CN111518878B - Method for evaluating animal model in screening of neoantigen vaccine or drug and application - Google Patents

Abstract

The invention discloses a primer and a probe for evaluating an animal model when screening vaccines or drugs of neoantigens, a detection method is established, and the kit is manufactured into a kit.

Description

Method for evaluating animal model in screening of neoantigen vaccine or drug and application

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a method for evaluating an animal model in screening of a neoantigen vaccine or a medicament and application of the method.

Background

At present, in the process of screening neoantigen vaccines and drugs, luciferase animal imaging and circulating tumor cell quantitative detection results are generally adopted to evaluate the standardization and consistency of experimental animal models.

Luciferase animal imaging is to integrate luciferase genes into tumor cells, culture a tumor cell line capable of stably expressing luciferase, inoculate the marked tumor cells into an experimental animal body, and observe the position of exogenous tumor cells through detection equipment to observe the tumor state of the experimental animal model.

Circulating Tumor Cells (CTCs) refer to Tumor Cells that enter the peripheral blood circulation, either spontaneously or as a result of a diagnostic procedure. A large number of researches prove that the peripheral blood CTCs detection is simple to operate and accurate in height determination, and is considered to be one of the best and most objective detection means at present. The detection methods of CTCs are classified into two types, i.e., a slide-based Immunocytochemistry (ICC) technique and a PCR-based molecular biology technique (including RT-PCR, nested PCR, etc.).

The ICC method, known as the gold standard for evaluating CTC detection methods, has the advantages of: the method is visual and simple, but factors such as uneven expression of tumor cell surface antigens, partial cross reaction of lymphocytes and the like influence the specificity and sensitivity of detection.

The PCR-based detection technology utilizes the principle that the expression of tissue or tumor specific mRNA or the RNA level is abnormal after certain genes are changed, and the mRNA markers are not expressed in normal peripheral blood cells, thereby achieving the purpose of detecting CTC. The method is efficient, specific, sensitive, simple and convenient to operate, objective in result judgment and the most effective method for detecting CTC at present.

The tumor neoantigen is a neoantigen with immunogenicity generated by the induction or spontaneous mutation of somatic genes due to physicochemical damage factors. Because of these characteristics, tumor neoantigens can elicit effective, safe and highly specific anti-tumor immune responses. Therefore, personalized neo-antigen vaccines are currently considered to be an extremely effective and safe immunotherapy. By comparing the whole exon and transcriptome data of tumor tissues and normal tissues, and combining with a machine learning algorithm, a plurality of software tools for predicting new antigens, such as pVAC-seq, TSNAD, Neoppepsee and the like, have been developed internationally. The immunogenicity of the new antigen predicted by these software needs to be verified by various in vitro and in vivo immunological experiments, such as enzyme-linked immunospot assay (ELISPOT), intracellular factor staining assay (ICS), mutant-HLA tetramer (pHLAmultimer), target cell killing or animal model in vivo experiments, etc. Wherein, the tumor-bearing animal model such as a CDX model (cell-line-derived xenograde) for inoculating a human-derived tumor cell line into an immunodeficient mouse and a PDX model (patient-derived xenograde) for inoculating a patient-derived tumor tissue block into the immunodeficient mouse can more comprehensively evaluate the prediction accuracy of the new antigen prediction software.

Due to the heterogeneity of tumor cells, immunocytochemistry and molecular biology techniques can be used to identify circulating tumor cells in the heterogeneous tumor cell evaluation assay in the prior art. However, the immunocytochemistry technology depends on specific anti-tumor antigen antibodies, and due to the technical limitation, the immunocytochemistry technology can only use 2-3 tumor markers, and the judgment depends on manual work, so that subjective deviation is easy to occur; the molecular biology technology mainly evaluates the development condition of tumor cells through the specific expression of tumor cell genes, but the detection result is unstable due to the conditions of small base difference and unstable expression quantity of different sections of the specific expression genes.

Therefore, how to provide a method for evaluating the consistency of tumor-bearing animal models, which is simple to operate, high in sensitivity and strong in stability, is an urgent problem to be solved in the field.

Disclosure of Invention

The invention discloses a detection probe and a primer for humanized circulating tumor cells, which can be used for accurately and normatively evaluating a tumor-bearing animal model.

