CN111766286A

CN111766286A - Method for detecting content of hybrid protein in collagen

Info

Publication number: CN111766286A
Application number: CN202010654768.6A
Authority: CN
Inventors: 魏欣苗; 佘振定; 谭荣伟
Original assignee: Shenzhen Lando Biomaterials Co ltd
Current assignee: Shenzhen Lando Biomaterials Co ltd
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-10-13
Anticipated expiration: 2040-07-09
Also published as: CN111766286B

Abstract

The invention relates to a method for detecting the content of hybrid protein in collagen. A method for detecting the content of hybrid protein in collagen comprises the following steps: diluting the standard substance in a gradient manner to prepare a plurality of standard substance solutions with different concentrations; respectively carrying out Tricine-SDS-PAGE electrophoresis on a plurality of standard substance solutions with different concentrations and a sample to be detected, and acquiring the band gray values of the plurality of standard substance solutions with different concentrations and the band gray value of the sample to be detected; fitting a standard curve according to the content of the standard substance in the standard substance solution and the corresponding band gray value; and calculating the content of the impure protein according to the standard curve and the strip gray value of the sample to be detected. The method for detecting the content of the hybrid protein in the collagen is simple and can accurately detect the content of the hybrid protein in the collagen.

Description

Method for detecting content of hybrid protein in collagen

Technical Field

The invention relates to the technical field of biology, in particular to a method for detecting the content of hybrid protein in collagen.

Background

Collagen is the most abundant extracellular matrix protein existing in the animal kingdom, and at least 45 collagen genes encoding vertebrate species are now being found to form 28 different types of collagen. Collagen is the main structural component of connective tissues in the body, particularly load-bearing tissues such as tendons, bones, skin and cartilage, and can be mechanically transferred between tissues and provide a highly biocompatible environment for cells. It is this high degree of biocompatibility and low immunogenicity that makes collagen a perfect biomaterial for implanted medical products and scaffolds for in vitro test systems. As medical materials, collagen has been widely used in connection with wound healing, burn treatment, hemostasis, hernia repair, bone/cartilage defects, and a variety of dental applications including osteoinductive repair.

In the process of collagen production, foreign proteins other than type i collagen are easily left, including other proteins introduced into the raw material (not completely removed in the purification process) and inactivated collagen due to partial or complete destruction of the triple helical structure of collagen caused by chemical agents, mechanical agitation, etc. introduced during the production process. All the residual hetero-proteins in the collagen have immunogenicity, so the detection of the hetero-proteins in the collagen is one of important indexes for the safety performance detection of collagen-based products.

Appendix A of a method for characterizing type I collagen of a standard YY/T1453-2016 tissue engineering medical instrument product is the determination of the purity of the type I collagen, and the principle is that the total amount of impurity protein contained in a collagen sample is detected by determining the staining limit of Bovine Serum Albumin (BSA) by Coomassie brilliant blue by utilizing the triple helix structure of the collagen which is not contained in other proteins and matching with the action of specific collagenase. However, this method is limited by the dyeing limit of the film, and only the content of the hetero protein is detected to be lower than the dyeing limit, and the hetero protein with small molecular weight cannot be accurately quantified, which is cumbersome to operate.

Disclosure of Invention

Therefore, there is a need for a method for detecting the content of a foreign protein in collagen, which is simple and highly accurate in quantifying the foreign protein.

A method for detecting the content of hybrid protein in collagen comprises the following steps:

diluting the standard substance in a gradient manner to prepare a plurality of standard substance solutions with different concentrations;

respectively carrying out Tricine-SDS-PAGE electrophoresis on a plurality of standard substance solutions with different concentrations and a sample to be detected, and acquiring the band gray values of the plurality of standard substance solutions with different concentrations and the band gray value of the sample to be detected;

fitting a standard curve according to the content of the standard substance in the standard substance solution and the corresponding strip gray value; and

and calculating the content of the hybrid protein according to the standard curve and the strip gray value of the sample to be detected.

The method for detecting the content of the hybrid protein in the collagen realizes the accurate quantification of the hybrid protein in the collagen by combining Tricine-SDS-PAGE electrophoresis with a standard curve; in addition, compared with the traditional method for detecting the content of the hybrid protein in the collagen, the method for detecting the content of the hybrid protein in the collagen is simpler and more convenient.

