1. An ultrafiltration-high performance liquid chromatography method for detecting the antigen peptide in the pHLA compound is characterized by comprising the following steps:

(1) carrying out ultrafiltration treatment on the pHLA compound to remove free antigen peptide;

(2) treating the pHLA complex with an acidic solution;

(3) ultrafiltering the solution obtained in the step (2), and collecting an antigen peptide solution;

(4) the content of the antigen peptide in the antigen peptide solution was measured by HPLC.

2. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 1,

also comprises a pretreatment step: the HLA-I molecules and the antigen peptide containing anchoring residue sites are subjected to renaturation to generate interaction, and a pHLA complex is formed.

3. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 2,

the HLA-I molecule consists of recombinant HC protein and recombinant beta 2m protein, or the HLA-I molecule is recombinant beta 2m/HC fusion protein;

the co-renaturation of the HLA-I molecules and antigen peptides containing anchoring residue sites is carried out in renaturation buffer solution;

the renaturation buffer solution contains 100-500 mmol/L-Arginine, 10-500mmol/L Tris HCl, 0.1-10mmol/L EDTA, 0.1-5mmol/L oxidized glutathione and 1-10mmol/L reduced glutathione, and the pH value is 7-9;

the molar concentration of HLA-I molecules in the renaturation buffer solution is 0.5-10 mu mol/L;

the molar ratio of the recombinant HC protein to the recombinant beta 2m protein to the antigen peptide containing anchoring residue sites is 1: (1-10): (5-100); or recombinant beta 2m/HC fusion protein, the molar ratio of the antigen peptide containing anchoring residue sites is 1: (5-100);

the co-renaturation temperature is 10-14 ℃, and the renaturation time is 12-48 h.

4. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 1,

the ultrafiltration cut-off molecular weight in the steps (1) and (3) is 3kD-10 kD.

5. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 1,

in the step (2), the acid solution is trifluoroacetic acid aqueous solution with volume fraction of 2-10%, and the standing time is 10-30 min.

6. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 1,

in step (4), HPLC is performed using a C18 column, and the mobile phase is acetonitrile containing 0.005-0.1% v/v trifluoroacetic acid: water containing 0.005-0.1% v/v trifluoroacetic acid 15:85-40:60 at a flow rate of 0.5-1.2mL/min, a column temperature of 35 ± 3 ℃, and a detection wavelength of 214 nm.

7. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 1,

before the content of the antigen peptide in the antigen peptide solution is measured by HPLC in the step (4), the antigen peptide standard product is detected, a characteristic peak is determined, and a standard curve is drawn.

8. The ultrafiltration-high performance liquid chromatography for detecting an antigenic peptide in a pHLA complex of claim 1,

the antigen peptide determined in step (4) is an antigen peptide with equimolar combination of HLA-I molecules in the pHLA compound, and the amount of the pHLA compound can be calculated according to the amount of the antigen peptide.

Background

HLA class I molecules (human lymphocyte antigen class I molecules) are heterodimers composed of highly polymorphic heavy chain transmembrane glycoprotein (HC) and β 2 microglobulin (β 2 m). After endogenous antigens are treated by Antigen Presenting Cells (APCs), antigen peptides (also called antigen CTL epitope peptides) combined with HLA-I molecules are generated, and are specifically recognized by CD8+ T cell antigen receptor (TCR) through the antigen presenting function of the APCs, so that Cytotoxic T Lymphocytes (CTL) are induced to clone to generate immune response. The alpha 1 and alpha 2 structural domains of HLA-I molecule HC form an antigen peptide binding groove, and the bottom of the antigen peptide binding groove is provided with 6 small pocket-shaped depressions (A → F pocket), wherein conservative amino acids on the A pocket and the F pocket form hydrogen bonds with the N-terminal amino group and the C-terminal amino group of the polypeptide, and play a role in fixing the bound antigen peptide. HLA class I molecules of the same allelic type can bind to various antigen peptides by recognizing specific anchor amino acid residues (anchor amino acid residues) on the polypeptide sequence. The research of screening different peptides to interact with HLA-I molecules to form HLA-I molecules/antigen peptide complexes (pHLA complexes) is helpful to find new antigen CTL epitope peptides, and has important significance for the elucidation of CD8+ T cell specific immune recognition mechanisms, the development of polypeptide vaccines and the development of specific cellular immunotherapy. And the HLA tetramer technology developed on the basis of pHLA also provides a more direct and effective method for specific detection and research of CTL.

