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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (12): 14-20    DOI: 10.13523/j.cb.20171203
研究报告     
重组不可分型流感嗜血杆菌E蛋白与血浆脂蛋白(a)的相互作用
王瑜1,4,刘治1,4,白文成2,4,许丽萍3,韩润林2,4()
1 内蒙古农业大学生命科学学院 呼和浩特 010018
2 内蒙古农业大学兽医学院 呼和浩特 010018
3 内蒙古医科大学基础医学院 呼和浩特 010059
4 内蒙古农业大学血浆脂蛋白免疫学研究中心 呼和浩特 010018
The Interaction between Plasma Lipoprotein(a) and Recombinant Protein E Derived from Nontypeable Haemophilus influenza
Yu WANG1,4,Zhi LIU1,4,Wen-cheng BAI2,4,Li-ping XU3,Run-lin HAN2,4()
1 College of Life Science, Inner Mongolia Agriculture University, Hohhot 010018, China
2 College of Veterinary Medicine, Inner Mongolia Agriculture University, Hohhot 010018, China
3 College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010018, China
4 Research Center of Plasma Lipoprotein Immunology, Inner Mongolia Agriculture University, Hohhot 010018, China
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摘要： 目的

E蛋白(Protein E, PE)是不可分型流感嗜血杆菌(Nontypeable Haemophilus influenza, NTHi)表面的一种纤溶酶原(plasminogen, Plg)受体,其C末端含有两个赖氨酸残基。NTHi可通过其表面的PE与Plg结合,从而利用机体的纤溶系统深层入侵宿主。基于脂蛋白(a)[Lipoprotein(a), Lp(a)]中载脂蛋白(a)[Apolipoprotein(a), Apo(a)]与Plg高度的同源性,拟证明Lp(a)是否会与重组表达的PE(rPE)结合。

方法

原核表达并纯化rPE及敲除C末端两个赖氨酸残基的rPEΔKK,密度梯度离心结合阴离子交换层析分离人血浆Lp(a),通过ELISA、Pull down、Western blot等方法研究rPE与Lp(a)的相互作用。

结果

rPE与Lp(a)结合,但不与LDL结合,且rPEΔKK与Lp(a)的结合能力明显低于rPE;赖氨酸类似物6-氨基乙酸(EACA)能有效抑制rPE与Lp(a)的结合;Lp(a)对rPE与Plg的结合具有微弱的抑制作用。

结论

rPE能够与Lp(a)结合,其中rPE的C末端赖氨酸残基和Apo(a)的赖氨酸结合位点(lysine binding sites, LBS)是rPE与Lp(a)结合的主要位点。
关键词： 不可分型流感嗜血杆菌;  PE蛋白;  脂蛋白(a);  纤溶酶原;  赖氨酸结合位点    
Abstract: Objective:

Protein E (PE), with two lysine residues at its C-terminus, is a plasminogen (Plg) receptor on the surface of nontypeable Haemophilus influenza (NTHi). NTHi can recruit Plg on the cell surface by PE and utilize host fibrinolytic system to achieve its immune invasion. Based on the high homology of Plg and Apolipoprotein(a) [Apo(a)] of Lipoprotein(a) [Lp(a)], Lp(a) was supposed to bind to PE.

Methods:

The recombinant PE (rPE) and C-terminal lysine residues-deleted variant (rPEΔKK) were obtained by prokaryotic expression and further purified. Lp(a) was isolated and purified from human plasma by KBr density gradient centrifugation followed by Q SepharoseTM Fast Flow ion exchange chromatography. The interaction between rPE and Lp(a) was investigated by enzyme-linked immunosorbent assay(ELISA) and Pull down followed by Western blot.

Results:

The results indicated that rPE could bind to Lp(a) but not to LDL, and the interaction was significantly inhibited by EACA. The binding capacity of rPEΔKK to Lp(a) was obviously lower than that of rPE. In addition, Lp(a) could inhibit the binding of rPE to Plg slightly.

Conclusion:

In overall, Lp(a) could bind to rPE and the C-terminal lysine residues of rPE and the lysine binding site(LBS) of Apo(a) was responsible for this interaction.
Key words: Nontypeable    Haemophilus influenza    Protein E    Lipoprotein(a)    Plasminogen    Lysine binding sites
收稿日期: 2017-08-15 出版日期: 2017-12-16
ZTFLH:  R378.4  
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王瑜
刘治
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韩润林

引用本文:

王瑜,刘治,白文成,许丽萍,韩润林. 重组不可分型流感嗜血杆菌E蛋白与血浆脂蛋白(a)的相互作用[J]. 中国生物工程杂志, 2017, 37(12): 14-20.

Yu WANG,Zhi LIU,Wen-cheng BAI,Li-ping XU,Run-lin HAN. The Interaction between Plasma Lipoprotein(a) and Recombinant Protein E Derived from Nontypeable Haemophilus influenza. China Biotechnology, 2017, 37(12): 14-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171203    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I12/14

PrimerSequence (5'-3')Restrictive enzymes
rPEF: CGAGACCGCGGTCCCGAATTCAAGGCTAAACAAAATGATGTGAAGCEcoRⅠ
R: CCCCTGCAGGTCGACCTCGAGTTATTTTTTATCAACTGAAAATGCTTCXholⅠ
rPEΔKKF: CGAGACCGCGGTCCCGAATTCAAGGCTAAACAAAATGATGTGAAGCEcoRⅠ
R: CCCCTGCAGGTCGACCTCGAGTTAATCAACTGAAAATGCTTCACCAXholⅠ
表1  引物列表
图1  SDS-PAGE和Western blot检测Lp(a)纯度
图2  ELISA检测rPE、rPEΔKK与Lp(a)的结合
图3  Pull down结合Western blot 检测rPE、rPEΔKK与Lp(a)的结合
图4  ELISA检测rPE与LDL的结合
图5  EACA抑制rPE与Lp(a)的结合
图6  ELISA检测rPE、rPEΔKK与Plg的结合
图7  Lp(a)对rPE与Plg结合的抑制
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