In one embodiment, the standard is selected from at least one of bovine serum albumin, human serum albumin, and ovalbumin.

In one embodiment, before the step of subjecting the plurality of standard solutions with different concentrations and the sample to be tested to Tricine-SDS-PAGE electrophoresis, a step of heating the sample to be tested to uncoil collagen in the sample to be tested is further included.

In one embodiment, the heating temperature is 90-105 ℃, and the heating time is 2-15 min.

In one embodiment, the concentration of the standard substance in the standard substance solution is 50 ng/muL-300 ng/muL.

In one embodiment, the step of fitting a standard curve comprises: calculating the mass of each sample according to the concentration of each standard solution and the corresponding sample loading volume of Tricine-SDS-PAGE electrophoresis, wherein the sample loading volume of each standard solution is equal during Tricine-SDS-PAGE electrophoresis; and fitting a standard curve according to the quality of the loaded standard substance and the corresponding strip gray value.

In one embodiment, the step of calculating the content of the impure protein according to the standard curve and the band gray value of the sample to be detected comprises: substituting the strip gray value of the sample to be detected into the standard curve to obtain the quality of the impure protein in the sample to be detected; and calculating the content of the hybrid protein according to the mass of the hybrid protein in the sample to be detected and the mass of the total protein in the sample to be detected.

In one embodiment, the sample to be tested has a plurality of bands after Tricine-SDS-PAGE electrophoresis, and the step of calculating the content of the hybrid protein comprises the following steps:

substituting the band gray values corresponding to the plurality of bands into a standard curve to obtain the mass of the protein corresponding to the plurality of bands; and

and adding the masses of the proteins corresponding to the multiple bands to obtain the mass of the hybrid protein in the sample to be detected.

In one embodiment, the support medium for Tricine-SDS-PAGE electrophoresis consists of a concentrated gel, a separation gel and an interlayer gel between the concentrated gel and the separation gel, wherein the preparation of the separation gel comprises the following steps:

storing the first glue in liquid, ddH₂Mixing the gel buffer solution and the glycerol uniformly, adding the coagulant and the coagulant, mixing uniformly, adding the mixture into a gel maker, and covering a water layer on the separation gel; wherein the first glue stores liquid and ddH₂The volume ratio of O, gel buffer and glycerol is 1: (0.8-1.2): (1.3-1.5): (0.8 to 1.2), wherein each 50mL of the first gel liquid comprises: 20g to 30g of polyacrylamide and 0.5g to 2.5g of methylene bisacrylamide.

In one embodiment, the preparation step of the interlayer adhesive comprises the following steps:

removing the aqueous layer covering the separation gel, storing the second gel in a liquid, ddH₂And O and the gel buffer solution are uniformly mixed, then the coagulant and the coagulant are added into the gel making device after being uniformly mixed, and the water layer is covered on the separation gel, wherein each 50mL of the second gel liquid comprises: 22.5-28 g of polyacrylamide and 1.0-3.0 g of methylene bisacrylamide.

In one embodiment, the cathodic buffer in Tricine-SDS-PAGE electrophoresis comprises tris, tris (hydroxymethyl) methylglycine and SDS, wherein each 500mL of 8 × cathodic buffer comprises: 44.84g to 50.44g of Tris, 65.6g to 77.6g of Tris (hydroxymethyl) methylglycine and 4g to 6g of SDS.

Drawings

FIG. 1 is a Tricine-SDS-PAGE electrophoresis of example 1;

FIG. 2 is an SDS-PAGE electrophoresis chart of step (6) of example 1.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Appendix A of the method for characterizing type I collagen of the standard YY/T1453-2016 tissue engineering medical instrument product is the determination of type I collagen purity, wherein the content (%) of the impurity protein in the sample is 100% -the purity of the collagen. Wherein, when B-C is not equal to 0, the purity of the collagen is A- (B-C); when B-C is 0,

a is the sum of the optical densities of all bands in the sample dissolved with 3% acetic acid or 0.01mol/L hydrochloric acid (i.e., the amount of all proteins in the sample), B is the sum of the optical densities of all bands in the sample dissolved with collagenase digestion solution (i.e., the amount of proteins in the sample that cannot be hydrolyzed by collagenase), C is the sum of the optical densities of all bands in ultrapure water plus collagenase digestion solution (i.e., the amount of collagenase), and BSA limit is the staining limit of Bovine Serum Albumin (BSA) with Coomassie brilliant blue determined by SDS-PAGE (SDS-polyacrylamide gel).

