AU2021103860A4 - ULTRAFILTRATION-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETECTING ANTIGEN PEPTIDES IN pHLA COMPLEX - Google Patents
ULTRAFILTRATION-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETECTING ANTIGEN PEPTIDES IN pHLA COMPLEX Download PDFInfo
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- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 27
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 27
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- 238000004153 renaturation Methods 0.000 claims description 53
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
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- 235000014852 L-arginine Nutrition 0.000 claims description 3
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- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 claims description 3
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 claims description 3
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- 102100033400 4F2 cell-surface antigen heavy chain Human genes 0.000 description 1
- 102100031547 HLA class II histocompatibility antigen, DO alpha chain Human genes 0.000 description 1
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- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6878—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids in eptitope analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70539—MHC-molecules, e.g. HLA-molecules
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Abstract
An ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in a pHLA
complex, comprising the following steps: (1) performing ultrafiltration on the pHLA complex; and removing
free antigen peptides; (2) treating the pHLA complex with an acid solution; (3) performing ultrafiltration on
the solution obtained in the step (2); and collecting an antigen peptide solution; (4) determining antigen
peptide content in the antigen peptide solution by HPLC.
Drawings of Description
-pHLA
(A) (B)
FIG. 3
** **
**
30
25
X 20
15- M UIRAE VYF
10
HC+P HC+p2m+P p2m+P
FIG. 4
3
Description
Drawings of Description
-pHLA
FIG. 3
** 30
25
X 20 15- M UIRAE VYF
10
HC+P HC+p2m+P p2m+P
FIG. 4
Description
ULTRAFILTRATION-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR DETECTING ANTIGEN PEPTIDES IN pHLA COMPLEX
Technical Field
The present invention belongs to the technical field of protein and peptide interaction researches, and particularly relates to an ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in a pHLA complex.
Background
HLA-I molecules (human lymphocyte antigen class I molecules) are heterodimers composed of high-polymorphism heavy-chain transmembrane glycoproteins (HC) and 2 microglobulins (f2m). After an endogenous antigen is treated with antigen presenting cells (APCs), an antigen peptide (also called an antigen CTL epitope peptide) bound with the HLA-I molecules is produced. By virtue of an antigen presenting effect of the APCs, the antigen peptide is specifically recognized by a CD8+ T cell receptor (TCR), and then induced to form cytotoxic T lymphocyte (CTL) clones to produce immune response. Structural domains al and a2 of the HLA-I molecules, that is, HC, form an antigen peptide-binding cleft; and there are 6 pocket depressions (A-F pocket) at the bottom of the antigen peptide-binding cleft, wherein conservative amino acids on the A pocket and the F pocket form hydrogen bonds with N-terminal amino and C-terminal amino of the peptide, thereby achieving an effect of fixing the bound antigen peptide. The HLA-I molecules of the same allelotype may be bound with multiple antigen peptides by recognizing specific anchor amino acid residues on a peptide sequence. A research of screening interactions of different peptides and HLA-I molecules to form an HLA-I molecule/antigen peptide complex (pHLA
Description
complex) contributes to discovering a new antigen CTL epitope peptide, and has significances for illustrating a CD8+ T cell-specific immune recognition mechanism, developing a peptide vaccine and developing specific cell immunotherapy. Moreover, an HLA tetramer technology developed on the basis of the pHLA also provides a more direct and effective method for specific detection and study of the CTL. At present, a common method of obtaining a pHLA complex is as follows: the HC andf2m are respectively subjected to recombinant expression; and an antigen peptide is bound in a renaturation buffer, thereby obtaining the pHLA complex. However, an artificial renaturation process of the pHLA complex is affected by a molar ratio of HC,f2m and the antigen peptide included in the renaturation buffer system, a pH value, a temperature and electrolyte conditions; a renaturation effect is difficult to be controlled; time consumption is high; and HC dimmers and extremely unstable no-load HLA molecules are easily formed. Therefore, in the preparation process, a preparation product needs to be detected so as to optimize renaturation conditions, thereby facilitating correct folding of the HC and 2m and interacting with the antigen to form the pHLA complex. In addition, due to the high polymorphism of the HLA, the HLA-I molecules of different types may be bound with multiple antigen peptides by recognizing peptide sequence motifs; and countless pHLA complexes may be formed theoretically. In screening the antigen peptides interacting with the HLA-I molecules, researching biological functions of the pHLA complex or exploring preparation conditions of the pHLA complex, a preparation system of a small amount of renaturation buffer is often used; and the preparation system is low in cost and easy to operate. At present, common methods for verifying whether preparation of the pHLA complex is successful or not include Western blot, ELISA, chromatographic separation and other methods. Methods such as Western blot and ELISA by using an HLA-I molecular conformation specific antibody W6/32 are the most common