The currently common method for obtaining the pHLA complex is to respectively express HC and beta 2m in a recombinant mode and obtain the pHLA complex after combining antigen peptides in a renaturation buffer solution. However, the artificial renaturation process of the pHLA complex is influenced by renaturation buffer system comprising HC, beta 2m, the mole ratio of antigen peptide, pH value and temperature, and electrolyte conditions, the renaturation effect is not easy to control, the time consumption is long, and HC dimer and extremely unstable unloaded HLA molecule are easily formed. Therefore, during the preparation process, the prepared product needs to be tested to optimize renaturation conditions, promote the correct folding of HC and beta 2m and interact with antigen peptide to form pHLA complex. In addition, due to the high polymorphism of HLA, HLA class I molecules of different types can be combined with various antigen peptides by recognizing polypeptide sequences, and theoretically, countless pHLA complexes can be formed. Screening antigen peptide interacting with HLA-I type molecule, researching biological function of pHLA compound, or exploring preparation condition of pHLA compound, and adopting small amount of renaturation buffer solution preparation system, it is not only low in cost, but also easy to operate.

At present, Western blot, ELISA, chromatographic separation and other methods are commonly used to verify the success of pHLA compound preparation. Western blot, ELISA and other methods using HLA-I class molecule conformation specific antibody W6/32 are the most common methods for verifying the success of small-amount preparation of pHLA complexes, wherein W6/32 is a monoclonal conformation antibody against HLA-I class molecules, can recognize specific epitopes of HC and beta 2m, can not be combined with the independent beta 2m, and can only be weakly combined with the independent HC, and the specific epitopes are combined with antigen peptides to form HC-alpha 2 regions after folding. However, Western blot and ELISA based on the conformation specific antibody W6/32 can only qualitatively detect whether the pHLA complex is formed, but cannot quantitatively detect the antigen peptide bound in the pHLA complex. Furthermore, studies have shown that pHLA complexes obtained by dilution renaturation in vitro may have different conformations but the same biological activity. This conformational difference may not be specifically recognized by the conformation-specific antibody W6/32, resulting in uncertainty in the identification result. Therefore, other methods need to be found for detecting and identifying a small amount of products prepared from the pHLA complex in the processes of quality detection and preparation condition exploration of the pHLA complex, and a method capable of quantitatively detecting the antigen peptide in the recombinant pHLA complex is urgently needed to be established.

Disclosure of Invention

The invention discloses an ultrafiltration-high performance liquid chromatography for detecting antigen peptide in a pHLA compound, which can be used for quantitatively detecting the antigen peptide combined in the pHLA compound.

In order to achieve the purpose, the invention adopts the following technical scheme:

an ultrafiltration-high performance liquid chromatography method for detecting the antigen peptide in the pHLA compound comprises the following steps:

(1) carrying out ultrafiltration treatment on the pHLA compound to remove free antigen peptide;

(2) treating the pHLA complex with an acidic solution;

(3) ultrafiltering the solution obtained in the step (2), and collecting an antigen peptide solution;

(4) the content of the antigen peptide in the antigen peptide solution was measured by HPLC.

Preferably, the method further comprises the following pretreatment steps: the HLA-I molecules and the antigen peptide containing anchoring residue sites are subjected to renaturation to generate interaction, and a pHLA complex is formed.

Preferably, the HLA-class I molecule consists of recombinant HC protein and recombinant β 2m protein, or the HLA-class I molecule is recombinant β 2m/HC fusion protein;

the co-renaturation of HLA-I molecules and antigen peptides containing anchoring residue sites is carried out in renaturation buffer solution;

the renaturation buffer solution contains 100-500 mmol/L-Arginine, 10-500mmol/L Tris HCl, 0.1-10mmol/L EDTA, 0.1-5mmol/L oxidized glutathione and 1-10mmol/L reduced glutathione, and the pH value is 7-9;

the molar concentration of HLA-I molecules in the renaturation buffer solution is 0.5-10 mu mol/L;

the molar ratio of the recombinant HC protein to the recombinant beta 2m protein to the antigen peptide containing anchoring residue sites is 1: (1-10): (5-100); or recombinant beta 2m/HC fusion protein, the molar ratio of the antigen peptide containing anchoring residue sites is 1: (5-100);

the co-renaturation temperature is 10-14 ℃, and the renaturation time is 12-48 h.

