[1]王立霞,孟庆玲,蔡扩军,等.单核细胞增生李斯特菌核糖核酸酶RNaseⅢ RncS的表达及其生物学活性研究[J].中国预防兽医学报,2018,(07):581-585.[doi:0.3969/j.issn.1008-0589.201711034]
 WANG Li-xia,MENG Qing-ling,CAI Kuo-jun,et al.Expression and biological activity of RNaseⅢ RncS of Listeria monocytogenes[J].Chinese journal of preventive veterinary medicine,2018,(07):581-585.[doi:0.3969/j.issn.1008-0589.201711034]
点击复制

单核细胞增生李斯特菌核糖核酸酶RNaseⅢ RncS的表达及其生物学活性研究()
分享到:

《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年07
页码:
581-585
栏目:
出版日期:
2018-08-15

文章信息/Info

Title:
Expression and biological activity of RNaseⅢ RncS of Listeria monocytogenes
文章编号:
1008-0589(2018)7-0581-05
作者:
 

王立霞1孟庆玲1蔡扩军2王登峰3伍晔晖1王熙凤1郭 晶1乔 军1*才学鹏4

 (1. 石河子大学 动物科技学院,新疆 石河子 832003;2. 乌鲁木齐市动物疾病控制与诊断中心,新疆 乌鲁木齐 830063;
3. 新疆畜牧科学院 兽医研究所,新疆 乌鲁木齐 830000;4. 中国兽医药品监察所,北京 100081)
Author(s):
 

WANG Li-xia1 MENG Qing-ling1 CAI Kuo-jun2 WANG Deng-feng3 WU Ye-hui1 WANG Xi-feng1GUO Jing1 QIAO Jun1* CAI Xue-peng4

 (1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China; 2. Animal Disease Control and Diagnosis Center in Urumqi, Urumqi 830063, China; 3. Institute of Veterinary Medicine, Xinjiang Academy of Animal Science, Urumqi 830000;
4. China Institute of Veterinary Drugs Control, Beijing 100081, China)
关键词:
单核细胞增生李斯特菌核糖核酸酶RNaseⅢrncS基因原核表达生物学活性
Keywords:
 Listeria monocytogenes  ribonuclease RNase Ⅲ  rncS gene  prokaryotic expression  biological activity
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201711034
文献标志码:
A
摘要:
为了解单增李斯特菌(LM)核糖核酸酶RNaseⅢ RncS的生物学活性,本研究通过PCR方法对该菌LM-SB5 野毒株中编码RNaseⅢ的rncS基因进行扩增、克隆及测序并对其进行生物信息学分析;将rncS基因克隆至表达载体pET-32a(+),转化至大肠杆菌中进行诱导表达。通过体外酶活实验研究目的蛋白对RNA的降解活性。序列分析结果显示,rncS基因全长690 bp,编码229个氨基酸;生物信息学分析结果显示,该基因编码的蛋白含有由9个保守氨基酸(ERLEFLGDA)组成的基序,2个N-酰基化位点、3个酪蛋白激酶Ⅱ磷酸化位点、2个蛋白激酶C磷酸化位点,提示该酶蛋白活性受磷酸化的调控。同源性分析显示,LM-SB5 rncS基因编码的蛋白氨基酸序列与LM标准株CAC99883.1的同源性为99.13 %。SDS-PAGE检测结果显示,表达的RncS蛋白相对分子量约为42.5 ku,与理论值相符;western blot分析表明重组RncS蛋白具有较强的反应原性。体外酶活实验表明,RncS是一种依赖于二价金属离子的核酸酶,且二价金属离子浓度不同其RNaseⅢ的切割活性则不同。本研究为进一步研究RncS在LM中调控ncRNAs的分子机制奠定前期基础。
  
Abstract:
To investigate the biological activity of the Listeria monocytogenes ribonuclease RNase III RncS, the rncS gene encoding Rnase III in L.monocytogenes SB5 wild-type strain was amplified by PCR, and cloned, sequenced and analyzed for the molecular characteristics. The rncS gene was subcloned into the expression vector pET-32a(+) and expressed in Escherichia coli. The biological activity of target protein was studied by enzymatic activity in vitro. Sequence analysis showed that the rncS gene had a length of 690bp and encoded 229 amino acid. Bioinformatics analysis revealed that the RNase III protein had 1 motif consisting of 9 conserved amino acids (ERLEFLGDA), 2 N-acylation sites, 3 casein kinaseⅡ phosphorylation sites and 2 protein kinase C phosphorylation sites. The result showed that the protein activity was regulated by phosphorylation. Homology analysis showed that Rncs protein of LM-SB5 shared 99.13% identities with the standard strain CAC99883.1. The expressed recombinant protein was about 42.5ku. In vitro enzymatic activity test showed that RncS was a nuclease depending on divalent metal ions. The cleavage activity of the RNase III was different due to the different concentrations of divalent metal ion. This study laid foundation for further study of the molecular mechanism of RncS regulating ncRNAs in L.monocytogenes.

