嗜酸乳杆菌GGDEF和EAL结构域相关蛋白的表达、纯化及活性分析
目的 鉴定嗜酸乳杆菌（Lactobacillus acidophilus）中含GGDEF和EAL结构域相关蛋白的功能。为进一步研究环二鸟苷酸（c-di-GMP）在该菌株中的调控机制奠定基础。方法 从嗜酸乳杆菌ATCC4356基因组中PCR扩增出NH1307045-GGDEF、NH1307050和NH1307055这3个基因片段,分别构建其重组表达质粒。经鉴定后,转入大肠杆菌表达宿主中,用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白,经直链淀粉树脂纯化后进行体外酶活性实验,再通过高效液相色谱检测反应产物。结果 通过PCR扩增的目的 基因片段经重组质粒双酶切后其大小与预期相符,测序...
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| Published in | 南方医科大学学报 Vol. 37; no. 5; pp. 633 - 639 |
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| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
南方医科大学南方医院口腔科,广东广州,510515%南方医科大学基础医学院神经生物学教研室,广东广州,510515
2017
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1673-4254 |
| DOI | 10.3969/j.issn.1673-4254.2017.05.11 |
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| Abstract | 目的 鉴定嗜酸乳杆菌（Lactobacillus acidophilus）中含GGDEF和EAL结构域相关蛋白的功能。为进一步研究环二鸟苷酸（c-di-GMP）在该菌株中的调控机制奠定基础。方法 从嗜酸乳杆菌ATCC4356基因组中PCR扩增出NH1307045-GGDEF、NH1307050和NH1307055这3个基因片段,分别构建其重组表达质粒。经鉴定后,转入大肠杆菌表达宿主中,用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白,经直链淀粉树脂纯化后进行体外酶活性实验,再通过高效液相色谱检测反应产物。结果 通过PCR扩增的目的 基因片段经重组质粒双酶切后其大小与预期相符,测序结果 与Gen Bank中嗜酸乳杆菌ATCC4356基因序列一致,表明重组质粒构建成功。SDS-PAGE和Western Blot检测表达的重组蛋白相对分子质量分别约为59000（含GGDEF结构域）、67000（含EAL结构域）和72000（含EAL结构域）,与预期大小吻合。经树脂亲和柱纯化及浓缩获得目的 蛋白。高效液相色谱分析结果 显示,NH1307045-GGDEF的表达产物体外无明显双鸟苷酸环化酶（DGC）活性,NH1307050的表达产物体外具有磷酸二酯酶（PDE）活性,而NH1307055的表达产物在体外无明显PDE活性。结论 在本研究的3种基因中,NH1307050的表达产物在体外具有磷酸二酯酶（PDE）的活性。为研究环二鸟苷酸在嗜酸乳杆菌中的调控机制提供了实验依据。 |
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| AbstractList | 目的 鉴定嗜酸乳杆菌（Lactobacillus acidophilus）中含GGDEF和EAL结构域相关蛋白的功能。为进一步研究环二鸟苷酸（c-di-GMP）在该菌株中的调控机制奠定基础。方法 从嗜酸乳杆菌ATCC4356基因组中PCR扩增出NH1307045-GGDEF、NH1307050和NH1307055这3个基因片段,分别构建其重组表达质粒。经鉴定后,转入大肠杆菌表达宿主中,用异丙基-β-D-硫代吡喃半乳糖苷（IPTG）诱导表达重组蛋白,经直链淀粉树脂纯化后进行体外酶活性实验,再通过高效液相色谱检测反应产物。结果 通过PCR扩增的目的 基因片段经重组质粒双酶切后其大小与预期相符,测序结果 与Gen Bank中嗜酸乳杆菌ATCC4356基因序列一致,表明重组质粒构建成功。SDS-PAGE和Western Blot检测表达的重组蛋白相对分子质量分别约为59000（含GGDEF结构域）、67000（含EAL结构域）和72000（含EAL结构域）,与预期大小吻合。经树脂亲和柱纯化及浓缩获得目的 蛋白。高效液相色谱分析结果 显示,NH1307045-GGDEF的表达产物体外无明显双鸟苷酸环化酶（DGC）活性,NH1307050的表达产物体外具有磷酸二酯酶（PDE）活性,而NH1307055的表达产物在体外无明显PDE活性。结论 在本研究的3种基因中,NH1307050的表达产物在体外具有磷酸二酯酶（PDE）的活性。为研究环二鸟苷酸在嗜酸乳杆菌中的调控机制提供了实验依据。 目的 鉴定嗜酸乳杆菌(Lactobacillus acidophilus)中含GGDEF和EAL结构域相关蛋白的功能.为进一步研究环二鸟苷酸(c-di-GMP)在该菌株中的调控机制奠定基础.方法 从嗜酸乳杆菌ATCC4356基因组中PCR扩增出NH 13_07045-GGDEF、NH13_07050和NH13 07055这3个基因片段,分别构建其重组表达质粒.经鉴定后,转入大肠杆菌表达宿主中,用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达重组蛋白,经直链淀粉树脂纯化后进行体外酶活性实验,再通过高效液相色谱检测反应产物.结果 通过PCR扩增的目的基因片段经重组质粒双酶切后其大小与预期相符,测序结果与GenBank中嗜酸乳杆菌ATCC4356基因序列一致,表明重组质粒构建成功.SDS-PAGE和Western Blot检测表达的重组蛋白相对分子质量分别约为59000(含GGDEF结构域)、67000(含EAL结构域)和72000(含EAL结构域),与预期大小吻合.经树脂亲和柱纯化及浓缩获得目的蛋白.高效液相色谱分析结果显示,NH13_07045-GGDEF的表达产物体外无明显双鸟苷酸环化酶(DGC)活性,NH1307050的表达产物体外具有磷酸二酯酶(PDE)活性,而NH13 07055的表达产物在体外无明显PDE活性.结论 在本研究的3种基因中,NH13_07050的表达产物在体外具有磷酸二酯酶(PDE)的活性.为研究环二鸟苷酸在嗜酸乳杆菌中的调控机制提供了实验依据. |