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

a primer and a probe for detecting human-derived circulating tumor cells, wherein the primer comprises PF 1: SEQ ID NO.1, PR 1: SEQ ID NO.2, PF 2: SEQ ID NO.4, PR 2: the nucleotide sequence of SEQ ID NO.5, and the probe comprises P1: SEQ ID No.3, P2: a nucleotide sequence shown as SEQ ID NO.6, wherein the 5 'end of the probe is provided with a fluorescent group, and the 3' end of the probe is provided with a quenching group;

the PF 1: CGGTCTGTACTTCTGTAC, SEQ ID NO. 1;

the PR 1: GACAGAGATCTGGTAATTCA, SEQ ID NO. 2;

the P1: CACTGTCTTGCTCCACGCTG, SEQ ID NO. 3;

the PF 2: AGCCTAAGATGAGAGTTC, SEQ ID NO. 4;

the PR 2: CACAGAACTAGAACATTGATA, SEQ ID NO. 5;

the P2: ATCTGGAGTCCTATTGACATCGCC, SEQ ID NO. 6;

wherein, PF1, PR1 and P1 are used in a matching way, and PF2, PR2 and P2 are used in a matching way;

a method for detecting circulating tumor cells, which is characterized in that the primers and the probe of claim 1 are used for detecting the circulating tumor cells in an animal experimental model;

a method of detecting circulating tumor cells, comprising:

(1) extracting RNA in an animal blood sample;

(2) reverse transcribing the extracted RNA into cDNA, and taking the cDNA as a template; PF1/PR1 is used as a primer, P1 is used as a probe or PF2/PR2 is used as a primer, and P2 is used as a probe; carrying out real-time fluorescent PCR amplification reaction;

(3) calculating the number of the human tumor cells in the blood sample according to the result of the fluorescent PCR amplification instrument;

a method for evaluating animal model when screening new antigen vaccine or medicine, establishes animal model in screening new antigen vaccine or medicine process, utilizes the above-mentioned primer and probe to detect circulating tumor cell in model animal, evaluates standardization and consistency of animal model;

the animal model comprises a human tumor cell line xenograft animal model and a human tumor tissue source transplantable tumor model;

the new antigen vaccine comprises polypeptide vaccine, nucleic acid vaccine and new antigen reactive cell;

a kit for detecting circulating tumor cells of human origin, comprising: the primer and the probe which are used in a matched way also comprise: RNA reverse transcription reaction solution and RT-PCR reaction solution;

the RNA reverse transcription reaction solution comprises: reverse transcription primer, dNTPs, reverse transcription buffer solution, DTT and MgCl₂RNase inhibitors, reverse transcriptase, RNase H;

preferably, the reverse transcription primer is oligo (dT);

preferably, the rnase inhibitor is a recombinant rnase inhibitor;

the RT-PCR reaction solution comprises: an upstream primer, a downstream primer, a probe, Taqman Universal Master Mix II and DEPC water;

the concentration of the upstream primer and the downstream primer is 400M, and the concentration of the probe is 200 nM;

a method of using a kit for detecting circulating tumor cells of human origin, comprising the steps of:

(1) extracting RNA in a detection sample;

(2) carrying out reverse transcription on the extracted sample RNA to obtain cDNA, and carrying out real-time fluorescence PCR amplification reaction by using the cDNA as a template;

(3) the number of the human tumor cells in the sample is determined according to the result of the fluorescence PCR amplification instrument;

the reaction conditions are as follows: pre-running for 2-15 minutes at 94-98 ℃; running at 94-98 ℃ for 5-20 seconds, running at 58-60 ℃ for 40-80 minutes, and repeating the running for 40-45 cycles;

preferably, the reaction conditions are: pre-running for 10 minutes at 95 ℃; running at 95 ℃ for 15 seconds, running at 60 ℃ for 1 minute, and repeating the running for 40 cycles;

and establishing a standard curve and a standard curve according to the relation between the Ct values of the tumor cells with different numbers and the detection primer. Based on the standard curve, the number of tumor cells in the sample is deduced by real-time quantitative PCR detection of Ct values.

