脂多糖/脂多糖结合蛋白复合物的初步构建

doi:10.3877/cma.j.issn.1674-6902.2017.02.012

目的

采用温孵法构建脂多糖/脂多糖结合蛋白(LPS/LBP)复合物，研究LPS炎症信号通路。

方法

将LPS与LBP按15︰1、10︰1、5︰1的比例混匀。把LPS/LBP混合物、LBP(浓度分别为200 μg/ml、100 μg/ml和50 μg/ml)、LPS(浓度分别为100 μg/ml、50 μg/ml)过夜孵育。将孵育后的LPS/LBP混合物、LBP、LPS行凝胶电泳，经考马斯亮蓝染色后将各染色条带切取，行蛋白回收，并测定内毒素。

结果

凝胶电泳后，LPS/LBP复合物、LBP泳道于相对分子质量52×10³处可见考马斯亮兰染色的条带。样本中LBP浓度越高、条带染色越浓，LPS泳道对应于相对分子质量52×10³处无染色条带出现。各浓度比例的LPS/LBP复合物电泳后凝胶条带回收物中均能检测出内毒素。10︰1 LPS/LBP复合物组、15︰1 LPS/LBP复合物组复合物内毒素浓度显著高于5︰1 LPS/LBP复合物组内毒素浓度(P<0.05)。10︰1 LPS/LBP复合物组与15︰1 LPS/LBP复合物组内毒素浓度无统计学差异(P>0.1)。各浓度LBP组均未检出内毒素，各LPS组内毒素检测也为阴性。

结论

LPS︰LBP为10︰1是较为合理的构建LPS/LBP复合物的比例，通过温孵及凝胶电泳可获得纯度较高的LPS/LBP复合物。

关键词: 脂多糖, 脂多糖结合蛋白, 复合物, 构建

Objective

To construct lipopolysaccharide/lipopolysaccharide binding protein(LPS/LBP) complexes by incubation for further study of LPS inflammatory signaling pathway.

Methods

LPS and LBP were mixed at following ratio: 15︰1, 10︰1 and 5︰1. The LPS/LBP mixtures, LBP (200 μg/ml, 100 μg/ml, 50 μg/ml) and LPS (100 μg/ml, 50 μg/ml) were incubated in constant temperature shaker overnight. The LPS/LBP mixtures, LBP and LPS were separate by gel electrophoresis. All the bands were harvested after coomassie blue staining for protein recovery, and endotoxin test.

Results

At relative molecular mass 52×10^3, LPS/LBP mixture groups and LBP groups appeared Coomassie blue staining bands, LPS groups didn′t show it. The higher concentration of LBP had the darker band . The endotoxin tests of all LPS/LBP mixture groups were positive. The endotoxin concentration of 15︰1 and 10︰1 LPS/LBP mixture groups were significantly higher than that of 5︰1 LPS/LBP mixture group(P<0.05). There was no significant difference between the endotoxin concentrations of 15︰1 and 10︰1 LPS/LBP mixture groups(P>0.1). The endotoxin tests of all LBP groups were negative. LPS groups aslo showed negative results for endotoxin test.

Conclusions

The ideal ratio of LPS︰LBP to construct LPS/LBP complex is 10︰1. Purified LPS/LBP complex can be obtained by incubation and gel electrophoresis.

Key words: Lipopolysaccharide, Lipopolysaccharide binding protein, Complexes, Construction

图1 LPS/LBP复合物、LBP和LPS的凝胶显像结果；注：泳道1：3 000 μg/ml︰200 μg/ml LPS/LBP；泳道2：1 000 μg/ml︰100 μg/ml LPS/LBP；泳道3：250 μg/ml︰50 μg/ml LPS/LBP；泳道4：200 μg/ml LBP；泳道5：100 μg/ml LBP；泳道6：50 μg/ml LBP；泳道7：200 μg/ml LPS；泳道8：100 μg/ml LPS

图2 各实验组凝胶回收物内毒素水平；^aP<0.05，与5︰1LPS/LBP组比较；各LBP组、LPS组内毒素测定均为阴性

表1 LPS/LBP复合物组凝胶回收物内毒素水平(n＝5，±s)

组　别	内毒素浓度(EU/ml)
15︰1 LPS/LBP组	900.00±140.20^a
10︰1 LPS/LBP组	823.33±232.09^a
5︰1 LPS/LBP组	499.17±111.60

表1 LPS/LBP复合物组凝胶回收物内毒素水平(n＝5，±s)

组　别	内毒素浓度(EU/ml)
15︰1 LPS/LBP组	900.00±140.20^a
10︰1 LPS/LBP组	823.33±232.09^a
5︰1 LPS/LBP组	499.17±111.60