Description
methods for verifying whether preparation of a small amount of the pHLA complex is successful or not. The W6/32 is a monoclonal conformation antibody resistant to HLA-I molecules, can be bound with the antigen peptide to form a specific epitope in an HC-a2 after recognizing folding of the HC and 2m, cannot be bound with separate p2m, and is extremely weakly bound with separate HC only. However, the methods such as Western blot and ELISA by using the conformation-based 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. Moreover, researches show that, pHLA complexes obtained by in-vitro dilution refolding may have different conformations, but have the same biological activity. The conformation differences may not be able to be specifically recognized by the conformation-based specific antibody W6/32, so that the identification result has uncertainty. Therefore, during mass detection of the pHLA complex and exploration of the preparation conditions, other methods need to be searched for detecting and identifying the preparation products of the small amount of pHLA complex; and a method that can quantitatively detect the antigen peptide in the recombinant pHLA complex urgently needs to be established.
Summary
The present invention discloses an ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in a pHLA complex. The method can be used for quantitatively detecting the antigen peptide bound in the pHLA complex. To achieve the above purpose, technical solutions of the present invention are as follows: The ultrafiltration-high performance liquid chromatography method for detecting the antigen peptides in the pHLA complex includes the following steps:
Description
(1) performing ultrafiltration on the pHLA complex; and removing free antigen peptides; (2) treating the pHLA complex with an acid solution; (3) performing ultrafiltration on the solution obtained in the step (2); and collecting an antigen peptide solution; (4) determining antigen peptide content in the antigen peptide solution by HPLC. Preferably, the method further includes a pretreatment step: performing co-renaturation on the HLA-I molecules and the antigen peptide containing anchor amino acid residues to produce interaction to form the pHLA complex. Preferably, the HLA-I molecules are composed of recombinant HC proteins and recombinant p2m proteins, or the HLA-I molecules are recombinant 2m/HC fusion proteins; co-renaturation of the HLA-I molecules and the antigen peptide containing anchor amino acid residues is conducted in the renaturation buffer; the renaturation buffer includes 100-500 mmol/L of L-Arginine, 10-500 mmol/L of Tris HCl, 0.1-10 mmol/L of EDTA, 0.1-5 mmol/L of oxidized glutathione and 1-1Ommol/L of reduced glutathione and has a pH value of 7-9; a molar concentration of the HLA-I molecules in the renaturation buffer is 0.5-10 pmol/L; a molar ratio of the recombinant HC proteins to the recombinant p2m proteins to the antigen peptide containing anchor amino acid residues is 1:(1-10):(5-100); or a molar ratio of the recombinant P2m/HC fusion proteins to the antigen peptide containing anchor amino acid residues is 1:(5-100); a co-renaturation temperature is 10-14°C; and renaturation time is 12-48 h. The recombinant HLA-I molecule/antigen peptide complex (pHLA complex) has important use of researching human T cell specific immune response. Based on genetic engineering and protein in-vitro dilution folding renaturation, the key of
Description
preparation of the pHLA complex is to correctly fold the recombinant HLA-I molecules in the renaturation system and bind with the antigen peptide to form the complex. In the present invention, by treating the recombinant HLA-I molecules and the antigen peptide before loading, heavy chain HC and light chain p2m in the recombinant HLA-I molecules are subjected to renaturation folding in the renaturation buffer and then interact with the antigen peptide containing the anchor residues to form the pHLA complex. The unbound free antigen peptides are removed by ultrafiltration, and the complex is remained. Finally, the interaction of the pHLA complex is destructed by virtue of acid treatment so as to release the antigen peptides; and then the antigen peptides are collected by ultrafiltration and subjected to HPLC detection. The determined amount of the antigen peptides is an amount of antigen peptides bound by virtue of interactions of the recombinant HLA-I molecules and the antigen peptides. The prepared recombinant pHLA complex may be recognized by the HLA-I molecule conformation specific antibody W6/32, which indicates that the HLA-I molecules have correct folding conformation; and the pHLA complex may be identified to exist. Meanwhile, the ultrafiltration-HPLC method detects that the pHLA complex contains the antigen peptides. Therefore, the ultrafiltration-HPLC method is feasible for detecting the pHLA complex formed by binding the antigen peptides and the HLA-I molecules. Preferably, the molecular weight cut-off during ultrafiltration in the steps (1) and (3) is 3-1OkD. Further preferably, an ultrafiltration tube having the molecular weight cut-off of 3-lOkD is passivated overnight with a BSA solution having a mass fraction of 1% before ultrafiltration.