The recombinant HLA-class I molecule/antigen peptide complex (pHLA complex) has important application in the research of human T cell specific immune response. The preparation of pHLA complex is based on gene engineering and protein in vitro dilution folding renaturation technology, and its key lies in that the recombinant HLA-I class molecule is correctly folded in renaturation system, and combined with antigen peptide to form complex. The method comprises the steps of carrying out sample loading pretreatment on recombinant HLA-I molecules and antigen peptides, carrying out refolding on heavy chain HC and light chain beta 2m of the recombinant HLA-I molecules in renaturation buffer solution, and carrying out interaction with the antigen peptides containing anchoring residues to form a pHLA compound; removing unbound free antigen peptide by ultrafiltration to retain the complex, treating the pHLA complex with acid to destroy the interaction to release antigen peptide, ultrafiltering to collect antigen peptide, and performing HPLC detection to obtain antigen peptide amount, which is the amount of antigen peptide bound by the interaction between the recombinant HLA-I molecules and the antigen peptide. The prepared recombinant pHLA compound can be identified by an HLA-I molecule conformation specific antibody W6/32, which shows that the folding conformation of the recombinant HLA-I molecule is correct and the existence of the pHLA compound can be identified; meanwhile, the pHLA complex is detected to contain the antigen peptide by the ultrafiltration-HPLC method, so the method for detecting the pHLA complex formed by combining the antigen peptide with HLA-I molecules by the ultrafiltration-HPLC method is feasible.

Preferably, the ultrafiltration cut-off in steps (1) and (3) is from 3kD to 10 kD.

Further preferably, the ultrafiltration tubes with a molecular weight cut-off of 3kD to 10kD are passivated overnight with a 1% by mass BSA solution before ultrafiltration.

Preferably, in the step (2), the acidic solution is 2-10% by volume of trifluoroacetic acid aqueous solution, and the standing time is 10-30 min.

Preferably, in step (4), the HPLC uses a C18 column and the mobile phase is acetonitrile containing 0.005-0.1% (v/v) trifluoroacetic acid: water containing 0.005-0.1% (v/v) trifluoroacetic acid at 15:85-40:60 flow rate of 0.5-1.2mL/min, column temperature of 35 ± 3 ℃, detection wavelength of 214 nm.

Preferably, before the content of the antigen peptide in the antigen peptide solution is measured by HPLC in step (4), the antigen peptide standard is detected, characteristic peaks are determined, and a standard curve is drawn (peak area is ordinate, antigen peptide concentration is abscissa).

Preferably, the method further comprises precision verification: multiple measurements were performed on the same sample, and the retention time and peak area of the peaks were recorded and the relative standard deviation RSD was calculated.

Preferably, the method also comprises the following verification of ultrafiltration recovery rate: two parts of antigen peptide standard solutions with the concentration of 1-10 mug/mL are respectively prepared, wherein one part of the solution is directly subjected to HPLC sample injection detection, and the other part of the solution is subjected to HPLC sample injection detection after ultrafiltration and centrifugation for antigen peptide recovery rate investigation.

Preferably, the antigenic peptide determined in step (4) is an antigenic peptide to which HLA-class I molecules in the pHLA complex are bound in equimolar amounts, and the amount of the pHLA complex can be calculated from the amount of the antigenic peptide.

In conclusion, the method can realize the quantitative detection of the antigen peptide combined in the pHLA compound, is suitable for the detection and identification of a small amount of products prepared from the pHLA compound in the process of exploring the preparation conditions, further conveniently optimizes different combination conditions according to the amount of the antigen peptide combined in the compound so as to improve the folding efficiency of HLA-I molecules and promote the HLA-I molecules to combine the antigen peptide, and can also calculate the preparation rate of the pHLA compound formed in a renaturation system according to the content of the antigen peptide combined by the pHLA compound. The ultrafiltration-HPLC method established by the invention can be used for quality control in the preparation process of the pHLA compound, and has advantages in the aspects of T cell specific immunity research, artificial APC and specific tetramer probe application development.

Drawings

FIG. 1 shows a chromatogram of a peptide standard;

wherein, the A is VYF antigen peptide standard product, and the B is IRA reference peptide standard product.

FIG. 2 shows a standard curve for a peptide standard;

wherein, the A is VYF antigen peptide standard product, and the B is IRA reference peptide standard product.

FIG. 3 shows the results of the co-renaturation verification;

wherein, the (A) is non-denaturing polyacrylamide gel electrophoresis, and the (B) is Western blot.

FIG. 4 shows the result of ultrafiltration-HPLC detection of the renaturation product.

FIG. 5 is a graph showing the effect of different pH and different peptide concentrations on the amount of pHLA complex bound;

wherein (A) is the influence of pH and (B) is the influence of peptide concentration.