The hybrid protein in the method for characterizing the type I collagen of the standard YY/T1453-2016 tissue engineering medical instrument product refers to other proteins except the type I collagen remained in the process of purifying the collagen. The method mainly comprises the following steps: extracellular matrix proteins such as elastin, contaminants such as cellular debris, and the like. However, for collagens with small molecular weight and damaged part of the triple helix structure of the collagen caused by factors such as chemical reagents, mechanical stirring and the like, the collagens with small molecular weight and the collagens with normal size are degraded under the action of the collagens, and the detection cannot be carried out according to the I-type collagen characterization method of the YY/T1453-2016 tissue engineering medical instrument product.

One embodiment of the invention provides a method for detecting the content of hybrid protein in collagen, which is not limited by the staining limit of Coomassie brilliant blue on bovine serum albumin, and when B-C is 0, the staining limit of Coomassie brilliant blue on bovine serum albumin does not need to be determined by SDS-PAGE electrophoresis which is complicated to operate. In addition, the method for detecting the content of the hybrid protein in the collagen can accurately and quantitatively detect the hybrid protein with the concentration of 1kDa to 100kDa, for example, a few uncoiled low molecular weight collagen peptide chains (incomplete collagen peptide chains) can also be detected.

Specifically, the method for detecting the content of the hybrid protein in the collagen comprises the following steps of a to d:

step a: and (3) diluting the standard substance in a gradient manner to prepare a plurality of standard substance solutions with different concentrations.

Specifically, the standard is selected from at least one of bovine serum albumin, human serum albumin and ovalbumin.

In this embodiment, the standard is bovine serum albumin. In the subsequent Tricine-SDS-PAGE electrophoresis, the color development of the bovine serum albumin and the coloring agent (Coomassie brilliant blue) is highly similar to the color development of the hybrid protein and the coloring agent (Coomassie brilliant blue), and the bovine serum albumin has high stability, is not easy to degrade no matter in solution or dry powder and is placed at room temperature.

In the present embodiment, the concentration of the standard substance in the standard substance solution is 5 ng/. mu.L to 30 ng/. mu.L. Further, the concentrations of the standards in the standard solution were 30 ng/. mu.L, 25 ng/. mu.L, 20 ng/. mu.L, 15 ng/. mu.L, 10 ng/. mu.L, and 5 ng/. mu.L, respectively. Through gradient dilution, a plurality of standard solutions with different concentrations are prepared to prepare a standard curve representing the relationship between the protein concentration and the band gray value.

Step b: and respectively carrying out Tricine-SDS-PAGE electrophoresis on the plurality of standard substance solutions with different concentrations and the sample to be detected, and acquiring the band gray values of the plurality of standard substance solutions with different concentrations and the band gray value of the sample to be detected.

SDS-PAGE can be classified into Glycine-SDS-PAGE and Tricine-SDS-PAGE, the former uses a Tris buffer system, and the latter uses a Tris buffer system. The resolution of the Glycine-SDS-PAGE is 20 kDa-200 kDa, the resolution of the protein below 20kDa is lower, and small molecular bands are dispersed and have no colored band. The resolution of Tricine-SDS-PAGE is 1 kDa-100 kDa, the small molecular band is clear, and the coloration is obvious. In this embodiment, Tricine-SDS-PAGE is used to allow the accurate quantitative detection of small molecular weight hetero-proteins.

In the present embodiment, the concentration of collagen in the sample to be tested is 1mg/mL to 6 mg/mL. In an alternative embodiment, the collagen concentration in the test sample is 4 mg/mL. Of course, in other embodiments, the protein in the sample to be tested can be adjusted to an appropriate concentration according to the effective range of the prepared standard curve.

In this embodiment, the support medium for Tricine-SDS-PAGE electrophoresis consists of a concentrated gel, a separation gel and an interlayer gel between the concentrated gel and the separation gel. The Tricine-SDS-PAGE electrophoresis gel preparation steps comprise: preparing separation gel, then preparing sandwich gel on the separation gel, and preparing concentrated gel on the sandwich gel.