Preferably, in the step (2), the acid solution is an aqueous solution of trifluoroacetic acid having a volume fraction of 2-10%; and standing time is 10-30 min.
Description
Preferably, in the step (4), a C18 chromatographic column is adopted during HPLC; a mobile phase includes acetonitrile containing 0.005-0.1% (v/v) of trifluoroacetic acid and water containing 0.005-0.1% (v/v) of trifluoroacetic acid according to a ratio of 15:85-40:60; a flow rate is 0.5-1.2 mL/min; a column temperature is 35±3°C; and a detection wavelength is 214 nm. Preferably, in the step (4), before the content of the antigen peptides in the antigen peptide solution is determined by HPLC, an antigen peptide standard substance is detected; a characteristic peak is determined; and a standard curve is drawn (a peak area is a vertical coordinate, and an antigen peptide concentration is a horizontal coordinate). Preferably, the method further includes precision verification as follows: the same sample is detected for many times; peak retention time and the peak area are recorded; and a relative standard deviation RSD is calculated. Preferably, the method further includes verification of an ultrafiltration recovery rate as follows: two parts of antigen peptide standard solutions having a concentration of 1-10 pg/mL are respectively prepared; one part of the solution is directly subjected to HPLC injection detection; the other part of the solution is subjected to ultrafiltration centrifugation and HPLC injection detection in sequence; and the recovery rate of the antigen peptide is investigated. Preferably, the antigen peptides determined in the step (4) are antigen peptides bound with the HLA-I molecules in the pHLA complex in an equimolar manner; and then the amount of the pHLA complex can be calculated according to the amount of the antigen peptides. To sum up, the method in the present invention may quantitatively detect the antigen peptides bound in the pHLA complex, is applicable to detection and identification of preparation products of a small amount of the pHLA complex in the process of exploring the preparation conditions, further brings convenience for optimizing different binding conditions according to the amount of the antigen
Description
peptides bound in the complex so as to increase the folding efficiency of the HLA-I molecules and promoting binding of the HLA-I molecules and the antigen peptides, and may further calculate the preparation rate of the pHLA complex in the renaturation system according to the content of the antigen peptides bound in the pHLA complex. The ultrafiltration-HPLC method established in the present invention may be used for quality control in the preparation process of the pHLA complex, and has advantages in aspects of T cell specific immune study, artificial APC and application and development of specific tetramer probes.
Description of Drawings
Fig. 1 shows a chromatogram of a peptide standard substance; wherein, (A) is a VYF antigen peptide standard substance; and (B) is an IRA control peptide standard substance. Fig. 2 shows a standard curve of a peptide standard substance; wherein, (A) is a VYF antigen peptide standard substance; and (B) is an IRA control peptide standard substance. Fig. 3 shows a co-renaturation verification result; wherein (A) is native polyacrylamide gel electrophoresis; and (B) is Western blot. Fig. 4 shows an ultrafiltration-HPLC detection result of a co-renaturation product. Fig. 5 shows effects of different pH values and different peptide concentrations on a binding amount of the pHLA complex; wherein (A) is an effect of the pH value; and (B) is the effect of the peptide concentrations. Fig. 6 shows comparison of different peptide binding amounts in the pHLA complex.
