CN110790815B - Method for extracting natural antigen peptide from gynecological tumor tissue - Google Patents
Method for extracting natural antigen peptide from gynecological tumor tissue Download PDFInfo
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Abstract
The invention discloses a method for extracting natural antigen peptide from gynecological tumor tissue, which comprises the following steps: s1: selecting a tissue sample; s2: tissue sample homogenization; s3: performing immunoaffinity column chromatography; s4: extracting a mixture of protein and polypeptide; s5: concentrating the polypeptide mixture; s6: and detecting and identifying polypeptide fragment information by LC-MS or LC-MS/MS. The method for extracting the natural presenting antigen peptide from the gynecological tumor tissue has obvious clinical significance for tumor immunotherapy, and the natural presenting antigen peptide extracted by the method does not need gene sequencing, so that the cost can be reduced to a greater extent, and the burden of a patient is relieved.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for extracting natural antigen peptide from gynecological tumor tissue.
Background
Tumors are currently one of the leading causes of death in humans. Despite the recent improvements and enhancements in the diagnosis and treatment of tumors, and the continued improvements in the regimens of chemotherapy and radiotherapy, it is ultimately difficult for most patients to escape the early forms of death. Among different treatment modes of malignant tumors, immunotherapy has attracted more and more attention because it kills tumor cells specifically, and has the characteristics of high efficiency and low toxic and side effects. In recent years, advances in molecular biology and further understanding of immune system function have brought the development and development of biotherapeutic approaches to a rapid stage. The development of tumor vaccines is the most important direction for the biological treatment of tumors at present.
Gynecological tumors are common diseases which harm the health of women, and the malignant tumors with high incidence rate comprise ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma and the like. For many gynecological tumors, the traditional treatment cannot improve survival rate, but rather increases the incidence of complications seriously. Although there are studies on the direct extraction of antigenic peptides from solid tumors, no report has been found on the direct extraction of antigenic peptides from gynecological tumors.
Therefore, by researching the method for extracting the natural presented antigen peptide from the gynecological tumor tissue, the obtained antigen peptide has significant clinical significance for the current individualized tumor immunotherapy.
Disclosure of Invention
The invention aims to provide a method for extracting natural presented antigen peptide from gynecological tumor tissue, which does not need gene sequencing and has low cost so as to solve the problems in the background technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting natural antigen peptide from gynecological tumor tissue comprises the following steps:
s1: selecting a tissue sample;
s2: tissue sample homogenization;
s3: performing immunoaffinity column chromatography;
s4: extracting a mixture of protein and polypeptide;
s5: concentrating the polypeptide mixture;
s6: and detecting and identifying polypeptide fragment information by LC-MS or LC-MS/MS.
Further, in step S1, the tissue sample selection condition is as follows: the preoperative biopsy is confirmed to be cervical squamous cell carcinoma or cervical adenocarcinoma; the WBC number is more than or equal to 4 x 109/L before operation and after chemotherapy or neoadjuvant chemotherapy; the clinical stage is higher than Ib1 stage; the pathological examination of the postoperative tumor tissue and the paracancer normal cervical tissue conforms to the preoperative diagnosis; the weight of the obtained sample tissue is more than or equal to 1g, and meanwhile, in order to ensure that the content of cancer tissue in the tumor tissue sample is more than or equal to 80 percent, the content of normal cervical tissue in the tissue beside the cancer is more than or equal to 99 percent, the sample edge section HE staining is carried out for pathological examination.
Further, in step S2, the tissue sample homogenization includes the following steps:
a1: lysing the tissue sample;
a2: multi-step centrifugation of tissue lysates:
a3: and filtering the final solution.
Further, in step a1, when the tissue sample is lysed, the method comprises the following steps: the method comprises the following steps: placing the qualified tissue sample selected in the step S1 on ice and cutting; step two: the mixture is prepared according to the proportion of 1g of tissue sample and 10ml of lysis solution, and then the tissue sample is placed on a shaking table at 4 ℃ for 1-2 hours for full lysis.
Further, the lysis solution comprises CHAPS, a protease inhibitor and a phosphatase inhibitor, and the proportion is as follows: adding 1 protease inhibitor tablet and 1 phosphatase inhibitor tablet into 1% CHAPS per 10ml, wherein the protease inhibitor is Rockville protease inhibitor and does not contain EDTA; the phosphatase inhibitor is Cocktail, a Roche phosphatase inhibitor, and does not contain EDTA.