FIG. 6 shows a comparison of the binding capacity of peptides of different pHLA complexes.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Preparation of a peptide standard product:

human melanoma-associated antigen peptide gp100-intron 4 (with the amino acid sequence of NH2-VYFFLPDHL-COOH, VYF antigen peptide for short, containing corresponding anchor amino acid residues as a binding peptide) and NH2-IRAWVAWRNR-COOH (IRA control peptide for short, containing no corresponding anchor amino acid residues as a binding peptide) restricted by HLA-A2402 were selected, dissolved in renaturation buffer (with the components of 400 mmol/L-arginine, 100 mmol/L-Tris HCl, 2 mmol/L-DTA, 0.5mmol/L oxidized glutathione, 5mmol/L reduced glutathione, and pH of 8.0) to prepare a peptide standard solution.

Chromatographic conditions are as follows: using an LC-10AT high performance liquid chromatography pump (Shimadzu, Japan), SPD-20A high performance liquid chromatography detector (Shimadzu, Japan) RP-HPLC, a C18 column (4.6 mm. times.250 mm, 5 μm) was used. The mobile phase was acetonitrile containing 0.1% (v/v) trifluoroacetic acid: water containing 0.1% (v/v) trifluoroacetic acid ═ 31: 69(v/v), a flow rate of 1mL/min, a column temperature of 35 ℃, a detection wavelength of 214nm, and a loading volume of 20. mu.L.

Under the chromatographic conditions, the chromatogram of the VYF antigen peptide standard solution and the IRA reference peptide standard solution is shown in figure 1. VYF the standard solution of antigen peptide has an absorption peak at 14-15 min, as shown in FIG. 1(A), therefore the absorption peak at 14-15 min is selected to be the characteristic peak of VYF antigen peptide; the IRA control peptide has an absorption peak at 8-9 min, so that the absorption peak at 8-9 min is selected as a characteristic peak of the IRA control peptide, as shown in FIG. 1 (B).

VYF antigen peptide standard solution and IRA reference peptide standard solution with the concentrations of 1, 2, 3, 4, 5, 6, 7, 8 and 9 mu g/ml are precisely prepared and sequentially measured. Standard curves were plotted with peak area as ordinate and peptide concentration (. mu.g/ml) as abscissa. As shown in FIG. 2, the standard curves for the VYF antigen peptide and the IRA control peptide were well linear over the range of 0-9. mu.g/ml. The regression equations are respectively: 13.031x, R²＝0.9963；y＝9.4913x，R²＝0.9921。

A2. mu.g/ml peptide standard solution was prepared, and the detection was performed a plurality of times under the above-mentioned chromatographic conditions, and the retention time and peak area of the peak were recorded, and the Relative Standard Deviation (RSD) was calculated to verify the precision of the method. The results are shown in table 1, and the retention time and peak area RSD of VYF antigen peptide and IRA control peptide are less than 2%, which proves that the method has good precision.

TABLE 1

VYF antigen peptide and IRA control peptide are respectively prepared into standard solutions with the concentration of 1, 5 and 9 mu g/ml, 20 mu L of standard solution is taken for sample injection detection in one group of experiments, 20 mu L of standard solution is taken for sample injection detection after the standard solution is ultrafiltered by using an ultrafiltration tube with the cut-off molecular weight of 3kD (BSA solution with the mass fraction of 1 percent is used for overnight passivation in advance) in the other group of experiments, and the recovery rate of the ultrafiltered peptide is calculated according to the detection result. The detection result is shown in a table 2, the ultrafiltration recovery rate of VYF antigen peptide is in the range of 96.91% -98.14%, and the ultrafiltration recovery rate of IRA control peptide is in the range of 95.10% -98.48%. The experimental result shows that the ultrafiltration method has better recovery rate and can be used for determining the interaction between VYF antigen peptide and IRA control peptide and HLA molecules.

TABLE 2

Example 2

preparation of pHLA complexes: selecting HLA-a 2402 heavy chain protein (HC), β 2 m; VYF antigen peptide and IRA control peptide as raw materials.

The HC + beta 2m + VYF antigen peptide is diluted and renatured according to the molar ratio of about 1:2:11 to prepare pHLA complex: adding 10 mu g of VYF antigen peptide (8.69nmol) into each milliliter of precooled renaturation buffer solution, uniformly mixing the mixture at a high speed of 10 ℃ for 30min, slowly dropwise adding 25 mu g of beta 2m (1.45nmol), uniformly mixing the mixture at a high speed of 10 ℃ for 1h, finally adding 30 mu g of HC (0.77nmol) in total amount for 3 times, and carrying out vibration renaturation at 10 ℃ until the renaturation is finished at intervals of 12 h.

