from Escherichia coli S17-1λpir by conjugation. BPSL1549 gene in wild strain was knocked out by homologous recombination,and the target gene knockout mutant strain was selected by sucrose screening. PCR and Western blotting were used to identify BPSL1549 gene knockout mutant strain. Mouse model was established to evaluate the phenotypic variation of the mutant strain. Results BPSL1549 gene homologous arm fragments of B. pseudomallei mutant strain（ △BPSL1549） had a lack of 600 bp compared with the wild strain. Western blotting showed that the mutant strain did not express BPSL1549 proteins,indicating that B. pseudomallei BPSL1549 gene knockout mutant strain was successfully constructed. In the animal experiments,the virulence of the mutant strain was significantly lower than that of the wild strain（ P 〈0. 05）. Conclusion B. pseudomallei BPSL1549 gene knockout mutant strain is successfully constructed by homologous recombination,which improves B. pseudomallei gene knockout platform and evaluation system.

关 键 词：类鼻疽伯克霍尔德菌 基因敲除 BPSL1549基因 

分 类 号：R372[医药卫生—病原生物学] R378.99[医药卫生—基础医学]