CN108362884B

CN108362884B - Serum and tissue molecular index for evaluating tumor risk and treatment effect

Info

Publication number: CN108362884B
Application number: CN201710206641.6A
Authority: CN
Inventors: 谢君; 朱乃硕; 朱嗣博
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2020-12-01
Anticipated expiration: 2037-03-31
Also published as: CN108362884A

Abstract

The invention belongs to the technical field of molecular indexes for tumor diagnosis, treatment curative effect and relapse judgment, and particularly relates to a diagnostic reagent for specifically detecting tumor risk degree and treatment curative effect by applying epitope peptide of ApoA1 protein closely related to tumors in serum and preparing a specific antibody thereof. According to the invention, the content change of serum protein before and after treatment of tumors such as prostate cancer is firstly detected by using iTRAQ in a large quantity, and the ApoA1 protein is found to be closely related to the risk classification and the treatment curative effect of the tumors. Then screening and artificially synthesizing functional epitope peptides in APOA1, and immunizing animals with the peptides as antigens to prepare corresponding antibodies, wherein the obtained antibodies can sensitively detect low-risk, medium-risk and high-risk tumor patients and have better evaluation effect on the curative effect of tumors such as prostate cancer; the molecular marker can be used as a detection standard control and for preparing an antibody for producing a corresponding tumor diagnosis and research kit.

Description

Serum and tissue molecular index for evaluating tumor risk and treatment effect

Technical Field

The invention belongs to the technical field of tumor diagnosis and treatment effect evaluation, and particularly relates to an epitope peptide in ApoA1 protein serving as a molecular index in serum and tissues and a specific antibody prepared by the epitope peptide, and the content of ApoA1 in the serum is detected by using the antibody, so that the tumor risk classification and treatment effect are evaluated.

Background

More than 400 million new tumor patients occur in China every year, only prostate cancer occurs in the world every year and reaches up to 110 ten thousand, in China, prostate cancer is the second-ranked male malignant tumor, and in the past twenty years, the fatality rate is increased by 10 times. The high sensitivity and diagnosis of tumors, risk classification for taking corresponding therapeutic measures and effective evaluation of the therapeutic effect of tumors require the discovery and application of specific and highly sensitive molecular markers of specific pathologies specific to tumors, which is a problem to be solved urgently at present. However, the pathological molecular markers which can be used for tumor diagnosis and treatment evaluation at present are very limited, for example, prostate cancer is classified into three categories of low, medium and high risk according to patient serum PSA, Gleason score and clinical staging, and the grading method relates to three indexes, wherein the Gleason score and the clinical staging index are diagnosed according to various forms such as tumor shape and boundary and are difficult to grasp. More importantly, many tumors (such as prostate cancer) have no unified index for evaluating the treatment effect at present, and are very unfavorable for the implementation of accurate treatment.

Disclosure of Invention

The invention aims to provide a serum molecular index which can conveniently and accurately diagnose the risk degree of tumors and can be used for evaluating the treatment effect on the tumors.

The technical scheme is as follows:

(1) in the present study, the expression changes of serogroups in different stages of tumor risk and different stages of treatment are first detected by iTRAQ, and it is found that ApoA1 has important differences (p < 0.001) in the expression of prostate cancer in different stages of risk, and the content of the molecular index is significantly different (p < 0.001) in the process of improving tumor symptoms (based on tumor volume and pathological index) in different stages of surgery, chemotherapy, radiotherapy such as heavy ion treatment (table 1 and fig. 1).

(2) The effect of heavy ion therapy was affected by platelet degranulation by differential protein enrichment analysis (figure 2).

(3) Four peptide epitope sequences are obtained by methods of bioinformatics and structural biology and a B cell epitope analysis method, the required peptide is artificially synthesized by a mixed anhydride method, and immune animals generate high-affinity antibodies against APO1 protein epitopes. The diagnostic reagent prepared from the antibody can diagnose the risk degree and therapeutic effect of tumors such as prostate cancer (FIG. 3).

TABLE 1 expression profile of APOA1 in different graded tumors (prostate cancer)

The serum molecular index ApoA1 gene sequence and amino acid sequence provided by the invention are SeqNo.1 and SeqNo.2 respectively.

The epitope peptides provided by the invention are SeqNo.3, SeqNo.4, SeqNo5 and SeqNo6 (sequence attachments), and the four peptides are found to cause strong immune reaction and generate specific antibodies, so that the epitope peptides can be used for detecting the content and the existence of APOA 1.

