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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2017, Vol. 10 ›› Issue (02) : 173 -177. doi: 10.3877/cma.j.issn.1674-6902.2017.02.012

所属专题： 文献；

论著

脂多糖/脂多糖结合蛋白复合物的初步构建
郑珍容1, 贺斌峰1, 魏征华1, 王关嵩1, 戢福云1, 徐智1,†()   
  1. 1. 400037　重庆，第三军医大学新桥医院呼吸内科　全军呼吸内科研究所　全军呼吸病研究重点实验室
  • 收稿日期:2017-03-09 出版日期:2017-04-20
  • 通信作者: 徐智
  • 基金资助:
    国家自然科学基金资助项目(81370169)

Preliminary constructing of lipopolysaccharide/lipopolysaccharide complexes

Zhenrong Zheng1, Binfeng He1, Zhenghua Wei1, Guansong Wang1, Fuyun Ji1, Zhi Xu1,†()   

  1. 1. Institute of Respiratory Diseases, Key Laboratory of Respiratory Diseases Research, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China
  • Received:2017-03-09 Published:2017-04-20
  • Corresponding author: Zhi Xu
  • About author:
    Corresponding author: Xu Zhi, Email:
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引用本文：

郑珍容, 贺斌峰, 魏征华, 王关嵩, 戢福云, 徐智. 脂多糖/脂多糖结合蛋白复合物的初步构建[J]. 中华肺部疾病杂志(电子版), 2017, 10(02): 173-177.

Zhenrong Zheng, Binfeng He, Zhenghua Wei, Guansong Wang, Fuyun Ji, Zhi Xu. Preliminary constructing of lipopolysaccharide/lipopolysaccharide complexes[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2017, 10(02): 173-177.

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 52×103, 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±140.20a
10︰1 LPS/LBP组 823.33±232.09a
5︰1 LPS/LBP组 499.17±111.60
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