Further, the step of preparing the separation gel comprises: storing the first glue in liquid, ddH₂Mixing the gel buffer solution and the glycerol uniformly; then adding a coagulant and a coagulant, mixing uniformly, adding into a gel making device, and covering a water layer of 0.3-2 cm on the separation gel; standing until the gel polymerization is finished. The preparation method of the interlayer adhesive comprises the following steps: removing the aqueous layer covering the separation gel, storing the second gel in a liquid, ddH₂Mixing the O and the gel buffer solution uniformly; then adding coagulant and coagulant to mixUniformly adding the mixture into a gel maker, and covering a water layer of 0.3-2 cm on the separation gel; standing until the gel polymerization is finished. The step of preparing the concentrated gum comprises: removing the water layer covering the interlayer glue, storing the second glue in a liquid, ddH₂Mixing the O and the gel buffer solution uniformly; then adding a coagulant and a coagulant, mixing uniformly, adding into a gel maker, inserting a comb into the gel, standing until the gel polymerization is completed, and pulling out the comb.

Specifically, each 50mL of the first gel stock solution comprises: 20g to 30g of polyacrylamide and 0.5g to 2.5g of methylene bisacrylamide. Polyacrylamide and methylene bisacrylamide as monomers. Preferably, each 50mL of the first gum reservoir comprises: 22.5-28 g of polyacrylamide and 0.5-1 g of methylene bisacrylamide. In an alternative specific example, each 50mL of the first gel reservoir comprises: 24.0g of polyacrylamide and 0.75g of methylenebisacrylamide.

Specifically, each 100mL of gel buffer included: 18.3-36.5 g of Tris and 0.2-0.8 g of SDS (sodium dodecyl sulfate), and the pH value of the gel buffer solution is 8.35-8.5. Preferably, every 100mL of gel buffer comprises: 21.4g to 29.4g of Tris and 0.2g to 0.6g of SDS. In an alternative specific example, each 100mL of gel buffer comprises: 36.4g Tris and 0.3g SDS.

Specifically, the coagulant, also referred to as a catalyst, is an ammonium persulfate solution. The concentration of the ammonium persulfate in the ammonium persulfate solution is 0.08 g/mL-0.16 g/mL. Preferably, the coagulant is ammonium persulfate solution with the concentration of 0.08 g/mL-0.13 g/mL of ammonium persulfate.

Specifically, the coagulant is Tetramethylethylenediamine (TEMED). Of course, in other embodiments, other set accelerating accelerators commonly used in the art may also be used.

Specifically, the first glue reservoir, ddH₂The volume ratio of O, gel buffer and glycerol is 1: (0.8-1.2): (1.3-1.5): (0.8 to 1.2).

Specifically, each 50mL of the second gel stock solution comprises: 22.5-28 g of polyacrylamide and 1.0-3.0 g of methylene bisacrylamide. Polyacrylamide and methylene bisacrylamide as monomers. Preferably, each 50mL of the second gum reservoir comprises: 22.5g to 27g of polyacrylamide and 1.0g to 2.0g of methylenebisacrylamide. In an alternative specific example, each 50mL of the second gel reservoir comprises: 23.2g of polyacrylamide and 1.5g of methylenebisacrylamide.

In this embodiment, the first gel stock, ddH in preparing the separation gel₂O, gel buffer and Glycerol, second gel stock in preparation of Sandwich gel, ddH₂Mixing with gel buffer and second gel stock, ddH in preparing concentrated gel₂The mixing modes of O and the gel buffer solution are vortex mixing, and the mixing modes of adding the coagulant and the coagulant after the step of mixing are vortex mixing. Of course, in other embodiments, the mixing method of the reagents is not limited to vortex mixing, and may be a mixing method commonly used in the art, such as stirring, centrifugation, and the like, as long as the reagents can be uniformly mixed.

The steps of Tricine-SDS-PAGE electrophoresis include electrophoresis, staining, visualization and image analysis. Specifically, the method comprises the following steps:

the electrophoresis step of Tricine-SDS-PAGE electrophoresis comprises the following steps: and putting the electrophoresis tank into an ice water bath for 20-40V pre-electrophoresis for 8-16 min, adding 5-20 mu L of marker, sample to be detected and standard solution into the sample hole respectively, carrying out 20V-40V electrophoresis for 0.5-1.5 h, and stopping electrophoresis after 40V-100V electrophoresis until bromophenol blue reaches the bottom of the gel.