1	牛莎，张睢扬. 脓毒症和脓毒症休克抗感染治疗进展[J/CD]. 中华肺部疾病杂志(电子版), 2013, 6(3): 202-204.
2	Oblak A, Jerala R. The molecular mechanism of species-specific recognition of lipopolysaccharides by the MD-2/TLR4 receptor complex[J]. Mol Immunol, 2015, 63(2): 134-142.
3	Li CH, Zuo HL, Zhang Q, et al. Analysis of soluble proteins in natural cordyceps sinensis from different producing areas by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis[J]. Pharmacognosy Res, 2017, 9(1): 34-38.
4	Kopp F, Kupsch S, Schromm AB. Lipopolysaccharide-binding protein is bound and internalized by host cells and colocalizes with LPS in the cytoplasm: Implications for a role of LBP in intracellular LPS-signaling[J]. Biochim Biophys Acta, 2016, 1863(4): 660-672.
5	Stasi A, Intini A, Divella C, et al. Emerging role of lipopolysaccharide binding protein in sepsis-induced acute kidney injury[J]. Nephrol Dial Transplant, 2016, pii: gfw250.
6	Dauphinee SM, Karsan A. Lipopolysaccharide signaling in endothelial cells[J]. Lab Invest, 2006, 86(1): 9-22.
7	Fujihara M, Muroi M, Tanamoto K, et al. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex[J]. Pharmacol Ther, 2003, 100(2): 171-194.
8	Park BS, Lee JO. Recognition of lipopolysaccharide pattern by TLR4 complexes[J]. Exp Mol Med, 2013, 45(12): e66.
9	Miyake K. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2[J]. Int Immunopharmacol, 2003, 3(1): 119-128.
10	Ryu JK, Kim SJ, Rah SH, et al. Reconstruction of LPS transfer cascade reveals structural determinants within LBP, CD14, and TLR4-MD2 for Efficient LPS Recognition and Transfere[J]. Immunty, 2017, 46(1): 38-50.
11	Jiao YL, Wu MP. Apolipoprotein A-I diminishes acute lung injury and sepsis in mice induced by lipoteichoic acid[J]. Cytokine, 2008, 43(1): 83-87.
12	Chen YY, Lien JM, Peng YS, et al. Lipopolysaccharide binding protein in cirrhotic patients with severe sepsis[J]. J Chin Med Assoc, 2014, 77(2): 68-74.
13	Café-Mendes CC, Garay-Malpartida HM, Malta MB, et al. Chronic nicotine treatment decreases LPS signaling through NF-κB and TLR-4 modulation in the hippocampus[J]. Neurosci Lett, 2016, doi: 10.1016/j.neulet.2016.10.056.
14	Zweigner J, Schumann RR, Weber JR. The role of lipopolysaccharide-binding protein in modulating the innate immune response[J]. Microbes Infect, 2006, 8(3): 946-952.
15	Fang L, Xu Z, Wang GS, et al. Directed evolution of an LBP/CD14 inhibitory peptide and its anti-endotoxin activity[J]. PLoS One, 2014, 9(7): e101406.
16	Vreugdenhil AC, Rousseau CH, Hartung T, et al. Lipopolysaccha-ride (LPS)-binding protein mediates LPS detoxification by chylomicrons[J]. J Immunol, 2003, 170(3): 1399-1405.
17	冯起甲，徐智，吴国明，等. 脂多糖结合蛋白与CD14结合位点模拟肽的初步筛选[J]. 第三军医大学学报，2007, 29(16): 1572-1575.
18	Chen KF, Chaou CH, Jiang JY, et al. Diagnostic accuracy of lipopolysaccharide-binding protein as biomarker for sepsis in adult patients: a systematic review and Meta-analysis[J]. PLoS One, 2016, 11(4): e0153188.
19	Park BS, Lee JO. Recognition of lipopolysaccharide pattern by TLR4 complexes[J]. Exp Mol Med, 2013, 45: e66.
20	Sheen H. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading[J]. Anal Biochem, 2016, 498: 95-97.
21	Manabe T, Jin Y. Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro-2-DE followed by 3-D SDS-PAGE and MS[J]. Electrophoresis, 2007, 12(28): 2065-2079.
22	Brunelle JL, Green R. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE)[J]. Methods Enzymol, 2014, 541: 151-159.
23	Alu′datt MH, Rababah T, Alhamad MN, et al. Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species[J]. Food Chem, 2017, 230: 125-134.
24	Ilavenil S, Al-Dhabi NA, Srigopalram S, et al. Removal of SDS from biological protein digests for proteomic analysis by mass spectrometry[J]. Proteome Sci, 2016, 14(1): 11.
25	Pant P, Singh RG, Usha, et al. Serum sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of patients with membranous nephropathy and focal and segmental glomerulosclerosis[J]. Saudi J Kidney Dis Transpl, 2016, 27(3): 539-545.
26	Hempelmann E, Krafts K. The mechanism of silver staining of proteins separated by SDS polyacrylamide gel electrophoresis[J]. Biotech Histochem, 2017, 92(2): 79-85.

