CN105273080A

CN105273080A - Preparation method of reduced beta2-glycoprotein I

Info

Publication number: CN105273080A
Application number: CN201410330324.1A
Authority: CN
Inventors: 于珮; 于德民; 周赛君; 汤云昭; 陈睿; 孟振兴; 王洁
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-07-14
Filing date: 2014-07-14
Publication date: 2016-01-27

Abstract

The invention discloses a preparation method for obtaining stable reduced beta2-glycoprotein I (beta2GPI). The method comprises the following steps: carrying out in vitro reduction on purified beta2-glycoprotein I by using thioredoxin-1 and dithiothreitol, identifying the reduced beta2GPI by using polyacrylamide gel electrophoresis and liquid chromatography-mass spectrometry, and protecting a free sulfhydryl group by using reduced glutathione (GSH) in order to obtain the reduced beta2GPI existing in vitro. The method lays a foundation for further researches of the biological functions of the reduced beta2GPI.

Description

A kind of preparation method of reduced form β2-glycoproteinⅰ

Technical field

The present invention relates to the preparation method of reduced form beta 2 glycoprotein I.

Background technology

Beta 2 glycoprotein I (β 2-glycoproteinI, β 2GPI), also known as Apolipoprotein H, being the plasma proteins (concentration is about 4 μm of ol/L) being rich in phosphatide binding site, is also the dominant autoantigen of antiphospholipid syndrome.This molecular weight of albumen is about 50KD, is made up of 326 amino acid, includes 5 short consensus repeats (being called I-V structural domain successively) and the chain glycosylation site of 4 N, forms a fish hook shape 3-D solid structure.Front four domain constructs of this albumen are similar, respectively have two disulfide linkage, and the 5th structural domain are at Cys ²⁸⁸and Cys ³²⁶between form an extra disulfide linkage, therefore containing three disulfide linkage.

In recent years research finds, the Cys of β 2GPI the 5th structural domain ²⁸⁸-Cys ³²⁶disulfide linkage can be opened by Trx-1 (TRX-1) and protein disulfide isomerase (PDI), and form two free sulfydryls, the β 2GPI of this form is called reduced form β 2GPI by investigator.In healthy population, reduced form β 2GPI accounts for major portion.And in antiphospholipid syndrome patient body, the ratio shared by β 2GPI (oxidized form) significantly raises.Preliminary research finds, reduced form β 2GPI can alleviate H ₂o ₂the damage of the Human umbilical vein endothelial cells of induction, the foamed of the scavenger cell that ox-LDL can also be suppressed to induce and apoptosis.Study supposition according to this, reduced form β 2GPI may be a kind of protective protein of body, and plays a significant role in the processes such as oxidative stress, blood coagulation, atherosclerosis.

At present, the research about reduced form β 2GPI is not still enriched, and to its concrete function, we still know little about it.The free sulfhydryl groups character of reduced form β 2GPI is enlivened, in vitro can not stable existence in environment.At present, to be extracted by blood plasma or the method for recombinating can obtain purer β 2GPI (oxidized form), but all can not obtain the reduced form β 2GPI of stable existence.Therefore, prepare the reduced form β 2GPI of stable existence, to studying further and being familiar with the β 2GPI of this newfound special shape, have very important significance.

Summary of the invention

The object of this invention is to provide a kind of method obtaining the reduced form β 2GPI of stable existence.

Technical scheme of the present invention is summarized as follows:

A kind of method obtaining the reduced form β 2GPI of stable existence.

The present invention utilize TRX-1 and DTT reduce in vitro purify β 2GPI; utilize the technical evaluation such as polyacrylamide gel electrophoresis and liquid chromatograph mass spectrography reduced form β 2GPI; and utilize reduced glutathion (GSH) to protect free sulfhydryl groups; thus obtain the reduced form β 2GPI of stable existence in vitro, for the biological function studying reduced form β 2GPI further lays the foundation.

Accompanying drawing explanation

Fig. 1 is the SDS-PAGE result figure (non-reduced do not heat) of β 2GPI and reduced form thereof.This figure illustrates that reduced form β 2GPI relatively lags behind than β 2GPI.

Fig. 2 is the WesternBlot result figure of β 2GPI and reduced form thereof.This figure conforms to SDS-PAGE result.

Fig. 3 is the 318-326 peptide section LC-MS information table of β 2GPI.This table provides essential information for following LC-MS interpretation of result.

Fig. 4 is the LC-MS ion flow graph (non-alkylation) of β 2GPI after TRX-1+DTT reduction.In this figure, main peak is the 318-326 peptide section containing free sulfhydryl groups.

Fig. 5 is the LC-MS mass spectrum (non-alkylation, M2H+=468.21) of β 2GPI after TRX-1+DTT reduction.This figure is the mass spectroscopy figure of Fig. 4 main peak, and the non-alkylation of this peptide section is described.

Fig. 6 is the LC-MS ion flow graph of β 2GPI after TRX-1+DTT reduction.This figure is composite diagram, and after alkylation, the main peak of 318-326 peptide section offsets.

