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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 an IBD1549 gene knockout strain [BP (△ BPSL1549)] and establish an effective GJL virulence gene knockout platform. Methods Primers were designed to amplify the upstream and downstream homologous arms of BPSL1549 gene and ligated into the pK18mob Sac B suicide plasmid. The recombinant plasmid was transformed into Helicobacter pylori by E. coli S17-1λpir. The wild-type BPSL1549 gene was replaced by homologous recombination. The target gene knockout strain was screened by sucrose. The knockout strain was identified by PCR and Western blot. The phenotype of knockout strain was evaluated by animal model. Results The BP (△ BPSL1549) strain lacked 600 bp in comparison with the wild-type BPSL1549 gene and the BPSL1549 gene knockout strain was successfully constructed by Western blot. Animal experiments confirmed that the virulence of knockout strain was significantly lower than that of wild strain (P <0.05). Conclusion The homologous recombination principle was successfully used to construct the BPSL1549 gene knockout strain, which improved the platform and evaluation system of GJL knockout.