In an alternative embodiment, the step of electrophoresis by Tricine-SDS-PAGE comprises: and (3) putting the electrophoresis tank into an ice water bath, adding 700mL of anode buffer solution into an outer tank, adding 550mL of cathode buffer solution into an inner tank, performing 30V pre-electrophoresis for 15min, adding 20 mu L of marker, a sample to be detected and a standard solution into the sample hole, performing 30V electrophoresis for 1 h, and performing 100V electrophoresis until bromophenol blue reaches the bottom of the gel.

Specifically, every 500mL of 8 × Anode buffer included: 96.92g to 181.71g of Tris. Preferably, every 500mL of 8 × anode buffer comprises: 96.92g to 145.37g of Tris. In an alternative specific example, every 500mL of 8 × anode buffer comprises: 121.14g of Tris, pH 8.9.

Specifically, every 500mL of 8 × cathode buffer included: 40g to 62g of Tris, 65g to 90g of Tris (hydroxymethyl) methylglycine (Tricine) and 4g to 6g of SDS. Preferably, every 500mL of 8 × cathode buffer comprises: 44.84 g-60.55 g of Tris, 65.6 g-89.5 g of Tricine and 4 g-6 g of SDS. In an alternative specific example, every 500mL of 8 × cathode buffer comprises: 60.55g Tris, 89.50g Tricine and 5g SDS.

In one embodiment, before loading the plurality of standard solutions with different concentrations and the sample to be tested, the method further comprises the step of heating the plurality of standard solutions with different concentrations to uncoil collagen in the sample to be tested. Partially and/or completely disrupted peptide chains in the triple helix structure are more easily detected after the collagen has been uncoiled.

In one embodiment, before the loading of the plurality of standard solutions with different concentrations and the sample to be tested, the method further comprises the step of heating the plurality of standard solutions with different concentrations and the sample to be tested. Specifically, the volume ratio of the standard solution and the sample to be detected with different concentrations to the loading buffer is 1: 1; the heating condition is 1 min-2 min in boiling water. Each 50mL of loading buffer included: 11 to 14mL of a 7% tris-HCl solution (pH 6.8), 12 to 15g of glycerol, 0.5 to 2g of SDS, 5 to 15mg of bromophenol blue, and 0.6 to 1.2g of dithiothreitol.

The staining and visualization step in Tricine-SDS-PAGE electrophoresis included: placing the film after electrophoresis into a watch glass, washing with pure water, and flattening; then adding the stationary liquid to immerse the film, and shaking the film in a shaking table at a low speed for 50-80 min. And then, replacing the liquid with a dyeing solution, dyeing for 50-60 min, and finally, decoloring with a decoloring solution until the background of the film is nearly colorless, so that the strip is clearly visible.

Specifically, every 500mL of fixative solution comprises: 3 to 6g of trichloroacetic acid and 150 to 250mL of methanol. Every 500mL of staining solution comprises: 0.4g to 0.8g of Coomassie Brilliant blue R250 and 150mL to 250mL of methanol.

After the development is finished, the strip gray value analysis is carried out on the strip on the film by using an imaging system, so that the strip gray values of a plurality of standard substance solutions with different concentrations and the strip gray value of the sample to be detected are obtained. Specifically, a gel imaging analyzer and related software thereof are opened, and the gel imaging analyzer and related software are photographed after adjusting the focal length and the brightness to obtain the stripe gray values of a plurality of standard solution with different concentrations and the stripe gray value of the sample to be detected.

Step c: and fitting a standard curve according to the content of the standard substance in the standard substance solution and the corresponding band gray value.

In one embodiment, the mass of the standard and the corresponding band gray value are used to fit a standard curve. More specifically, the mass of each sample of the standard substance is calculated according to the concentration of each standard substance solution and the corresponding sample loading volume of Tricine-SDS-PAGE electrophoresis; and fitting a standard curve according to the quality of the loaded standard substance and the corresponding strip gray value. Of course, in other embodiments, a standard curve may be fit using the concentrations of the standard solutions and the corresponding band gray values, and the loading volumes of Tricine-SDS-PAGE electrophoresis of the standard sample solutions are equal.

Step d: and calculating the content of the hybrid protein according to the standard curve and the band gray value of the sample to be detected.