参考文献/References:

[1] 王少辉,刘萍萍,魏建超,等. 单增李斯特菌inlK基因缺失株的构建及其生物学特性分析[J]. 中国动物传染病学报,2017,25(4):19-23.
[2]Good J A, Andersson C, Hansen S, et al. Attenuating listeria monocytogenes virulence by targeting the regulatory protein PrfA [J]. Cell Chem Biol, 2016, 23(3): 404-414.
[3]卢海亭,乔军,孟庆玲,等. 分子伴侣hfq基因缺失对单核细胞增生李斯特菌毒力及生物被膜生成的影响[J]. 华北农学报,2017,32(1):94-98.
[4]卢海亭,乔军,孟庆玲,等. ncRNA伴侣分子hfq基因缺失对单核细胞增生李斯特菌环境适应能力的影响[J]. 应用与环境生物学报,2015,21(6):1174-1178.
[5]李森,热依汉古丽·麦麦提力. 单增李斯特菌srtA基因敲除菌株的构建及其生物学特性[J]. 食品科学,2017,38(4):113-117.
[6]张再超,彭叶龙,孟庆玲,等. 单核细胞增生李斯特菌ncRNA研究进展[J]. 生命的化学,2013,53(4):87-91.
[7]许先进. 布鲁氏菌核糖核酸酶RibonucleaseⅢ的酶活性研究 [D]. 武汉:华中农业大学,2014.
[8]Macrae I J, Doudna J A. Ribonuclease revisited: structural insights into ribonuclease III family enzymes [J]. Curr   Opin Struct Biol, 2007, 17(1): 138-145.
[9]Conrad C, Evguenieva-Hackenberg E, Klug G. Both N-terminal catalytic and C-terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III [J]. Febs Letters, 2001, 509(1): 53-58.
[10]Lim B, Sim S, Sim M, et al. RNase III controls the Degradation of corA mRNA in Escherichia coli [J]. J Bacteriol, 2012, 194(9): 2214-2220.
[11]Efthimia L, Sharma C M, Isabelle C, et al. Global regulatory functions of the Staphylococcus aureus endoribonuclease III in gene expression [J]. PLoS Genet, 2012, 8(6): 3202-3212.
[12]Durand S, Gilet L, Bessières P, et al. Three essential ribonucleases-RNase Y, J1, and III-control the abundance of a majority of Bacillus subtilis mRNAs [J]. PLoS Genet, 2012, 8(3): e1002520.
[13]Darfeuille F, Unoson C, Vogel J, et al. An antisense RNA inhibits translation by competing with standby ribosomes [J]. Mol Cell, 2007, 26(3): 381-392.
[14]Dunn J J. RNase III cleavage of single-stranded RNA. Effect of ionic strength on the fideltiy of cleavage [J]. J Biol Chem, 1976, 251(12): 3807-3814.
[15]Kindler P, Keil T U, Hofschneider P H. Isolation and characterization of a ribonuclease 3 deficient mutant of Escherichia coli [J]. Mol Gen Genet, 1973, 126(1): 53-59.
[16]Dunn J J, Studier F W. T7 Early RNAs and Escherichia coli Ribosomal RNAs are cut from large precursor RNAs in vivo by ribonuclease III [J]. Proc Natl Acad Sci USA, 1973, 70(12): 3296-3300.
[17]Shi Zhong-jie, Nicholson R H, Jaggi R, et al. Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates [J]. Nucleic Acids Res, 2011, 39(7): 2756-2768.
[18]Robertson H D. Escherichia coli ribonuclease III cleavage sites [J]. Cell, 1982, 30(3): 669-672.
                                          (本文编辑:李 娜)

相似文献/References:

[1]丁 波,孟庆玲,乔 军*,等.单核细胞增生李斯特菌prfA基因重组菌的构建及其生物学特性研究[J].中国预防兽医学报,2012,(09):677.[doi:doi: 10.3969/j.issn.1008-0589.2012.09.01]
 DING Bo,MENG Qing-ling,QIAO Jun*,et al.Construction and partial biological characterization of prfA gene recombinant of Listeria monocytogenes strain[J].Chinese journal of preventive veterinary medicine,2012,(07):677.[doi:doi: 10.3969/j.issn.1008-0589.2012.09.01]
[2]田秋丰*,于申业*,衣 菲,等.基于单核细胞增生李斯特菌prfA基因新型原核温控表达载体的构建[J].中国预防兽医学报,2014,(09):688.[doi:10.3969/j.issn.1008-0589.2014.09.06]
 TIAN Qiu-feng*,YU Shen-ye*,YI Fei,et al.The construction of a novel prokaryotic thermoregulated expression vector based on prfA gene of Listeria monocytogenes[J].Chinese journal of preventive veterinary medicine,2014,(07):688.[doi:10.3969/j.issn.1008-0589.2014.09.06]
[3]夏 叶,辛永萍,李肖梁,等.单核细胞增生李斯特菌σB启动子报告质粒的构建及其在应激研究中的应用[J].中国预防兽医学报,2016,(03):211.[doi:10.3969/j.issn.1008-0589.2016.03.10]
 XIA Ye,XIN Yong-ping,LI Xiao-liang,et al.Construction of a reporter plasmid carrying Listeria monocytogenes σB promoter and its application in stress studies[J].Chinese journal of preventive veterinary medicine,2016,(07):211.[doi:10.3969/j.issn.1008-0589.2016.03.10]

更新日期/Last Update: 2018-08-15