[1]	乔莉, 赵超, 孙昊, 陈洁, 王军, 张劲松. 参附注射液对脂多糖所致肺泡细胞损伤的保护作用[J]. 中华危重症医学杂志(电子版), 2021, 14(05): 362-367.
[2]	刘光磊, 杨军, 李成荣. 儿童特发性肾病综合征低IgG血症机制：哺乳动物雷帕霉素靶蛋白过表达对滤泡辅助性T细胞数量的可能影响[J]. 中华妇幼临床医学杂志(电子版), 2020, 16(02): 171-180.
[3]	徐燕群, 李平, 杨兴, 薛慧. 脂多糖通过促进透明质酸受体CD44向核转移介导牙周膜细胞白细胞介素6释放[J]. 中华口腔医学研究杂志(电子版), 2023, 17(05): 335-344.
[4]	程世芳, 毛永芸, 唐生业, 侯志斌. 胃癌外周血TWA1 mRNA的表达与预后价值[J]. 中华普通外科学文献(电子版), 2020, 14(06): 421-424.
[5]	魏婷婷, 胡小红, 龚自强, 熊鹿. 老年非小细胞肺癌组织ARPC2表达及与预后关系[J]. 中华肺部疾病杂志(电子版), 2023, 16(04): 584-586.
[6]	李埝, 赵建军, 张建勇, 赵睿桢. hAMSCs调控MAPK信号通路对急性肺损伤AQP1的影响[J]. 中华肺部疾病杂志(电子版), 2023, 16(02): 156-163.
[7]	周莹芊, 汪振星, 张一帆, 孙家明, 曹谊林. 模块化与血管化组织工程技术[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 161-166.
[8]	岳影星, 杨舟鑫, 卢艳, 严静. 脂多糖调控对大鼠心脏微血管内皮细胞的转录组分析[J]. 中华细胞与干细胞杂志(电子版), 2020, 10(06): 328-335.
[9]	梅亚波, 张万巧, 陈冲, 韩涛, 封志纯. 人脐带间充质干细胞对脂多糖活化的小胶质细胞BV-2表型的影响[J]. 中华细胞与干细胞杂志(电子版), 2020, 10(04): 213-218.
[10]	郑啟发, 李华, 袁泽南, 叶林森, 朱曙光, 夏龙. 脂多糖诱导小鼠肝癌YAP表达及其调控巨噬细胞极化的研究[J]. 中华肝脏外科手术学电子杂志, 2020, 09(03): 283-288.
[11]	张紫薇, 卢弘. 脂多糖受体复合体在急性前葡萄膜炎虹膜色素上皮细胞中作用的研究进展[J]. 中华眼科医学杂志(电子版), 2023, 13(03): 167-171.
[12]	张大涯, 陈世锔, 陈润祥, 张晓冬, 李达, 白飞虎. 肠道微生物群对代谢相关脂肪性肝病发展的影响[J]. 中华临床医师杂志(电子版), 2023, 17(07): 828-833.
[13]	张小玉, 郭伟, 吴秀丽, 汪蕊, 汪莹. ICU专科护士培训大纲的构建和应用[J]. 中华临床医师杂志(电子版), 2023, 17(03): 330-334.
[14]	苏程程, 马永强, 郎胜坤, 刘斌, 魏路清, 姬文婕. 盐皮质受体对脂多糖诱导的巨噬细胞NOD样受体热蛋白结构域相关蛋白3炎症复合体激活的作用及其机制[J]. 中华临床医师杂志(电子版), 2022, 16(05): 447-451.
[15]	李正达, 张艳兵, 刘茂霞, 李玉芳, 杨新静. 艾司洛尔对脓毒症肠损伤的保护作用及对自噬蛋白AMPK表达水平的影响[J]. 中华卫生应急电子杂志, 2023, 09(02): 90-95.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Preliminary constructing of lipopolysaccharide/lipopolysaccharide complexes

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Preliminary constructing of lipopolysaccharide/lipopolysaccharide complexes