CN109867717A

CN109867717A - The extracting method of 1 microglobulin of α in a kind of blood plasma

Info

Publication number: CN109867717A
Application number: CN201910257872.9A
Authority: CN
Inventors: 苏少博; 裴朝红
Original assignee: Shanxi Ruiya Force Biological Technology Co Ltd
Current assignee: Shanxi Ruiya Force Biological Technology Co Ltd
Priority date: 2019-04-01
Filing date: 2019-04-01
Publication date: 2019-06-11

Abstract

The invention discloses a kind of extracting method of 1 microglobulin of α in blood plasma, successively follow the steps below it is obtained, 1) take 1 microglobulin of α immune after sheep blood plasma, many kinds of substance such as initial gross separation haemocyte after sedimentation；2) centrifuging and taking supernatant reaches 33%(w/v using ammonium sulfate precipitation to solution ammonium sulfate end saturated concentration) precipitate serum sufficiently；3) precipitation and separation sufficiently redissolves, and dialyses and is concentrated；4) initial gross separation is carried out to the processed filtrate of step 3) using gel permeation chromatography column, collects the eluent containing 1 microglobulin of α；5) eluent being collected into using Protein G affinity column to step 4) carries out affinity chromatography, collects filtrate and carries out after desalination, concentration up to 1 microglobulin of α；6) 1 microglobulin purity of α is identified using SDS-PAGE.This method step is simple, highly-safe, and stability is high and acquisition purity of protein is higher, is suitble to laboratory and enlarged experiment production.

Description

The extracting method of 1 microglobulin of α in a kind of blood plasma

Technical field

The invention belongs to protein separation fields, more particularly, to a kind of extraction side of 1 microglobulin of α in blood plasma Method.

Background technique

1 microglobulin of α (alpha 1-microglobulin, α 1-MG) be relative molecular mass about 26000 in human body ~ The micro-molecule glucoprotein of 33000KDa is usually made of 182 amino acid residues, earliest by Ekstrom etc. from Chronic Cadmium It separated in the urine of malicious patient, purify and obtain, be located at 1 zone of α when electrophoresis and gain the name.Its unique size and charge it is inhomogenous Property, so that function is become particularly significant.It is widely distributed in vivo as the important indicator for judging liver and renal function, mainly by After liver cell or lymphocyte specific synthesis, it is assigned in internal various tissues with blood transportation.In blood, 1 microballoon egg of α The white form that is primarily present is with the presence of free and Immunoglobulin IgA or albumin combining form.When severe hepatic disorder, Since liver cell is impaired, reduce liver synthesis, metabolism and secretion α 1-MG, α 1-MG content reduces in hepatopathy patient serum. Sequestered α 1-MG can freely through glomerular filtration membrane, but at proximal convoluted tubule because reabsorption almost again be absorbed, together When combining form α 1-MG cannot may only detect sequestered α 1- in the urine of normal condition servant by glomerular filtration The presence of MG, once it will lead to combine since detection of glomeruli filtration function is impaired or reabsorption and metabolic capability reduce Form α 1-MG is more discharged into urine by blood, and the content of α 1-MG is caused to increase.Therefore, α 1-MG is as screening early stage Nephrosis, the important indicator of hepatopathy, in clinic, the various aspects such as outpatient service and Gernral Check-up are widely used.

Since α 1-MG is widely used in medical diagnosis on disease, developing efficient purification schemes has significantly Realistic meaning and application value.The relevant method of purification of existing report is more complicated, cumbersome and costly, sees Shen Xin Justice etc. " separation of human urine 1-microglobulin of α, purifying and identification " (The 2nd Army Medical College journal, 1992,13), biography used The fiber medium DE52/CM52 of system, flow velocity is slow, and effect of living again is poor, and the service life is short, is not suitable for quantization production.

Summary of the invention

Problem to be solved by this invention is just to provide a kind of extracting method of 1 microglobulin of α in blood plasma, for preparation diagnosis Antibody starting material provides basis in kit, and its technical solution is as follows:

The extracting method of 1 microglobulin of α in a kind of blood plasma, comprising the following steps:

1) the fresh sheep blood plasma after taking 1 microglobulin antigen of α immune, after waiting for blood to solidify, takes blood plasma to be centrifuged off fibrin Light yellow transparent liquid-serum is isolated after former and certain coagulation factors；

2) upper layer light yellow transparent liquid is sucked out in siphonage, is precipitated to solution ammonium sulfate end saturated concentration using ammonium sulfate precipitation method Reach 33%(w/v).After serum sufficiently precipitates, precipitating is redissolved using the kaliumphosphate buffer of pH7.5, using normal saline solution Dialysis, using PEG20000 protein concentrate.