Meanwhile, HC + VYF antigen peptide and beta 2m + VYF antigen peptide are set as control groups.

The same renaturation procedure was also performed using the IRA control peptide as the non-binding control peptide.

An overnight passivation of the 3kDa molecular weight cut-off ultrafiltration tube was performed using a 1% BSA solution. 2ml of renaturation products of each group are respectively subjected to primary ultrafiltration, then renaturation buffer solution is used for supplementing to 500 mu L, the ultrafiltration is carried out again after the gentle blowing and even mixing, and the ultrafiltration is carried out for three times to remove free peptide. HC and beta 2m are taken to respectively carry out non-denaturing polyacrylamide gel electrophoresis with VYF antigen peptide and IRA contrast peptide renaturation folding products, and Western blot verification is carried out by using a W6/32 antibody. The results are shown in fig. 3, only HC + β 2m + VYF antigen renaturation can generate correct conformation of HLA-a 2402/VYF complex, which indicates that HC and β 2m can be combined with VYF antigen peptide through renaturation folding to form pHLA complex with correct conformation.

Adding 3% trifluoroacetic acid solution into the prepared product of the pHLA compound obtained after ultrafiltration to make up to 100 μ L, standing for 20min, and performing ultrafiltration again to collect filtrate. And (5) carrying out sample injection detection according to the liquid phase condition. As shown in fig. 4, the concentration of VYF antigen peptide bound in pHLA complexes prepared by HC + β 2m + VYF antigen co-renaturation was significantly higher than in the remaining groups (P < 0.01).

Example 3

preparation of pHLA complexes: selecting recombinant HLA-A2402 heavy chain protein (HC) and recombinant beta 2 m; recombinant β 2m/HLA-a 2402 fusion protein (β 2m/HC), VYF antigen peptide.

The pHLA complexes were prepared by further optimizing the conditions by changing the renaturation conditions in example 2, including the pH of the renaturation buffer and the concentration of the antigen peptide in the renaturation system (the pH of the renaturation buffer was adjusted to 8.0, 7.5 and 7.0, respectively, and the concentration of the antigen peptide in the renaturation buffer was adjusted to 5, 10, 20 and 40. mu.g/mL, respectively).

Respectively diluting and renaturing HC + beta 2m + VYF antigen peptide and beta 2m/HC + VYF antigen peptide to prepare pHLA compound (wherein the dilution renaturation of the beta 2m/HC + VYF antigen peptide is based on example 2, after shaking and mixing VYF antigen peptide in renaturation buffer solution, 30 mu g (0.575nmol) of beta 2m/HC is added in 3 times, each time is 12h, and the shaking renaturation is carried out at 10 ℃ till the end of renaturation), and the binding capacity of the antigen peptide in the pHLA compound is detected by the method. Comparing the influence of different pH values and different peptide concentrations on the peptide binding capacity of the pHLA complex according to the detection result of the ultrafiltration-HPLC method. As shown in FIG. 5, the optimized HC + β 2m + VYF antigen peptide renaturation condition shows that the pH of the renaturation buffer solution is 8.0, the antigen peptide concentration is 20 μ g/mL, and the pHLA compound prepared under the condition has the highest peptide content. The optimized result of the beta 2m/HC + VYF antigen peptide renaturation condition is that the pH of the renaturation buffer solution is 7.0, the antigen peptide concentration is 20 mu g/mL, and the peptide content in the pHLA compound prepared under the condition is the highest.

The concentration of the antigen peptide was calculated using VYF standard curve of antigen peptide, and the preparation rate of the pHLA complex was calculated from the preparation rate of the number of moles of antigen peptide bound to HLA molecule/number of moles of total HLA heavy chain protein in the renaturation system × 100%. According to the optimized renaturation conditions, the maximum antigen peptide binding amount of the pHLA compound prepared by HC + beta 2m + VYF antigen peptides is 0.259 mu g, the corresponding mole number is 0.225nmol, namely the pHLA compound is 0.225nmol, and the preparation rate is about 14.6%. The maximum antigen peptide binding amount of the pHLA compound prepared by beta 2m/HC + VYF antigen peptide is 0.490 mug, the corresponding mole number is 0.426nmol, namely, folding renaturation is carried out to obtain 0.426nmol antigen peptide-HLA compound, and the preparation rate is about 36.9%. The results showed that the pHLA complexes prepared with the β 2m/HC + VYF antigen peptide had a significantly higher preparation rate (P < 0.01) than the pHLA complexes prepared with the HC + β 2m + VYF antigen peptide, as shown in fig. 6.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.