Fig. 7 is the LC-MS mass spectrum (alkylation, M2H+=496.72) of β 2GPI after TRX-1+DTT reduction.This figure is the peptide section mass spectroscopy figure after alkylation, illustrates that this peptide section there occurs alkylation.Alkylating agent IAM is for free sulfhydryl groups, and β 2GPI albumen only has 326 may there is free sulfhydryl groups in this peptide section, and this figure confirms this point.In sum, LC-MS result illustrates that TRX-1+DTT can open the disulfide linkage of Cys288-Cys326, and then forms free sulfhydryl groups.

Embodiment

Material and reagent

The aminoacid sequence of natural β 2GPI is shown in http://www.ncbi.nih.gov/entrez/viewer.fcgi db=protein & id=28814, and sequence number is CAA40977.

The natural β 2GPI (n β 2GPI) in blood plasma source extracts by the method in applicant's earlier patents " natural type, breaking type or V structural domain deficient mutant Β 2 glycoprotein I are preparing the application in anti-angiogenic drugs ".TRX-1 is purchased from R & DSystems(America).DTT available from Sigma (America).GSH is purchased from Yao You Pharmaceutical Co (Chongqing, China).30KD ultra-filtration centrifuge tube is purchased from Milipore company (America).BCA protein assay kit is purchased from doctor's moral biotechnology company limited (Wuhan, China).Mouse anti human β 2GPI antibody is purchased from Milipore company (America).The rabbit anti-mouse IgG antibody of horseradish peroxidase-labeled is purchased from CellSignallingTechnology(America).Nitrocellulose filter, ECL luminescent solution and other WesternBlot reagent are purchased from Puli's lema gene Technology Co., Ltd. (Beijing, China).0.1%RapiGest is purchased from Waters company (America).Iodo-acetamide (IAM) and trifluoroacetic acid (TFA) all available from Sigma (America).

Preparation method

1. prepare TRX-1/DTT mixing solutions.First use the TRX-1 of DTT and 1mg/ml of HBS buffer 1mM, then with the proportions TRX/DTT mixing solutions of HBS:DTT (1mM): TRX-1 (1mg/ml)=21:2:1, after mixing, hatch 45 minutes in 3 DEG C.

2. dilute β 2GPI solution to final concentration 0.2 μ g/ μ l with HBS damping fluid.

3.TRX-1/DTT reduction β 2GPI.Isopyknic TRX-1/DTT mixing solutions and β 2GPI (0.2 μ g/ μ l) are mixed, hatches 1 hour for 37 DEG C, then 4 DEG C of termination reactions.Now the concentration of reduced form β 2GPI is about 0.1 μ g/ μ l.

4.GSH protects sulfydryl.In above-mentioned reaction system, adding GSH to final concentration is 40mM, and 37 DEG C are spent the night.

5. super filter tube protein concentrate solution.Above-mentioned reaction system moved in MILIPORE30KD super filter tube, 4 DEG C, 2500g is centrifugal, removes unreacted TRX-1/DTT and GSH with concentrated reduced form β 2GPI.Ultra-filtration process repeats 2-3 time.Final acquisition final concentration is about the reduced form β 2GPI solution of 1mg/ml.Albumen actual concentrations is measured by BCA method.

6.WesternBlot identifies.Get purifying β 2GPI and the reduced form β 2GPI albumen of 10 μ l (1 μ g/ μ l), carry out SDS-PAGE electrophoresis, and forward to and nitrocellulose filter hatches film by anti-β 2GPI monoclonal antibody spend the night.Two of horseradish peroxidase-labeled resists hatches 2 hours, ECL colour developing, photograph.

7.LC/MS method qualification free sulfhydryl groups.With the β 2GPI purifying protein (1 μ g/ μ l) of 20 μ l, be handled as follows respectively: sample A:10 μ l albumen (10 μ g), (60 DEG C are dissolved with 0.1%RapiGest, 15 minutes) make it sex change, with TRX-1/DTT mixing solutions reduction (60 DEG C, 30 minutes), by 20mMIAM alkanisation (room temperature, 30 minutes) and 20:1 (w/w, albumen: enzyme) trypsin digestion digestion (37 DEG C, 4 hours).Sample B:10 μ l albumen (10 μ g), (60 DEG C are dissolved with 0.1%RapiGest, 15 minutes) make it sex change, with TRX-1/DTT mixing solutions reduction (60 DEG C, 30 minutes), and 20:1 (w/w, albumen: enzyme) trypsin digestion digestion (37 DEG C, 4 hours).IAM can with sulfydryl generation irreversible reaction, thus checking free sulfhydryl groups existence.Sample B compared with sample A without alkylation process.80 μ l0.1% trifluoroacetic acid (TFA) termination reactions are added in sample respectively to A, B two.Carry out LC/MS qualification respectively.

Claims

1. the preparation method of a reduced form beta 2 glycoprotein I.