The kit is used for evaluating the standardization and consistency of an animal model used when a new antigen vaccine or a medicament is screened;

the neoantigen vaccine comprises: polypeptide vaccines, nucleic acid vaccines, neoantigen-reactive cells, and the like;

a construction method of a kit for detecting human-derived circulating tumor cells comprises the following steps:

(1) selecting a human-derived efficient and stable expression gene and an animal model genome to perform differential analysis to obtain differential sites;

(2) screening primers and probes of fluorescent quantitative PCR through differential sites;

(3) optimizing a fluorescent quantitative PCR reaction system, and establishing a detection reaction kit;

(4) performing fluorescent quantitative PCR detection analysis on the species model tissue sample by using the kit to detect the effect of the kit;

in the heterogenous tumor cell experiment model, human cells existing in a species model only have artificially inoculated tumor cells, and the human specific gene with high expression efficiency is selected, has the characteristics of high and stable expression quantity, and has stable result and high accuracy by taking the expression quantity as an evaluation standard.

In conclusion, the detection kit can detect the human tumor cells in the animal model, has simple experimental operation and high sensitivity, and the sensitivity can reach the single cell level, thereby providing a method for evaluating the standardization and the consistency of the tumor animal model.

Drawings

FIG. 1: GUSB primer concentration optimization experiment; the abscissa is the cycle number, and the ordinate is the value of delta Rn;

FIG. 2: GUSB probe concentration optimization experiment; the abscissa is the cycle number, and the ordinate is the value of delta Rn;

FIG. 3: HPRT1 primer concentration optimization experiments; the abscissa is the cycle number, and the ordinate is the value of delta Rn;

FIG. 4: HPRT1 probe concentration optimization experiments; the abscissa is the cycle number, and the ordinate is the value of delta Rn;

FIG. 5: amplification curves for different numbers of human tumor cells; the abscissa is the cycle number, and the ordinate is the value of delta Rn; the amplification curve is from left to right, and the cell number is 10⁴、10³、10²、10¹、10⁰And 10^-1；

FIG. 6: ct values for GUSB and HPRT1 versus cell number; a is a correlation curve of the Ct value and the cell number of GUSB, the abscissa is log (cell number), and the ordinate is the Ct value; b is a curve relating the Ct value of HPRT1 and the cell number, the abscissa is log (cell number), and the ordinate is the Ct value;

FIG. 7: imaging a luciferase small animal and quantitatively detecting circulating tumor cells; a is a live imaging diagram of a triple negative breast cancer cell MDA-MB-231 tumor-bearing mouse; the B picture is the real-time quantitative PCR detection result of the HPRT1 gene, the ordinate is the number of circulating tumor cells, the abscissa B1 is the non-metastatic group, and B2 is the lung metastatic group.

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

Designing a primer:

1) selecting a human gene with high and stable expression, and copying a cDNA sequence of GUSB (NM _000181) and HPRT1(NM _000194) reference genes;

2) selecting an amplicon with the length of 70-150bp, a primer with the length of 18-25bp and a Tm value of 59 +/-5 ℃; the length of the probe is 20-27bp, and the Tm value is the candidate primer and the probe with the primer Tm value plus 10 +/-5 ℃;

3) the candidate primers and probes were subjected to sequence-specific alignment to determine candidate primers and probes (table 1).

TABLE 1 primer and Probe sequences

Optimizing the concentration of the primer:

20 μ L of reaction system, 2 XTaqman Universal Master Mix II is 10 μ L; the probe concentration was 250 nM; the template concentration is 100 ng; the upstream and downstream concentrations of GUSB and HPRT1 gene primers (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5) were set as follows: performing an orthogonal experiment at 100nM, 200nM, 400nM and 800nM for primer concentration optimization; according to the amplification curve, when the concentration of the upstream primer and the downstream primer is at least 400nM, the obtained Ct value is minimum and the obtained Delta Rn is maximum. Therefore, the upstream and downstream primer concentrations of 400nM are optimal primer pairs (FIG. 1, FIG. 3).

Optimizing the concentration of the probe:

20 mu L of reaction system, 10 mu L of 2 XTaqman Universal Master Mix II and 400nM final concentration of primers; the template concentration is 100 ng; the concentrations of GUSB and HPRT1 gene probes (SEQ ID NO.3, SEQ ID NO.6) were set to 50nM, 100nM, 150nM, 200nM, and 250nM, and changes in Δ Rn were evaluated at different probe concentrations with constant primer concentration and template amount. As shown in fig. 2 and 4: at 200nM probe concentration, the maximum Δ Rn and minimum Ct values were obtained, which are the optimal probe concentration.