Description
Detailed Description
Technical solutions in the embodiments of the present invention are described clearly and fully below. Apparently, the described embodiments are merely part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative labor will belong to the protection scope of the present invention. Embodiment 1 Preparation of a peptide standard substance: An HLA-A*2402 restrictive human melanoma-associated antigen peptide gp100-intron 4 (having an amino acid sequence of NH2-VYFFLPDHL-COOH; called a VYF antigen peptide for short; containing corresponding anchor amino acid residues; serving as a binding peptide) and NH2-IRAWVAWRNR-COOH (called an IRA control peptide; containing no corresponding anchor amino acid residues; serving as an unbinding peptide) were selected to be dissolved into a renaturation buffer (including components such as 400 mmol/L of L-Arginine, 100 mmol/L of Tris HCl, 2 mmol/L of EDTA, 0. 5 mmol/L of oxidized glutathione and mmol/L of reduced glutathione; and having a pH value of 8.0); and a peptide standard solution was prepared. Chromatographic conditions are as follows: an LC-1OAT high-performance liquid chromatographic pump (Shimadzu), a SPD-20A high performance liquid chromatography detector (Shimadzu) RP-HPLC and a C18 chromatographic column (4.6mmx250mm, 5 pm) are used; a mobile phase includes acetonitrile containing 0.1% (v/v) of trifluoroacetic acid and water containing 0.1% (v/v) of trifluoroacetic acid according to a ratio of 31:69 (v/v); a flow rate is 1 mL/min; a column temperature is 35°C; a detection wavelength is 214 nm; and a loading volume is 20 pL.
Description
Under the chromatographic conditions, the chromatogram of the VYF antigen peptide standard solution and the IRA control peptide standard solution is shown as Fig. 1. The VYF antigen peptide standard solution has an absorption peak at 14-15 min, as shown in Fig. 1(A); and therefore, the absorption peak at 14-15 min is a characteristic peak of the VYF antigen peptide. The IRA control peptide has an absorption peak at 8-9 min; and therefore, the absorption peak at 8-9 min is selected as a characteristic peak of the IRA control peptide, as shown in Fig. 1(B). The VYF antigen peptide standard solution and the IRA control peptide standard solution having concentrations of 1, 2, 3, 4, 5, 6, 7, 8 and 9 pg/ml respectively were precisely prepared and then determined in sequence. A standard curve was drawn by taking the peak area as a vertical coordinate and taking the peptide concentration (pg/ml) as a horizontal coordinate. As shown in Fig. 2, the standard curve of the VYF antigen peptide and the IRA control peptide has excellent linearity in a range of 0-9 pg/ml. Regression equations are respectively as follows: y=13.031x, R2 =0.9963; y--9.4913x, R2 =0.9921. A 2 pg/ml of peptide standard solution was prepared; the solution was detected for many times according to the above chromatographic conditions; peak retention time and the peak area were recorded; a relative standard deviation (RSD) was calculated; and preciseness of the method was verified. The results are shown as Table 1; and the RSD of the peak retention time and the peak area of the VYF antigen peptide and the IRA control peptide is respectively less than 2%, which indicates that the method has excellent preciseness. Table 1
Peptide standard Retention time (min) Peak area substance x SD RSD (%) SD RSD(%) VYF 14.97 0.098 0.651 32.48 1.241 0.764 IRA 8.41 0.027 0.326 19.46 1.419 1.458
The VYF antigen peptide and the IRA control peptide were respectively prepared into standard solutions having concentrations of 1, 5 and 9 pg/ml. 20pL
Description
of the standard solution was subjected to injection detection in one experiment. In another experiment, the standard solution was subjected to ultrafiltration by an ultrafiltration tube having a molecular weight cut-off of3kD (passivated overnight with a BSA solution having a mass fraction of 1% in advance); and recovery of the peptides subjected to ultrafiltration was calculated according to detection results. Detection results are shown as Table 2; the ultrafiltration recovery of the VYF antigen peptide is in a range of 96.91%-98.14%; and the ultrafiltration recovery of the IRA control peptide is in a range of 95.10%-98.48%. The experimental results show that, the ultrafiltration method has excellent recovery, and may be used for determining interactions of the VYF antigen peptide and the IRA control peptide and the HLA molecules. Table 2 Peptide Concentration Peak area before Peak area after sustanced (pg/ml) ultrafiltration ultrafiltration Recovery substance 1 14.57 14.12 96.91 VYF 5 64.15 62.96 98.14 9 120.62 117.73 97.60 1 9.79 9.31 95.10 IRA 5 48.65 47.91 98.48 9 83.32 80.19 96.24
Embodiment 2 Preparation of pHLA complex: HLA-A*2402 heavy chain proteins (HC) and P2m, a VYF antigen peptide and an IRA control peptide were selected as preparative raw materials. A HC+2m+VYF antigen peptide was subjected to dilution refolding according to a molar ratio of about 1:2:11 so as to prepare the pHLA complex as follows: 10 pg of the VYF antigen peptide (8.69 nmol) was added into pre-cooled renaturation buffer per milliliter; the solution was shaken at a high speed and uniformly mixed at 10°C for 30 min; then 25 pg of p2m (1.45 nmol) was slowly
Description
dropped into the mixture; the mixture was shaken at a high speed and uniformly mixed at 10°C for 1 h; finally HC (0.77 nmol) having a total amount of 30pg was added in 3 times at an interval of 12 h each time; and the solution was shaken at °C for renaturation until renaturation was ended.