Further, in step a2, when the tissue lysate is centrifuged in multiple steps, the tissue lysate is centrifuged 3 times by using a centrifuge under the following conditions: the temperature is 3-5 ℃, the rotating speed is 17000rpm, the time is 20-30min, and the supernatant is taken after each centrifugation.
Further, the immunoaffinity column chromatography in step S3 includes the following steps:
p1: preparing a reagent: the reagent comprises: coupling buffer, 1mM HCl, buffer A and buffer B, PBS;
p2: affinity column-coupled antibody: the method comprises the following steps: b1: ligand coupling; b2: eluting and passivating; b3: coupling efficiency is checked.
Further, in step B1, during ligand coupling, 1 drop of ice 1mM HCl was added rapidly to prevent bubbles by opening the top of the immunoaffinity column; eluting isopropanol with 1-2ml syringe, 1mM HCl and ice injection, flow rate less than or equal to 1 ml/min; injecting 1ml of ligand dissolving solution, namely diluting the ligand dissolving solution to 1mg/ml of anti-HLA-A/B/C antibody by using coupling buffer solution; connecting another syringe at the outlet for 1 hour repeatedly, and repeatedly circulating at 4 ℃ overnight; in the step B2, during elution and passivation, sequentially injecting a buffer solution A, a buffer solution B and a buffer solution A into the immunoaffinity column, sealing the immunoaffinity column, standing at room temperature for 30min or at 4 ℃ for 4 hours, sequentially injecting the buffer solution B, the buffer solution A and the buffer solution B, then adjusting the pH value of the immunoaffinity column to 7.2 by using a PBS buffer solution, and finally injecting 1% of CHAPS with the pH of 8; in the step B3, when the coupling efficiency is detected, 3 times of coupling buffer solution is used for washing the immunoaffinity column, 2M glycine is added into the waste liquid in the proportion of 1:1, and the absorbance is detected at 280 nm.
Further, in step S4, the extraction of the mixture of protein and polypeptide includes the following steps:
m1: preparing a reagent: the reagent comprises: a first washing buffer solution, a second washing buffer solution, a third washing buffer solution, a fourth washing buffer solution and an elution buffer solution.
M2: connecting the immunoaffinity column coupled with the ligand to a connector of a micro pump, and repeatedly circulating the tissue lysate to the immunoaffinity column at a low speed by the micro pump, wherein the flow rate is 0.2-1ml/min, the temperature is 4 ℃, and the circulation is carried out for 12 hours; then, sequentially using a first cleaning buffer solution, a second cleaning buffer solution, a third cleaning buffer solution and a fourth cleaning buffer solution for cleaning, and then eluting by using an elution buffer solution;
m3: ultrafiltering, wherein the upper layer is protein, and the lower layer is polypeptide complex.
Further, in step M3, the ultrafiltration comprises the following steps: n1: pretreating an ultrafiltration tube: pre-washing the ultrafiltration tube with 0.2M acetic acid and 10% acetonitrile, and then pre-washing with 10% acetic acid; 10000g, centrifuging for 10min and pouring out;
n2: the ultrafiltration tube was mounted and the sample eluted in step M2 was added to the tube at 4 ℃ and 12000rpm for 30 min.
In conclusion, compared with the prior art, the method for extracting the natural presented antigen peptide from the gynecological tumor tissue has the following beneficial technical effects: the natural presented antigen peptide is extracted from the solid tumor, gene sequencing is not needed, the cost is reduced, and the method has obvious clinical significance for the current individualized tumor immunotherapy.
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FIG. 1 is a flow chart of the present invention.
Detailed Description
The invention will be further elucidated with reference to specific embodiments and with reference to the drawing of the description. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, which are not specified under specific conditions, are generally performed under conventional conditions. 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. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
The first embodiment is as follows:
as shown in figure 1, a method for extracting natural antigen-presenting peptide from gynecological tumor tissue comprises the following steps:
s1: selecting a tissue sample; the tissue sample selection conditions are as follows: the preoperative biopsy is confirmed to be cervical squamous cell carcinoma or cervical adenocarcinoma; the WBC number is more than or equal to 4 x 109/L before operation and after chemotherapy or neoadjuvant chemotherapy; the clinical stage is higher than Ib1 stage; the pathological examination of the postoperative tumor tissue and the paracancer normal cervical tissue conforms to the preoperative diagnosis; the weight of the obtained sample tissue is more than or equal to 1g, and meanwhile, in order to ensure that the content of cancer tissue in the tumor tissue sample is more than or equal to 80 percent, the content of normal cervical tissue in the tissue beside the cancer is more than or equal to 99 percent, the sample edge section HE staining is carried out for pathological examination. The protein and polypeptide finally extracted by the quality-controlled sample have sufficient quantity, high purity and good specificity.