3) chromatographic column equipped with sephadex G100 filler is taken, uses the kaliumphosphate buffer of pH7.5 as equilibration buffer The chromatographic column is balanced, then loading is eluted with same buffer with 0.5 mL/min, collect eluent, dense with PEG20000 Contracting collection liquid.

4) chromatography that the homogenate of Protein G affinity chromatography medium is pre-loaded with 1mL Binding Buffer to one is drawn In column, allows filler filler natural subsidence to bottom of the pillar, 5 mL Binding Buffer are added into column, make Buffer slow Outflow, flow velocity 1mL/min.Concentrate in step 3) Binding Buffer 1:1 is diluted, starts to be loaded, flow velocity is about For 0.2-0.5 mL/min, efflux is collected, it is subsequent to detect purification efficiency with SDS-PAGE.Use about 30 mL Binding Buffer washing, flow velocity are about 2 mL/min, and the absorbance at frontier inspection survey 280nm is washed on side, until there is stationary value.Washing After, it is initially added into 10-15 mL Elution Buffer, using 1 mL/min compared with low flow velocity, collects eluent, then adjust PH to 7.4, as pure 1 microglobulin of α.Wherein Binding Buffer are as follows: 20Mm NaHPO4,0.15M NaCl, pH 8.0.Elution Buffer are as follows: 0.1 M glycine pH 2.5.

5) 1 microglobulin purity of α is identified using SDS-PAGE.Using 12% separation gel, 5% concentration glue takes 50 μ L samples molten Liquid adds 10 μ L 10x sample buffers to be uniformly mixed, and boils 5min, and 12000rpm is centrifuged 5min loading.

The beneficial effects of the present invention are:

In method provided by the invention, the sephadex G100 used, this glue pH range is wide, and carrying capacity is big, is more suitable for α 1-MG Purifying, and integrated operation is easy, and repeatability is strong, and single operation output is big.

Detailed description of the invention

Fig. 1 is 1 microglobulin SDS-PAGE map of α after purification of the embodiment of the present invention.

Specific embodiment

With reference to the accompanying drawing and specific embodiment is further elaborated the contents of the present invention, but embodiment is only this The better embodiment of invention, therefore all equivalence changes done according to feature described in present patent application range and principle, It is included in the scope of the patent application of the present invention.

Embodiment 1:

The first step, the sheep blood plasma after taking 1 microglobulin of α immune, natural subsidence is stayed overnight under room temperature, next day at 4 DEG C, 4000xg from Heart 15min, removal red blood cell, leucocyte, blood platelet go out etc. a variety of haemocytes；

Second step is sucked out upper layer light yellow transparent liquid, is precipitated to solution ammonium sulfate end saturated concentration using ammonium sulfate precipitation method Reach 33%(w/v), it is placed in 4 DEG C of overnight precipitations.After serum sufficiently precipitates, next day, 8000xg was centrifuged 20min, in abandoning at 4 DEG C Clearly, precipitating is collected.Precipitating is redissolved using the kaliumphosphate buffer of pH7.5, and saturating overnight at 4 DEG C using normal saline solution Analysis, to remove a large amount of ammonium sulfate having in serum deprivation, after dialysis, using PEG20000 protein concentrate；

Third step takes the chromatographic column (2cm*60cm) equipped with sephadex G100 filler, is made with the kaliumphosphate buffer of pH7.5 Then use same buffer with 0.5 mL/ the concentrate loading in second step for chromatographic column described in equilibration buffer The flow velocity of min elutes, while detecting the absorbance at 280nm, collects eluent until the absorbance appearance at 280nm is constant Collection liquid is concentrated with PEG20000 in value.

4th step draws the homogenate of Protein G affinity chromatography medium to one and is pre-loaded with 1mL Binding Buffer Chromatographic column in, allow filler filler natural subsidence to bottom of the pillar, 5 mL Binding Buffer be added into column, allow Buffer slowly flows out, flow velocity 1mL/min.Concentrate in step 3) Binding Buffer 1:1 is diluted, is started Sample-adding, flow velocity is about 0.2-0.5 mL/min, collects efflux, subsequent to detect purification efficiency with SDS-PAGE.Use about 30 ML Binding Buffer washing, flow velocity are about 2 mL/min, and the absorbance at frontier inspection survey 280nm is washed on side, until occurring Stationary value.After washing, it is initially added into 10-15 mL Elution Buffer, using 1 mL/min compared with low flow velocity, collection is washed De- liquid, then pH is adjusted to 7.4.Wherein Binding Buffer are as follows: 20Mm NaHPO4,0.15M NaCl, pH 8.0.Elution Buffer are as follows: 0.1 M glycine pH 2.5.It is concentrated later with the ultrafiltration membrane that interception is 10KD, as pure 1 microballoon of α Albumen.