| Abstract_FL | Objective To identify the functions of the proteins containing the GGDEF or EAL domain in Lactobacillus acidophilus for investigation of the regulatory mechanism of c-di-GMP in this strain.Methods The DNA fragments of NH13_07045-GGDEF,NH13_07050 and NH13_07055 from Lactobacillus acidophilus ATCC4356 were amplified by PCR and cloned into the expression vector pMAL-His-c2.After sequencing,the recombinant plasmids were transformed into competent Escherichia coli cells,which were induced by IPTG to express the recombinant proteins fused with maltose binding protein (MBP).The fusion proteins were purified using amylose resin column for diguanylate cyclase (DGC) or phosphodiesterase (PDE) activity assays invitro followed by analysis with high-performance liquid chromatography (HPLC).Results The target DNA fragments were obtained by PCR,and their sequences were all identical to that in GenBank.The purified and concentrated fusion proteins,which were identified by SDS-PAGE and Western blotting,had relative molecular masses of 59 kD,67 kD and 72 kD.HPLC analysis showed no DGC activity in NH13_07045-GGDEF,while PDE activity was found in NH13_07050 but not in NH13_07055.Conclusion We obtained the protein encoded by NH13_07050 that possesses PDE activity in vitro.This protein may facilitate the evaluation of the regulatory function of c-di-GMP in Lactobacillus acidophilus. |
| Author | 何嘉辉 孙洁丽 闫文娟 王方 |
| AuthorAffiliation | 南方医科大学南方医院口腔科;南方医科大学基础医学院神经生物学教研室 |
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| Author_FL | YAN Wenjuan WANG Fang SUN Jieli HE Jiahui |
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| Keywords | 嗜酸乳杆菌 酶活性检测 c-di-GMP EAL结构域 环二鸟苷酸 EAL domain GGDEF结构域 high-performance liquid chromatography 高效液相色谱 Lactobacillus acidophilus GGDEF domain enzymatic activity assays |
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| Publisher | 南方医科大学南方医院口腔科,广东广州,510515%南方医科大学基础医学院神经生物学教研室,广东广州,510515 |
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| Snippet | 目的 鉴定嗜酸乳杆菌（Lactobacillus acidophilus）中含GGDEF和EAL结构域相关蛋白的功能。为进一步研究环二鸟苷酸（c-di-GMP）在该菌株中的调控机制奠定基础。方法 从嗜酸乳... 目的 鉴定嗜酸乳杆菌(Lactobacillus acidophilus)中含GGDEF和EAL结构域相关蛋白的功能.为进一步研究环二鸟苷酸(c-di-GMP)在该菌株中的调控机制奠定基础.方法 从嗜酸乳杆... |
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| Title | 嗜酸乳杆菌GGDEF和EAL结构域相关蛋白的表达、纯化及活性分析 |
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