S2: tissue sample homogenization;
s3: performing immunoaffinity column chromatography;
s4: extracting a mixture of protein and polypeptide;
s5: concentrating the polypeptide mixture;
s6: and detecting and identifying polypeptide fragment information by LC-MS or LC-MS/MS.
Specifically, in step S2, the tissue sample homogenization includes the following steps:
a1: tissue sample lysis: the method comprises the following steps: the method comprises the following steps: placing the qualified tissue sample selected in the step S1 on ice and cutting; step two: mixing 1g of tissue sample with 10ml of lysis solution, and placing in a shaking table at 4 ℃ for 1-2 hours for full lysis;
a2: multi-step centrifugation of tissue lysates: centrifuging for 3 times by using a centrifuge under the following centrifugation conditions: the temperature is 3-5 ℃, the rotating speed is 17000rpm, the time is 20-30min, and supernatant is taken after each centrifugation;
a3: and (3) final liquid filtration: filtration was done using an Acrodisc needle filter (0.22 μm, 25mm, sterile).
The sample after homogenization has high efficiency of extracting polypeptide.
Specifically, the lysis solution comprises CHAPS, a protease inhibitor Cocktail which is a Rockwell protease inhibitor and does not contain EDT, a phosphatase inhibitor which is a Rockwell phosphatase inhibitor and does not contain EDTA, and the proportion is as follows: 1 tablet of protease inhibitor and 1 tablet of phosphatase inhibitor were added per 10ml of 1% CHAPS.
Specifically, the immunoaffinity column chromatography in step S3 includes the following steps:
p1: preparing a reagent: the reagent comprises: coupling buffer, 1mM HCl, buffer A and buffer B, PBS;
p2: affinity column-coupled antibody: the method comprises the following steps: b1: ligand coupling; b2: eluting and passivating; b3: coupling efficiency is checked.
Specifically, in step B1, during ligand coupling, 1 drop of ice 1mM HCl was added rapidly to prevent bubbles by opening the top of the immunoaffinity column; eluting isopropanol with 1-2ml syringe, 1mM HCl and ice injection, flow rate less than or equal to 1 ml/min; injecting 1ml of ligand dissolving solution, namely diluting the ligand dissolving solution to 1mg/ml of anti-HLA-A/B/C antibody by using coupling buffer solution; connecting another syringe at the outlet for 1 hour repeatedly, and repeatedly circulating at 4 ℃ overnight; in the step B2, during elution and passivation, sequentially injecting a buffer solution A, a buffer solution B and a buffer solution A into the immunoaffinity column, and repeatedly washing the immunoaffinity column through different pH values of the buffer solution A and the buffer solution B to achieve the purpose of passivation; sealing the immunoaffinity column, standing at room temperature for 30min or 4 ℃ for 4 hours, sequentially injecting a buffer solution B, a buffer solution A and a buffer solution B, then adjusting the pH value of the immunoaffinity column to 7.2 by using a PBS (phosphate buffer solution), and finally injecting 1% of CHAPS with the pH value of 8; in the step B3, when the coupling efficiency is detected, 3 times of coupling buffer solution is used for washing the immunoaffinity column, 2M glycine is added into the waste liquid in the proportion of 1:1, and the absorbance is detected at 280 nm.