Meanwhile, HC+VYF antigen peptide and f2m+VYF were set as control groups.
Additionally, the IRA control peptide served as an unbinding control peptide; and the same renaturation was conducted.
The ultrafiltration tube having the molecular weight cut-off of 3kD was passivated overnight with the 1% of BSA solution. 2 ml of the renaturation product in each group was respectively subjected to first ultrafiltration; then the solution was complemented to 500 pL with renaturation buffer; the solution was slightly blown and uniformly mixed and then subjected to ultrafiltration again; the operation was repeated for three times so as to remove free peptides; the HC and p2m were respectively subjected to native polyacrylamide gel electrophoresis with renaturation folding products of the VYF antigen peptide and the IRA control peptide; and Western blot verification was performed by utilizing an antibody W6/32. The results are shown as Fig. 3; and only the HC+2m+VYF antigen co-renaturation may produce the correct HLA-A*2402/VYF complex conformation, which indicates that the HC and f2m may be bound with the VYF antigen peptide by virtue of the renaturation folding and then the pHLA complex of the correct conformation can be formed.
A 3% of trifluoroacetic acid solution was added into the preparation product of the pHLA complex obtained by ultrafiltration so as to reach a total volume of 100 pL; the solution was stood for 20 min; and then the filtrate was collected by performing ultrafiltration again. Sample injection detection was conducted according to the above liquid phase conditions. As shown in Fig. 4, the concentration of the VYF antigen peptide bound in the pHLA complex prepared by
Description
HC+f2m+VYF antigen co-renaturation is significantly higher than that in the rest groups (P <0.01).
Embodiment 3
Preparation of a pHLA complex: recombinant HLA-A*2402 heavy chain proteins (HC) and recombinant 2m, recombinant 2m/HLA-A*2402 fusion proteins (p2m/HC), and a VYF antigen peptide were selected.
By changing renaturation conditions in embodiment 2, including pH values of renaturation buffer and concentrations of antigen peptides in the renaturation system (the pH values of the renaturation buffer were respectively regulated as 8.0, 7.5 and 7.0; and the concentrations of the antigen peptides in the renaturation buffer were respectively adjusted to 5, 10, 20 and 40 pg/mL), and the conditions were further optimized so as to prepare the pHLA complex.
Dilution refolding was respectively conducted on an HC+2m+VYF antigen peptide and a $2m/HC+VYF antigen peptide so as to prepare the pHLA complex (wherein the dilution refolding of the p2m/HC+VYF antigen peptide was conducted on the basis of embodiment 2; the VYF antigen peptide was shaken and uniformly mixed in the renaturation buffer; 30pg (0.575nmol) of $2m/HC was added in 3 times at an interval of 12 h each time; and the solution was shaken at °C for renaturation until the renaturation was ended); and the binding amount of the antigen peptide in the pHLA complex was detected by the method in the present invention. The effects of different pH values and different peptide concentrations on the peptide binding amount of the pHLA complex were compared according to the detection results of the ultrafiltration-HPLC method. The results are shown as Fig. 5; and optimization results of the renaturation conditions of the HC+f2m+VYF antigen peptide are as follows: the pH value of the renaturation buffer is 8.0; the concentration of the antigen peptide is 20 pg/mL; and the pHLA complex prepared under the condition has the highest peptide content. Optimization results of the renaturation conditions of the p2m/HC+VYF
Description
antigen peptide are as follows: the pH value of the renaturation buffer is 7.0; the concentration of the antigen peptide is 20 pg/nL; and the pHLA complex prepared under the condition has the highest peptide content.