Specifically, the strip gray of the sample to be detected is substituted into the standard curve to obtain the content of the hybrid protein. Of course, in some embodiments, after the concentration of the standard substance and the corresponding strip are set by using software matched with the imaging system, a standard curve is automatically drawn by using the software, and the content of the impure protein in the sample to be detected is output.

In one embodiment, the standard curve is a function of the mass of the standard and the corresponding band gray value. At this time, the step of calculating the content of the impure protein according to the standard curve and the strip gray value of the sample to be detected comprises the following steps: substituting the strip gray value of the sample to be detected into the standard curve to obtain the quality of the impure protein in the sample to be detected; and then dividing the mass of the hybrid protein in the sample to be detected by the mass of the total protein in the sample to be detected to obtain the content of the hybrid protein.

In one embodiment, the standard curve is a function of the relationship between the quality of the standard substance and the corresponding band gray value, and the sample to be detected has a plurality of bands after Tricine-SDS-PAGE electrophoresis. At this time, the step of calculating the content of the hetero protein includes: substituting the band gray values corresponding to the plurality of bands into the standard curve to obtain the mass of the protein corresponding to the plurality of bands; and adding the masses of the proteins corresponding to the multiple bands, and dividing the sum by the mass of the total protein in the sample to be detected to obtain the mass percentage content of the hybrid protein.

In one embodiment, the standard curve is a function of the concentration of the standard substance and the gray value of the corresponding strip, the sample to be detected has a plurality of strips after Tricine-SDS-PAGE electrophoresis, and the sample loading volumes of the Tricine-SDS-PAGE electrophoresis of the standard substance solution and the sample to be detected are equal. At this time, the step of calculating the content of the hetero protein includes: substituting the band gray values corresponding to the plurality of bands into the standard curve to obtain the concentrations of the proteins corresponding to the plurality of bands; and then adding the concentrations of the proteins corresponding to the multiple bands, and dividing the sum by the concentration of the total protein in the sample to be detected to obtain the content of the hybrid protein.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

The reagents used in the following examples were prepared as follows:

first glue storage: dissolving 24.0g of polyacrylamide and 0.75g of methylene bisacrylamide in deionized water, and then using the deionized water to fix the volume to 50 mL.

Storing liquid of the second glue: 23.2g of polyacrylamide and 1.5g of methylene bisacrylamide are dissolved by adding deionized water, and then the volume is adjusted to 50ml by using the deionized water.

Gel buffer: dissolving 36.4g of Tris and 0.3g of SDS in deionized water, diluting to 100mL with deionized water, and adjusting the pH to 8.5 with hydrochloric acid.

A coagulant: 0.1g of ammonium persulfate was dissolved in 1mL of deionized water.

Fixing liquid: 5.0g of trichloroacetic acid, 200mL of deionized water and 200mL of methanol were mixed, and the volume was adjusted to 500mL with deionized water.

Dyeing liquid: mixing 0.5g of Coomassie brilliant blue R25, 200mL of deionized water and 200mL of methanol, and then diluting the mixture to 500mL by using the deionized water;

decoloring liquid: 400mL of deionized water, 400mL of methanol and 100mL of acetic acid were mixed and then the volume was adjusted to 1000mL with deionized water.

Loading buffer solution: mixing 12.5mL of 7% Tris-HCl solution (pH 6.8), 12.5g of glycerol, 1.0g of SDS, 10mg of bromophenol blue and 1.0g of dithiothreitol, and adding deionized water to the mixture to reach 50 mL;

8 × anode buffer: 121.14g Tris was dissolved in 400mL deionized water, adjusted to 8.9 with hydrochloric acid, and made up to 500mL with deionized water.

8 × cathode buffer: 60.55g Tris, 89.50g Tricine and 5g SDS were dissolved in 400mL deionized water and made up to 500mL with deionized water.

Standard solution: 100mg BSA was added with deionized water to prepare 1mg/mL stock solution, which was then diluted 10-fold with deionized water to prepare 100 ng/. mu.L BSA solution, which was then sequentially diluted to 30 ng/. mu.L, 25 ng/. mu.L, 20 ng/. mu.L, 15 ng/. mu.L, 10 ng/. mu.L and 5 ng/. mu.L.