The invention provides a detection kit for diagnosing tumor risk degree and treatment effect for the first time based on the four epitope peptide sequences and the prepared specific antibody thereof, the kit can distinguish low-risk, medium-risk and high-risk tumor patients, and verifies the diagnosis of the tumor such as prostate cancer patients (P is less than 0.05) and the detection evaluation of the treatment effect (obvious difference between before treatment and after treatment, P is less than 0.05).

Drawings

FIG. 1 is a graph of the expression (iTRAQ measurements) of different fragments of APOA1 before and after heavy ion therapy in tumor (prostate cancer) cases of different risk stratification.

Fig. 2 is a schematic of ApoA1 being expelled from secretory granules to the outside of cells by platelet degranulation.

FIG. 3 is a line graph showing the efficacy of heavy ion therapy for prostate cancer using polyclonal antibodies prepared from four epitope peptides.

Detailed Description

1. Peripheral blood was sampled. The treatment was carried out immediately within 2 hours after the blood withdrawal.

2. After the blood is naturally coagulated for 10-20 minutes at room temperature, the supernatant is taken after centrifugation for about 20 minutes (3000 rpm at 2000-

3. The test solution is diluted in 150 mul small test tube according to the ratio of 1: 1-1: 500.

4. Blank holes (blank reference holes are not added with samples and enzyme labeling reagents, and the operation of the rest steps is the same), standard holes and sample holes to be detected are respectively arranged on a 96-or 48-pore plate. Accurately adding 50 mul of standard sample on an enzyme-labeled coating plate, and measuring 50 mul of sample in a sample hole to be measured. Adding a sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall; the standard hole is used for respectively adding target proteins and epitope peptides (with sequences of SEQ ID No.2, No.3, No.4, No5 and No6) with certain concentrations;

5. incubation, solution preparation, washing and enzyme addition: sealing the plate with a sealing plate film, and then incubating for 30 minutes at 37 ℃; diluting 30 times (20 times of 48T) of the concentrated washing liquid with 30 times of distilled water for later use; carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry; adding 50 mul of enzyme-labeled reagent into each hole except for blank holes;

6. color development and termination: adding 50 μ l of color-developing agent A into each well, adding 50 μ l of color-developing agent B, shaking gently, mixing, and developing at 37 deg.C in dark for 10 min; and (4) terminating: stop the reaction by adding 50. mu.l of stop solution to each well (blue color immediately turns yellow);

7. the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with blank air conditioning of zero. The measurement should be performed within 15 minutes after the addition of the stop solution. Drawing a standard curve on coordinate paper by taking the concentration of the standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of the sample; multiplying by the dilution times; or calculating a linear regression equation of the standard curve by using the concentration and OD value of the standard substance, substituting the OD value of the sample into the equation to calculate the concentration of the sample, and multiplying the concentration by the dilution factor to obtain the actual concentration of the sample.

8. Parameters for diagnostic indicators in the case of prostate cancer: taking normal as a control, and if the sample concentration is 1.2-2 times of the normal concentration, determining the patient as a low-risk patient; 2-3 times of the total weight of the composition, which is suitable for middle-risk patients; more than 3 times of the total weight of the composition is high-risk patients. Evaluation of treatment effect: and (4) for middle-risk and high-risk patients, if the concentration of the sample returns to below 2 times after treatment, the treatment is improved. Such as returning to normal level, i.e., substantially recovering.

Sequence listing

The nucleotide of the gene of SEQ ID No.1,

Claims

1. the application of a protein or peptide prepared from a nucleotide sequence shown in SeqNo.1, a protein sequence shown in SeqNo.2 and an epitope peptide sequence based on ApoA1 protein as a standard protein molecule control and a specific antibody molecule prepared from SeqNo.3, SeqNo.4, SeqNo.5 and SeqNo.6 epitope peptides as a probe core component in preparing an immunoassay kit for analyzing and diagnosing the risk, the treatment effect and the recurrence condition of the prostate cancer is characterized in that the immunoassay kit is used for measuring the content of the ApoA1 protein in serum and tissues so as to analyze and diagnose the risk, the treatment effect and the recurrence condition of the prostate cancer;

the epitope peptide sequence of the ApoA1 protein is characterized by SeqNo.3, SeqNo.4, SeqNo.5 and SeqNo.6;

the diagnostic index parameter is: taking normal as a control, and if the sample concentration is 1.2-2 times of the normal concentration, determining the patient as a low-risk patient; 2-3 times of the total weight of the composition, which is suitable for middle-risk patients; more than 3 times of the total weight of the composition is high-risk patients;

evaluation of the treatment effect: for the middle-risk and high-risk patients, if the concentration of the sample returns to below 2 times after treatment, the treatment is improved; such as returning to normal level, i.e., substantially recovering.