Specifically, in step S4, the extraction of the mixture of proteins and polypeptides includes the following steps:
m1: preparing a reagent: the reagent comprises: a first washing buffer solution, a second washing buffer solution, a third washing buffer solution, a fourth washing buffer solution and an elution buffer solution;
m2: connecting the immunoaffinity column coupled with the ligand to a connector of a micro pump, and repeatedly circulating the tissue lysate to the immunoaffinity column at a low speed by the micro pump, wherein the flow rate is 0.2-1ml/min, the temperature is 4 ℃, and the circulation is carried out for 12 hours; then, sequentially using a first cleaning buffer solution, a second cleaning buffer solution, a third cleaning buffer solution and a fourth cleaning buffer solution for cleaning, and then eluting by using an elution buffer solution;
m3: m3: ultrafiltering, wherein the upper layer is protein, and the lower layer is polypeptide compound; the ultrafiltration comprises the following steps: n1: pretreating an ultrafiltration tube: pre-washing the ultrafiltration tube with 0.2M acetic acid and 10% acetonitrile, and then pre-washing with 10% acetic acid; 10000g, centrifuging for 10min and pouring out;
n2: the ultrafiltration tube was mounted and the sample eluted in step M2 was added to the tube at 4 ℃ and 12000rpm for 30 min.
After ultrafiltration, concentrating the polypeptide liquid to be powder at-20 ℃ for storage; re-dissolving, and then passing through a C18 reverse phase column (Waters sep-pak C18 solid phase extraction column); concentrating again and drying. And detecting the peptide fragment information by LC-MS after redissolution.
Specifically, coupling buffer, 1mM HCl, buffer A, buffer B, first washing buffer, second washing buffer, third washing buffer, fourth washing buffer, elution buffer and PBS buffer are filtered by 0.45um filter.
It needs to be further explained that:
the coupling buffer solution comprises the following components in percentage by weight: 0.2M NaHCO3, 0.5M NaCl, pH 6.5-9(best for 8).
The buffer solution A is prepared from the following components in percentage by weight: ethanolamine 0.5M, NACl 0.5M, pH 8.3.
The buffer solution B comprises the following components in percentage by weight: 0.1M sodium acetate (sodium acetate), 0.5M NACl, pH 4.
The first washing buffer solution comprises the following components in percentage by weight: lysine buffer (20mM Tris-HCl, 150mM NaCl, 1% CHAPS), ph 8.0.
The second washing buffer solution comprises the following components in percentage by weight: 50mM Tris-HCl, 150mM NaCl, ph 8.0.
The third cleaning buffer solution is prepared from the following components in percentage by weight: 50mM Tris-HCl, 450mM NaCl, ph 8.0.
The fourth cleaning buffer solution is prepared from the following components in percentage by weight: 50mM Tris, ph 8.0.
The storage buffer solution comprises the following components in percentage by weight: 0.05M Na2HPO4, 0.1% NaN3, pH7.
The elution buffer solution is prepared from the following components in percentage by weight: 10% acetic acid, pH 2.4.
The PBS buffer was pH 7.2.
CHAPS is a non-denaturing zwitterionic detergent, a protein lysate, used to solubilize membrane proteins and lyse protein-protein interactions.
Example two: extracting natural antigen peptide from normal tissue sample.
The difference between the second embodiment and the first embodiment is that the tissue sample is selected from normal tissue, natural presented antigen peptide is extracted, and the total amount of peptide fragments of tumor tissue and normal tissue in the same sample is compared, and the result is shown in table 1.
TABLE 1 comparison of the total peptide content of tumor tissue and normal tissue in the same samples
As can be seen from Table 1, the total amount of the tumor tissue peptide fragments in the samples 2-6 is obviously higher than that of the normal tissue peptide fragments, so that the method can better extract the natural presented antigen peptide in the tumor tissue, does not need gene sequencing, reduces the cost, and has obvious clinical significance for the current individualized tumor immunotherapy.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (4)
1. A method for extracting natural antigen peptide from gynecological tumor tissue is characterized by comprising the following steps:
s1: selecting a tissue sample;
s2: tissue sample homogenization;
s3: performing immunoaffinity column chromatography;
s4: extracting a mixture of protein and polypeptide;
s5: concentrating the polypeptide mixture;
s6: detecting and identifying polypeptide segment information by LC-MS or LC-MS/MS;