Example 1

(1) Preparation of gel:

a. preparing separation gel: 0.95mL of the first gel stock, 1.13mL of ddH₂Mixing O, 1.4mL of gel buffer solution and 1.04mL of glycerol, adding 35 mu L of coagulant and 5 mu L of coagulant after vortex, immediately vortex and uniformly mixing, adding into a gel maker, covering a 1cm water layer on separation gel, and standing for 40-60 min until the gel polymerization is finished.

b. Preparing interlayer glue: after the gel polymerization was complete, the aqueous layer covering the gel was removed and 0.353mL of a second gel stock solution, 0.912mL of ddH₂Mixing O and 0.735mL of gel buffer solution, adding 30 mu L of coagulant and 4 mu L of coagulant after vortex, immediately adding the mixture into a gel maker after vortex and mixing uniformly, covering a 1cm water layer on the interlayer gel, and standing until the gel polymerization is finished.

c. Preparing concentrated glue: the aqueous layer covering the gel was removed and 0.24mL of the second gel stock, 1.2mL ddH₂O and 0.56mLMixing gel buffer solution, adding 30 mu L of coagulant and 4 mu L of coagulant after vortex, immediately vortex and uniformly mixing and adding into a gel maker, and inserting a comb into the gel to avoid generating bubbles. After the gel polymerized, the comb was pulled out.

(2) Sample preparation:

a. a sample to be detected: three samples to be tested (sample 1, sample 2 and sample 3) were diluted to a collagen content of about 4mg/mL, ensuring complete dissolution of collagen, and the concentration of collagen in each sample to be tested was determined.

b. Collagenase enzymolysis sample: using the diluted sample to be tested, adding 4mg/mL collagenase, wherein the volume ratio of the sample to be tested to the collagenase is 1: 1; a2 mg/mL collagenase control was also prepared and placed on a shaker at 37 ℃ until the collagen sample was completely digested.

c. Mixing the prepared sample to be detected, the collagenase enzymolysis sample and a plurality of sample standard solutions with different concentrations with a sample loading buffer solution in a ratio of 1:1 respectively, and then heating the mixture in boiling water (100 ℃) for 2 min.

(3) Electrophoresis: and (3) putting the electrophoresis tank into an ice water bath, adding 700mL of anode buffer solution into an outer tank, adding 550mL of cathode buffer solution into an inner tank, performing 30V pre-electrophoresis for 15min, adding 10 mu L of protein marker, the sample to be detected after being heated by boiling water in the step (2), the collagenase enzymolysis sample and a plurality of sample standard solution standard solutions with different concentrations into a sample application hole, performing 30V electrophoresis for 1 hour, and stopping electrophoresis after 100V electrophoresis is performed until bromophenol blue reaches the bottom of the gel.

(4) Dyeing and developing: and taking out the film after the electrophoresis, putting the film into a surface dish, washing the film with pure water, and flattening the film. Adding fixing liquid to immerse the film, and shaking in shaking table at low speed for 50 min. Then the liquid is changed into a dyeing liquid, the dyeing is carried out for 1 hour, and finally the decoloring liquid is used for decoloring until the background of the film is nearly colorless, and the strip is clearly visible.

(5) Imaging and analyzing: opening a gel imaging analyzer and related software thereof, adjusting the focal length and the brightness, then photographing, determining the direction of a film and the position of a lane, adjusting the range of the band, determining the band on each lane, calibrating the sample amount of a standard sample after calibrating the band and the related amount of each lane, performing curve fitting on the standard sample, wherein the meshing error is 3, obtaining the accurate content value of each impure protein band, and calculating the content of the impure protein. The results are shown in table 1 and fig. 1. In table 1, the mass of the sample loaded is the loading volume x the concentration of the sample loaded.

TABLE 1

Sample to be tested	Sample 1	Sample 2	Sample 3
				Heteroprotein Mass/ng at 75KD	93.934	74.486	92.451
Heteroprotein Mass/ng at 30KD	30.307	43.162	44.963
				Heteroprotein Mass/ng at 15KD	Not detected out	Not detected out	129.42
Total amount of hetero protein/ng	124.241	117.648	266.834
				Sample loading/ng	43000	38000	44000
Content of hetero protein/%)	0.29	0.31	0.61

In fig. 1,

lanes

1 and 15 are protein markers, lanes 2 to 7 are lanes corresponding to 50ng, 100ng, 150ng, 200ng, 250ng and 300ng of standard (BSA) solutions, lanes 8 to 10 are different samples to be detected (sample 1 to sample 3), lanes 11 to 13 are three different samples to be detected in lanes 8 to 10, and the three samples to be detected are samples to be detected after adding collagenase for enzymolysis, and lane 14 is a collagenase control.

