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利用λRed重组系统敲除鼠伤寒沙门氏菌sopB基因

, PP. 171-177

Keywords: 鼠伤寒沙门氏菌LT2,λRed,重组系统,sopB,基因敲除,同源重组

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Abstract:

:利用λRed重组系统敲除鼠伤寒沙门氏菌LT2的(SalmonellaentericaserovartyphimuriumLT2,S.typhimuriumLT2)sopB基因。以pKD4质粒为模板,扩增得到中间带有卡那霉素抗性基因且两端各带有59bp分别与sopB基因上下游序列同源的同源打靶片段,将其转化至表达Red重组酶的S.typhimuriumLT2感受态细胞中;在抗生素压力和λRed重组系统帮助下,同源片段和菌体sopB基因发生同源重组,通过卡那霉素筛选得到带有抗性标记的阳性重组菌;转入重组酶表达质粒pCP20以除去抗性标记,得到保留单一FRT位点的突变菌株;利用PCR技术鉴定重组菌,并通过检测沙门氏菌效应蛋白SopB的分泌以及沙门氏菌感染HeLa细胞后pAKT的激活反应来鉴定sopB基因是否被敲除。构建的ΔsopB突变菌株失去了分泌SopB蛋白的能力,且不能够像野生型菌株那样在感染HeLa细胞的过程中激活pAkt。本研究获得了S.typhimuriumLT2的sopB基因缺失突变株,为沙门氏菌感染宿主过程中SopB的功能研究提供工具,同时也为进一步探索其他类型细菌的基因敲除提供了线索。

References

[1]  Patel JC, Galán JE. Differential activation and function of Rho GTPases during Salmonella-host cell interactions[J]. J?Cell?Biol, 2006, 175(3):453-463.
[2]  H?nisch J, K?lm R, Wozniczka M, et al. Activation of a RhoA/Myosin II-dependent but Arp2/3 complex-independent pathway facilitates Salmonella invasion[J]. Cell Host Microbe, 2011, 9(4):273-285.
[3]  Mallo GV, Espina M, Smith AC, et al. SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34[J]. J?Cell?Biol, 2008, 182(4):741-752.
[4]  Bakowski MA, Braun V, Lam GY, et al. The phosphoinositide phosphatase SopB manipulates membrane surface Charge and trafficking of the Salmonella-containing vacuole[J]. Cell Host Microbe, 2010, 7(6):453-462.
[5]  Scallan E, Hoekstra RM, Angulo FJ, et al. Foodborne illness acquired in the United States-major pathogens[J]. ?Emerg Infect Dis, 2011, 17(1):7-15.
[6]  Norris FA, Wilson MP, Wallis TS, et al. SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase[J]. ?Proc Natl Acad Sci USA, 1998, 95(24):14057-14059.
[7]  Patel JC, Hueffer K, Lam TT, et al. Diversification of a Salmonella virulence protein function by ubiquitin-dependent differential localization[J]. Cell, 2009, 137(2):283-294.
[8]  Ruan HH, Li Y, Zhang XX, et al. Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB, a virulence effector protein secreted by Salmonella typhimurium[J]. Biochem Biophys Res Commun, 2014, 447(1):172-177.
[9]  Knodler LA, Finlay BB, Steele-Mortimer O. The Salmonella effector protein SopB protects epithelial cells from apoptosis by sustained activation of Akt[J]. J Biol Chem, 2005, 280(10):9058-9064.
[10]  Galán JE, Zhou D. Striking a balance:modulation of the actin cytoskeleton by Salmonella[J]. Proc Natl Acad Sci USA, 2000, 97(16):8754-8761.
[11]  阮海华, 赵霞, 张振奇. 过表达沙门氏菌效应蛋白 SopB 对 HeLa 细胞骨架的影响[J]. 安徽农业科学, 2013, 41(16):7067-7069.
[12]  陶永清, 刘崇基, 李健娇, 等. 沙门菌效应分子 SopB 激活 HeLa 细胞 MAPK 蛋白激酶的磷酸化[J]. 中国医药生物技术, 2011, 6(6):432-436.
[13]  Yang WL, Wang J, Chan CH, et al. The E3 ligase TRAF6 regulates?Akt?ubiquitination and activation[J]. Science, 2009, 325(5944):1134-1138.
[14]  Endale M, Park SC, Kim S, et al. Quercetin disrupts tyrosine-phosphorylated phosphatidylinositol 3-kinase and myeloid differentiation factor-88 association, and inhibits?MAPK/AP-1 and IKK/NF-κB-induced inflammatory mediators production in RAW 264.7 cells.[J]. Immunobiology, 2013, 218(12):1452-1467.
[15]  Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proc Natl Acad Sci USA, 2000, 97(12):6640-6645.
[16]  胡堃, 史兆兴, 赛道建, 等. Red 重组系统及在微生物基因敲除中的应用[J]. 遗传, 2003, 25(5):628-632.
[17]  姜娜, 王芃, 王艳春, 等. 利用 λRed 重组系统敲除伤寒沙门氏菌 rfaH 基因[J]. 生物技术通讯, 2009, 20(2):173-176.

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