Example 2

Establishing a kit:

a kit for detecting human-derived circulating tumor cells mainly comprises:

1) reverse transcription primer: oligo (dT)₂₀；

2) Specific primers: PF 1: CGGTCTGTACTTCTGTAC (SEQ ID NO.1), PR 1: GACAGAGATCTGGTAATTCA (SEQ ID NO. 2); PF 2: AGCCTAAGATGAGAGTTC (SEQ ID NO.4), PR 2: CACAGAACTAGAACATTGATA (SEQ ID NO. 5);

3) specific probes: p1: CACTGTCTTGCTCCACGCTG (SEQ ID NO.3), the 5 'end of the sequence is modified with FAM fluorophore, and the 3' end is modified with NFQ-MGB; p2: ATCTGGAGTCCTATTGACATCGCC (SEQ ID NO.6), the 5 'end of the sequence is modified with VIC fluorescent group, and the 3' end is modified with NFQ-MGB;

4) reverse transcription reaction solution: comprises RNA reverse transcriptase, 10 times buffer solution, DTT, dNTPs, RNaseOUT recombined RNase Inhibitor and RNase H;

5) qPCR reaction solution: AmpliTaq Gold DNA polymerase, dUTP, dNTPs, ROX fluorescence control, PCR reaction buffer.

Example 3

1) Extraction of RNA, digestion of DNA:

according to the sample properties, using a corresponding RNA extraction kit to extract sample RNA, eliminating genome, taking 1 mu L for nucleic acid quantification, carrying out nucleic acid electrophoresis identification on 5 mu L, and using DEPC water to dilute the extracted RNA to 10-100 ng/mu L for later use.

2) Preparation of cDNA:

respectively preparing reaction liquid from the extracted sample RNA according to the following systems:

3) real-time quantitative RT-PCR reaction:

the GUSB and HPRT1 primers and probes designed above were used to perform real-time quantitative PCR detection on the prepared human tumor cells MDA-MB-231, SK-BR-3, SMMC-7721, Hep G2, HT-29, Caco-2, A549, NCI-H2227 and murine cells RAW264.3, MEL, L7912 cDNA, and the following steps were performed:

mouse cells RAW264.3, MEL, L7912 showed no amplification after real-time quantitative PCR using GUSB, HPRT1 primers, whereas human tumor cells showed a clear amplification curve (FIG. 5). The GUSB and HPRT1 real-time quantitative PCR detection method designed by the application is proved to be capable of distinguishing human tumor cells from murine cells; the detection sensitivity can reach single cell (Table 2).

TABLE 2 GUSB and HRPT1 Gene transcript levels in various numbers of human tumor cells

Example 4: application of circulating tumor cell real-time quantitative RT-PCR method in screening tumor-bearing mice of tumor neoantigen vaccine

1) Establishment of CDX tumor-bearing metastasis mouse model:

6 week old SCID (Severe immunodeficiency mice, purchased from Witonglihua) female mice, 15, were kept for use. Trypsinization of luciferase Gene expressing MDA-MB-231 (purchased from Beijing coordination cell resource center) cells at 5X 10⁶Resuspended in 50. mu.L PBS and subcutaneously inoculated into the abdominal fat pad of SCID mice.

After 10 weeks of tumor cell inoculation, fluorescein Luciferin was intraperitoneally injected in an amount of 150mg/kg body weight, and after 5min, pentobarbital sodium was intraperitoneally injected for anesthesia (50mg/kg), and the fluorescence intensity and metastasis of tumors were observed using a live imaging system.

2) Preparation of total RNA of cells:

blood was collected from the mouse heart at 100. mu.L, and 0.9ml of PBS was added and mixed well. 0.125ml of Dynabeads CD45 was added to the centrifuge tube and mixed with a HulaMixer sample mixer at room temperature for 30 min. A10 ml fresh tube was added to the bottom of the tube 0.5ml of cell separation Ficoll-Paque Plus (from GE Healthcare), the whole blood sample was carefully transferred to the top of the separation liquid surface, and centrifuged at 350g for 5 min. Transferring the upper layer liquid into a 2ml centrifuge tube, centrifuging for 5min at 650g, sucking and removing the supernatant, rapidly adding 1ml TRIzol reagent, splitting at room temperature for 5min, centrifuging for 5min at 12000g, and taking the supernatant. Add 200. mu.L chloroform, vortex for 30 seconds, and let stand at room temperature for 3 minutes; centrifuging at 4 deg.C for 15min at 12000g, collecting upper water phase, adding 500 μ L of ice isopropanol, and mixing; standing at room temperature for 10min, centrifuging at 4 deg.C for 10min at 12000g, and removing supernatant; adding 1ml 75% ethanol, mixing, centrifuging at 4 deg.C 7500g for 5min, and removing supernatant; the precipitated RNA was dried at room temperature for 10 to 15min (incomplete drying), dissolved in RNase-free and DNase-free ultra pure water, and placed in a constant temperature metal bath at 58 ℃ for 15 min. Nucleic acid quantification was performed using NanoDrop 2000 at 1. mu.L.