As can be seen from lanes 11-14 of FIG. 1, there are no bands in the samples after the collagenase and the three different samples + collagenase have been digested. In this case, the conventional method for detecting a hybrid protein (standard YY/T1453-2016) requires the determination of the staining limit by SDS-PAGE to determine the content of the hybrid protein, which is a cumbersome operation.

(6) The sample 3 is detected according to the method for characterizing the type I collagen of the standard YY/T1453-2016 tissue engineering medical instrument product, and the result is shown in FIG. 2. In FIG. 2, lanes 1 to 5 are lanes corresponding to 50ng, 100ng, 150ng, 200ng, 250ng and 300ng of BSA solution in this order. Lanes 6-7 are in turn collagenase, collagenase + sample 3 and the lane corresponding to sample 3.

As shown in fig. 2, the samples after the collagenase, the measurement sample and the collagenase were digested showed no bands after the electrophoresis, and when the staining limit of coomassie brilliant blue on BSA was 50ng, the purity (%) of sample 3 was (10000-50)/10000 × 100%: 99.5%.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for detecting the content of hybrid protein in collagen is characterized by comprising the following steps:

2. The method for detecting the content of hetero proteins in collagen according to claim 1, wherein the standard substance is at least one selected from the group consisting of bovine serum albumin, human serum albumin and ovalbumin.

3. The method for detecting the content of hetero proteins in collagen according to claim 1, further comprising a step of heating the sample to be tested to uncoil the collagen in the sample to be tested, before the step of subjecting the plurality of standard solutions with different concentrations and the sample to be tested to Tricine-SDS-PAGE electrophoresis, respectively.

4. The method for detecting the content of the impurity proteins in the collagen according to claim 3, wherein the heating temperature is 90 ℃ to 105 ℃ and the heating time is 2min to 15 min.

5. The method for detecting the content of the hybrid protein in the collagen according to claim 1, wherein the concentration of the standard substance in the standard substance solution is 50ng/μ L to 300ng/μ L.

6. The method for detecting the content of the hybrid proteins in the collagen according to claim 1, wherein the step of fitting a standard curve comprises: calculating the mass of each sample according to the concentration of each standard solution and the corresponding sample loading volume of Tricine-SDS-PAGE electrophoresis, wherein the sample loading volume of each standard solution is equal during Tricine-SDS-PAGE electrophoresis; fitting a standard curve according to the quality of each loaded standard substance and the corresponding strip gray value;

and/or the step of calculating the content of the impure protein according to the standard curve and the strip gray value of the sample to be detected comprises the following steps: substituting the strip gray value of the sample to be detected into the standard curve to obtain the quality of the impure protein in the sample to be detected; and calculating the content of the hybrid protein according to the mass of the hybrid protein in the sample to be detected and the mass of the total protein in the sample to be detected.

7. The method for detecting the content of the hybrid proteins in the collagen according to claim 6, wherein the sample to be detected has a plurality of bands after Tricine-SDS-PAGE electrophoresis, and the step of calculating the content of the hybrid proteins comprises:

8. The method for detecting the content of the impurity proteins in the collagen according to any one of claims 1 to 7, wherein the supporting medium of Tricine-SDS-PAGE electrophoresis is composed of a concentrated gel, a separation gel and an interlayer gel between the concentrated gel and the separation gel, wherein the preparation of the separation gel comprises the following steps:

9. The method for detecting the content of the hybrid protein in the collagen according to claim 8, wherein the preparation step of the sandwich gel comprises the following steps:

10. The method for detecting the content of hetero-proteins in collagen according to claim 8, wherein the cathode buffer solution in Tricine-SDS-PAGE electrophoresis comprises Tris, Tris (hydroxymethyl) methylglycine and SDS, wherein the cathode buffer solution comprises per 500mL of 8X cathode buffer solution: 44.84g to 60.55g of Tris, 65.6g to 89.50g of Tris (hydroxymethyl) methylglycine and 4g to 6g of SDS.