类鼻疽伯克霍尔德菌BPSL1549基因敲除株的构建及鉴定
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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.
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.
引文
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[11]D’Cruze T,Gong L,Treerat P,et al.Role for the Burkholderia pseudomallei type three secretion system cluster 1bpsc N gene in virulence[J].Infect Immun,2011,79(9):3659-3664.10.1128/iai.01351-10
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[13]Wongtrakoongate P,Mongkoldhumrongkul N,Chaijan S,et al.Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis[J].Mol Cell Probes,2007,21(2):81-91.DOI:10.1016/j.mcp.2006.08.006
[14]任春艳,李倩.类鼻疽伯克霍尔德致死因子[J].微生物学免疫学进展,2015,43(3):47-50.DOI:10.13309/j.cnki.pmi.2015.03.011
[2]Leelarasamee A.Recent development in melioidosis[J].Curr Opin Infect Dis,2004,17(2):131-136.
[3]Currie B J,Fisher D A,Anstey N M,et al.Melioidosis:acute and chronic disease,relapse and re-activation[J].Trans R Soc Trop Med Hyg,2000,94(3):301-304.
[4]Stevens M P,Haque A,Atkins T,et al.Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa typeⅢsecretion mutant in murine models of melioidosis[J].Microbiology,2004,150(Pt 8):2669-2676.DOI:10.1099/mic.0.27146-0
[5]Campos C G,Borst L,Cotter P A.Characterization of Bca A,a putative classical autotransporter protein in Burkholderia pseudomallei[J].Infect Immun,2013,81(4):1121-1128.DOI:10.1128/IAI.01453-12
[6]Lazar Adler N R,Stevens J M,Stevens M P,et al.Autotransporters and Their Role in the Virulence of Burkholderia pseudomallei and Burkholderia mallei[J].Front Microbiol,2011,2:151.DOI:10.3389/fmicb.2011.00151
[7]Pumirat P,Broek C V,Juntawieng N,et al.Analysis of the prevalence,secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei[J].PLo S ONE,2014,9(5):e96298.DOI:10.1371/journal.pone.0096298
[8]Cruz-Migoni A,Hautbergue G M,Artymiuk P J,et al.A Burkholderia pseudomallei toxin inhibits helicase activity of translation factor e IF4A[J].Science,2011,334(6057):821-824.DOI:10.1126/science.1211915
[9]Fang Y,Huang Y,Li Q,et al.First genome sequence of a Burkholderia pseudomallei Isolate in China,strain BPC006,obtained from a melioidosis patient in Hainan[J].J Bacteriol,2012,194(23):6604-6605.DOI:10.1128/JB.01577-12
[10]毛旭虎.加强类鼻疽的研究[J].第三军医大学学报,2011,33(13):1315-1317.DOI:10.16016/j.1000-5404.2011.13.013
[11]D’Cruze T,Gong L,Treerat P,et al.Role for the Burkholderia pseudomallei type three secretion system cluster 1bpsc N gene in virulence[J].Infect Immun,2011,79(9):3659-3664.10.1128/iai.01351-10
[12]Schafer A,Tauch A,Jager W,et al.Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids p K18 and p K19:selection of defined deletions in the chromosome of Corynebacterium glutamicum[J].Gene,1994,145(1):69-73.
[13]Wongtrakoongate P,Mongkoldhumrongkul N,Chaijan S,et al.Comparative proteomic profiles and the potential markers between Burkholderia pseudomallei and Burkholderia thailandensis[J].Mol Cell Probes,2007,21(2):81-91.DOI:10.1016/j.mcp.2006.08.006
[14]任春艳,李倩.类鼻疽伯克霍尔德致死因子[J].微生物学免疫学进展,2015,43(3):47-50.DOI:10.13309/j.cnki.pmi.2015.03.011
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