in step S1, the tissue sample selection conditions are as follows: the preoperative biopsy is confirmed to be cervical squamous cell carcinoma or cervical adenocarcinoma; the WBC number before operation is not subjected to chemotherapy or after neoadjuvant chemotherapy is not less than 4 x 109L; the clinical stage is higher than Ib1 stage; the pathological examination of the postoperative tumor tissue and the paracancer normal cervical tissue conforms to the preoperative diagnosis; the weight of the obtained sample tissue is more than or equal to 1g, meanwhile, in order to ensure that the content of cancer tissue in the tumor tissue sample is more than or equal to 80 percent, the content of normal cervical tissue in the tissue beside the cancer is more than or equal to 99 percent, and the sample edge section HE staining is carried out for pathological examination;
in step S2, the tissue sample homogenization includes the following steps:
a1: lysing the tissue sample; when the tissue sample is cracked, the method comprises the following steps: the method comprises the following steps: placing the qualified tissue sample selected in the step S1 on ice and cutting; step two: mixing 1g of tissue sample with 10ml of lysis solution, and placing in a shaking table at 4 ℃ for 1-2 hours for full lysis;
a2: multi-step centrifugation of tissue lysate;
a3: filtering the final solution;
in step S3, the immunoaffinity column chromatography includes the following steps:
p1: preparing a reagent: the reagent comprises: coupling buffer, 1mM HCl, buffer A and buffer B, PBS;
p2: affinity column-coupled antibody: the method comprises the following steps: b1: ligand coupling; b2: eluting and passivating; b3: detecting coupling efficiency;
in step B1, during ligand coupling, the top of the immunoaffinity column is opened and 1 drop of ice 1mM HCl is rapidly added to prevent bubbles; eluting isopropanol with 1-2ml syringe, 1mM HCl and ice injection, flow rate less than or equal to 1 ml/min; injecting 1ml of ligand dissolving solution, namely diluting the ligand dissolving solution to 1mg/ml of anti-HLA-A/B/C antibody by using coupling buffer solution; connecting another syringe at the outlet for 1 hour repeatedly, and repeatedly circulating at 4 ℃ overnight; in the step B2, during elution and passivation, sequentially injecting a buffer solution A, a buffer solution B and a buffer solution A into the immunoaffinity column, sealing the immunoaffinity column, standing at room temperature for 30min or at 4 ℃ for 4 hours, sequentially injecting the buffer solution B, the buffer solution A and the buffer solution B, then adjusting the pH value of the immunoaffinity column to 7.2 by using a PBS buffer solution, and finally injecting 1% of CHAPS with the pH of 8; in the step B3, when the coupling efficiency is detected, 3 times of coupling buffer solution is used for washing the immunoaffinity column, 2M glycine is added into the waste liquid in the proportion of 1:1, and the absorbance is detected at 280 nm;
in step S4, the extraction of the mixture of proteins and polypeptides includes the following steps:
m1: preparing a reagent: the reagent comprises: a first washing buffer solution, a second washing buffer solution, a third washing buffer solution, a fourth washing buffer solution and an elution buffer solution;
m2: connecting the immunoaffinity column coupled with the ligand to a connector of a micro pump, and repeatedly circulating the tissue lysate to the immunoaffinity column at a low speed by the micro pump, wherein the flow rate is 0.2-1ml/min, the temperature is 4 ℃, and the circulation is carried out for 12 hours; then, sequentially using a first cleaning buffer solution, a second cleaning buffer solution, a third cleaning buffer solution and a fourth cleaning buffer solution for cleaning, and then eluting by using an elution buffer solution;
m3: ultrafiltering, wherein the upper layer is protein, and the lower layer is polypeptide complex.
2. The method of claim 1, wherein the lysis solution comprises CHAPS, protease inhibitor and phosphatase inhibitor, and the ratio of the components is as follows: adding 1 protease inhibitor tablet and 1 phosphatase inhibitor tablet into 1% CHAPS per 10ml, wherein the protease inhibitor is Rockville protease inhibitor and does not contain EDTA; the phosphatase inhibitor is Cocktail, a Roche phosphatase inhibitor, and does not contain EDTA.
3. The method for extracting natural antigen peptide from gynecological tumor tissue according to claim 1, wherein in step A2, the tissue lysate is centrifuged 3 times in multiple steps under the following conditions: the temperature is 3-5 ℃, the rotating speed is 17000rpm, the time is 20-30min, and the supernatant is taken after each centrifugation.
4. The method for extracting natural antigen-presenting peptide from gynecological tumor tissue according to claim 1, wherein the ultrafiltration comprises the following steps in step M3: n1: pretreating an ultrafiltration tube: pre-washing the ultrafiltration tube with 0.2M acetic acid and 10% acetonitrile, and then pre-washing with 10% acetic acid; 10000g, centrifuging for 10min and pouring out;
n2: the ultrafiltration tube was mounted and the sample eluted in step M2 was added to the tube at 4 ℃ and 12000rpm for 30 min.
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