3) Genome digestion:

preparing reaction solutions from the extracted sample RNA according to the following systems:

4) preparation of cDNA:

the same as in example 3.

5) Real-time quantitative RT-PCR reaction:

the same as in example 3.

After 15 tumor-bearing mice inoculated with triple negative breast cancer cells MDA-MB-231 were observed by a small animal living body imaging system for 10 weeks after the tumor cells were inoculated, 8 mice were found to have obvious lung metastasis, and 7 mice were found to have no obvious lung metastasis (FIG. 7A). After blood of each mouse is extracted, real-time quantitative PCR detection is carried out through HPRT1 gene,

based on the relationship between different numbers of MDA-MB-231 cells and Ct values of GUSB and HPRT1, a standard curve (FIGS. 5 and 6) was established, and the number of circulating tumor cells per microliter of blood was calculated by quantifying the Ct value detected by PCR in real time based on the standard curve, and as a result, as shown in FIG. 7B, the number of circulating tumor cells in the non-metastatic group was significantly different from that in the lung metastatic group. The result shows that the real-time quantitative PCR method based on GUSB and HPRT1 genes can be used for screening and evaluating whether the humanized tumor inoculated mice have metastasis, and the consistency of the mouse condition for evaluating the prediction efficiency of tumor neoantigen prediction software is ensured.

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.

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Claims (8)

1. A primer and probe for detecting human circulating tumor cells, wherein the primer comprises P F1: SEQ ID No.1, PR 1: SEQ ID NO.2, PF 2: SEQ ID NO.4, PR 2: the nucleotide sequence of SEQ ID NO.5, the probe comprises P1: SEQ ID No.3, P2: a nucleotide sequence shown as SEQ ID NO.6, wherein the 5 'end of the probe is provided with a fluorescent group, and the 3' end of the probe is provided with a quenching group;

the PF 1: CGGTCTGTACTTCTGTAC, SEQ ID NO. 1;

the PR 1: GACAGAGATCTGGTAATTCA, SEQ ID NO. 2;

the P1: CACTGTCTTGCTCCACGCTG, SEQ ID NO. 3;

the PF 2: AGCCTAAGATGAGAGTTC, SEQ ID NO. 4;

the PR 2: CACAGAACTAGAACATTGATA, SEQ ID NO. 5;

the P2: ATCTGGAGTCCTATTGACATCGCC, SEQ ID NO. 6.

2. A method for evaluating an animal model in screening a neoantigen vaccine or a drug, characterized in that the animal model is established in the course of screening the neoantigen vaccine or the drug, circulating tumor cells in the model animal are detected using the primer and the probe according to claim 1, and normalization and consistency of the animal model are evaluated.

3. The method for evaluating an animal model in screening a neoantigen vaccine or drug according to claim 2, wherein the animal model includes a human tumor cell line xenograft animal model and a human tumor tissue-derived transplantable tumor model.

4. The method for evaluating an animal model in screening for a neoantigen vaccine or drug according to claim 2, wherein the neoantigen vaccine comprises: polypeptide vaccine, nucleic acid vaccine, and neoantigen-reactive cell.

5. A kit for detecting a circulating tumor cell of human origin, comprising the primer and probe of claim 1, and further comprising: RT-PCR reaction solution and RNA reverse transcription reaction solution.

6. The kit according to claim 5, wherein the concentration of the upstream primer and the downstream primer is 400nM and the concentration of the probe is 200nM, respectively.

7. The kit according to claim 5 or 6, wherein the kit is used for evaluating the standardization and consistency of animal models used in screening for neoantigen vaccines or drugs.

8. The kit according to claim 7, wherein the neoantigen vaccine comprises: polypeptide vaccine, nucleic acid vaccine, and neoantigen-reactive cell.

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