5th step identifies 1 microglobulin purity of α using SDS-PAGE.Using 12% separation gel, 5% concentration glue takes 50 μ L Sample solution adds 10 μ L 10x sample buffers to be uniformly mixed, and boils 5min, samples 10 μ L addition after 12000rpm centrifugation 5min Gel loading wells.Electrophoresis apparatus is connected, adjustment voltage to 80 V uses 130 V instead after sample fully enters gel, starts electrophoresis Until bromophenol blue indicator reaches gel bottom.Blob of viscose is placed in distillation water washing 2 times after electrophoresis, then is placed in R-250 dye It is dyed overnight in color liquid, distillation water washing decoloration, testing goal albumen.

The 1 microglobulin molecule amount of α that the present embodiment obtains is 35KDa, and the purity of 1 microglobulin of α is 90%, and the rate of recovery is 85%。

1 microglobulin SDS-PAGE map of α is as shown in Figure 1 after purification for method provided by the invention.

In Fig. 1, the sample after respectively Maker, sheep blood serum sample, 33% ammonium sulfate precipitation sheep blood serum, sheep from left to right Sample after serum affinitive layer purification.

Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features. All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention Within protection scope.

Claims

1. the extracting method of 1 microglobulin of α in a kind of blood plasma, which is characterized in that comprise the following steps:

2) upper layer light yellow transparent liquid is sucked out in siphonage, precipitates serum sufficiently using ammonium sulfate precipitation method, and centrifuge separation is heavy It forms sediment, precipitating is redissolved in buffer, after sufficiently redissolving, dialyse, be concentrated；

3) gel permeation chromatography；

4) Protein G affinity chromatography；

5) SDS-PAGE method identifies 1 microglobulin purity of α.

2. the extracting method of 1 microglobulin of α in blood plasma according to claim 1, it is characterised in that: the centrifugal condition is At 4 DEG C, 4000xg is centrifuged 15min.

3. the extracting method of 1 microglobulin of α in blood plasma according to claim 1, it is characterised in that: the ammonium sulfate precipitation Method is precipitated to solution ammonium sulfate end saturated concentration and reaches 33%(w/v) precipitate serum sufficiently.

4. the extracting method of 1 microglobulin of α in blood plasma according to claim 1, it is characterised in that: the precipitating is redissolved slow Fliud flushing uses the kaliumphosphate buffer of pH7.5, and elution buffer uses normal saline solution, using PEG20000 protein concentrate.

5. the extracting method of 1 microglobulin of α in blood plasma according to claim 1, it is characterised in that: the step 3) is specific Are as follows: the chromatographic column equipped with sephadex G100 filler is taken, uses the kaliumphosphate buffer of pH7.5 as equilibration buffer institute Chromatographic column is stated, then loading is eluted with same buffer with 0.5 mL/min, collect eluent, be concentrated with PEG 20000 Collection liquid.

6. the extracting method of 1 microglobulin of α in blood plasma according to claim 1, it is characterised in that: the step 4) is specific Are as follows: it draws the homogenate of Protein G affinity chromatography medium and is pre-loaded with to one in the chromatographic column of 1mL Binding Buffer, allowed Filler filler natural subsidence is added 5 mL Binding Buffer into column, Buffer is allowed slowly to flow out, flow to bottom of the pillar Speed is 1mL/min；Concentrate in step 3) Binding Buffer 1:1 is diluted, starts to be loaded, flow velocity is about 0.2- 0.5 mL/min collects efflux, subsequent to detect purification efficiency with SDS-PAGE；Use about 30 mL Binding Buffer Washing, flow velocity are about 2 mL/min, and the absorbance at frontier inspection survey 280nm is washed on side, until there is stationary value；Washing finishes Afterwards, be initially added into 10-15 mL Elution Buffer, using 1 mL/min compared with low flow velocity, collect eluent, then adjust pH to 7.4, as pure 1 microglobulin of α；Wherein Binding Buffer are as follows: 20Mm NaHPO4,0.15M NaCl, pH 8.0； Elution Buffer are as follows: 0.1 M glycine pH 2.5.