The antigen peptide concentration was calculated by utilizing the standard curve of the VYF antigen peptide; and the preparation rate of the pHLA complex can be calculated according to the preparation rate=a mole number of the antigen peptide bound with the HLA molecules/a mole number of the total HLA heavy chain proteins in the renaturation systemx100%. According to the above optimized renaturation conditions, the pHLA complex prepared from the HC+2m+VYF antigen peptide has the highest antigen peptide binding amount of 0.259 pg and a corresponding mole number of 0.225 nmol, i.e., the obtained pHLA complex is 0.225 nmol and has a preparation rate of about 14.6%. The pHLA complex prepared from the p2m/HC+VYF antigen peptide has the highest antigen peptide binding amount of 0.490 pg and a corresponding mole number of 0.426 nmol, i.e., a 0.426 nmol of antigen peptide-HLA complex is obtained by folding renaturation; and a preparation rate of the antigen peptide-HLA complex is about 36.9%. Results show that, the pHLA complex is prepared from the p2m/HC+VYF antigen peptide; and the preparation rate of the pHLA complex is significantly higher than that of the pHLA complex prepared from the HC+2m+VYF antigen peptide (P <0.01), as shown in Fig. 6.
Each embodiment in the description is described in a progressive way. The difference of each embodiment from each other is the focus of explanation. The same and similar parts among all of the embodiments can be referred to each other.
The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications made to the above embodiments will be apparent to those skilled in the art. General principles defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be
Description
limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. An ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in a pHLA complex, comprising the following steps: (1) performing ultrafiltration on the pHLA complex; and removing free antigen peptides; (2) treating the pHLA complex with an acid solution; (3) performing ultrafiltration on the solution obtained in the step (2); and collecting an antigen peptide solution; (4) determining antigen peptide content in the antigen peptide solution by HPLC.
2. The ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in the pHLA complex according to claim 1, further comprising: a pretreatment step: performing co-renaturation on the HLA-I molecules and the antigen peptide containing anchor amino acid residues to produce interaction to form the pHLA complex.
3. The ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in the pHLA complex according to claim 2, wherein the HLA-I molecules are composed of recombinant HC proteins and recombinant p2m proteins, or the HLA-I molecules are recombinant 2m/HC fusion proteins; co-renaturation of the HLA-I molecules and the antigen peptide containing anchor amino acid residues is conducted in the renaturation buffer; the renaturation buffer comprises 100-500 mmol/L of L-Arginine, 10-500 mmol/L of Tris HCl, 0.1-10 mmol/L of EDTA, 0.1-5 mmol/L of oxidized glutathione and 1-1Ommol/L of reduced glutathione and has a pH value of 7-9; a molar concentration of the HLA-I molecules in the renaturation buffer is 0.5-10 pmol/L;
Claims
a molar ratio of the recombinant HC proteins to the recombinant p2m proteins to the antigen peptide containing anchor amino acid residues is 1:(1-10):(5-100); or a molar ratio of the recombinant P2m/HC fusion proteins to the antigen peptide containing anchor amino acid residues is 1:(5-100); a co-renaturation temperature is 10-14°C; and renaturation time is 12-48 h.
4. The ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in the pHLA complex according to claim 1, wherein the molecular weight cut-off during ultrafiltration in the steps (1) and (3) is 3-10kD; in the step (2), the acid solution is an aqueous solution of trifluoroacetic acid having a volume fraction of 2-10%; and standing time is 10-30 min.
5. The ultrafiltration-high performance liquid chromatography method for detecting antigen peptides in the pHLA complex according to claim 1, wherein in the step (4), a C18 chromatographic column is adopted during HPLC; a mobile phase comprises acetonitrile containing 0.005-0.1% (v/v) of trifluoroacetic acid and water containing 0.005-0.1% (v/v) of trifluoroacetic acid according to a ratio of 15:85-40:60; a flow rate is 0.5-1.2 mL/min; a column temperature is ±3°C; and a detection wavelength is 214 nm; in the step (4), before the content of the antigen peptides in the antigen peptide solution is determined by HPLC, an antigen peptide standard substance is detected; a characteristic peak is determined; and a standard curve is drawn; the antigen peptides determined in the step (4) are antigen peptides bound with the HLA-I molecules in the pHLA complex in an equimolar manner; and then the amount of the pHLA complex can be calculated according to the amount of the antigen peptides.
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