CN106701801B

CN106701801B - Detection markers, kits and applications for B lymphoma and leukemia

Info

Publication number: CN106701801B
Application number: CN201710086750.9A
Authority: CN
Inventors: 党永军; 蒋维; 李增霞; 周晓燕; 刘凯玉; 崔照盟; 谭仁可
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2017-02-17
Filing date: 2017-02-17
Publication date: 2021-07-23
Anticipated expiration: 2037-02-17
Also published as: CN106701801A

Abstract

The invention belongs to the technical field of medicine, in particular to a detection marker for B lymphoma and leukemia, a kit and application thereof. The present invention provides the use of P4HA2 gene or protein and Carabin protein in preparing diagnostic reagents or treating B lymphoma and leukemia. Studies have shown that the P4HA2 gene and protein are hardly expressed or lowly expressed in normal or reactive hyperplastic lymph nodes, but are significantly overexpressed in diffuse large B lymphoma; while the Carabin protein is lowly expressed in diffuse large B lymphoma. In addition, the expression of P4HA2 gene in acute myeloid leukemia blood samples was significantly higher than that of normal people; the proliferation of diffuse large B lymphoma cell lines with knockdown of P4HA2 expression slowed down, and the tumorigenic ability of nude mice was significantly reduced; As a diagnostic marker and therapeutic target for such hematopoietic and lymphoid tumors, Carabin can also be used as a diagnostic marker for such diseases. The present invention also provides corresponding detection methods and kits.

Description

Detection marker and kit for B lymphoma and leukemia and application of detection marker and kit

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a detection marker and a kit for B lymphoma or leukemia, and application of the detection marker and the kit in preparation of antitumor drugs.

Background

Catalyzing proline residues in mammalian cells is prolyl-4-hydroxylase (P4H), producing (2S,4R) -4-hydroxyproline. Prolyl-4-hydroxylase has a wide range of substrates, the most important of which is the catalysis of newly synthesized collagen precursors, critical and essential for their folding into the correct three-dimensional conformation. The substrate mammalian prolyl-4-hydroxylase is an alpha₂β₂The tetramer of (3), wherein the alpha subunit has a substrate binding domain and an enzyme active site. The alpha subunit HAs 3 subtypes, and the subunit type I (prolyl 4-hydrolase, alpha polypeptide I, abbreviated as "P4 HA 1") is the enzyme with the widest expression spectrum. Prolyl-4-hydroxylase type alpha subunit (prolyl 4-hydroxylase, alpha polypeptide II, abbreviated as "P4 HA 2"), consists of 535 amino acids and is expressed in certain specific tissues.P4ha1 ^-/-Knockout mice are embryonic but lethalP4ha1 ^+/-OrP4ha2 ^-/-No significant abnormal phenotype was observed in knockout mice, and knockout was observedP4ha1 ^+/-AndP4ha2 ^-/-the mouse has dysplasia. Recent studies have shown that abnormalities in the P4HA2 gene (rather than P4HA 1) are closely associated with disease, mutations in this gene are associated with non-syndromic high myopia disease, and that one intron region of the P4HA2 gene is among the chinese smokersSusceptible sites of lung cancer. In addition, P4HA1 and P4HA2 have been reported to be highly expressed in breast cancer and to be involved in malignant metastasis of breast cancer by modulating the conformation of collagen. However, no reports of association of P4HA1 and P4HA2 with diffuse large B lymphoma or leukemia have been found.

Carabin is a member of the TBC1 protein family, and previous studies have shown that Carabin is expressed in spleen and peripheral blood lymphocytes and negatively regulates the T cell antigen receptor (T cell antigen receptor TCR) signaling pathway. Further studies found that Carabin is an endogenous inhibitor of the Calcineurin. In addition, it inhibits the Ras signaling pathway by its Ras GAP activity. The regulation of Calcineurin and Ras pathways by Carabin is achieved by direct interaction with Ras and Calcineurin. In addition, Carabin can negatively regulate the BCR signal pathway of B cells through the same mechanism, and inhibition of the expression of Carabin can accelerate the early response of the B cells. More recently, Carabin was found to be associated with myocardial hypertrophy in mice knockout of Carabin, and also prevented myocardial hypertrophy by inhibiting Calcineurin, Ras and Ca2 +/calcium dependent protein kinase 2 signaling pathways. However, the expression of the Carabin protein in B lymphoma and the correlation thereof have not been reported in the literature.

B-cell lymphomas are solid tumors that develop in B-cells and are classified as hodgkin lymphomas and non-hodgkin lymphomas. Among them, diffuse large B lymphoma is the most common non-hodgkin lymphoma, accounts for over 1/3 of adult non-hodgkin lymphoma cases, and has strong invasive ability and high malignancy. Diffuse large B lymphoma is divided into GCB (gerinal centre B-cell), ABC and PMBL types, with 5-year overall survival rate of 76% for GCB type and only 34% for non-GCB type. Currently, the standard treatment regimen for CHOP or R-CHOP is used clinically in patients with B lymphoma, especially diffuse large B lymphoma. Although more than 50% of patients can be cured by this treatment regimen, at least over 1/3 patients are difficult to treat or relapse. Autologous hematopoietic stem cell transplantation will be used for such patients, making them sensitive to standard treatment regimens of chemotherapeutic drugs, but with little success. Therefore, the search for new specific high-expression genes or proteins with B lymphoma diagnosis or combined diagnosis value has important diagnosis and treatment significance.

Leukemias and lymphomas are among the same group of Tumors that affect the blood, bone marrow, and lymphatic systems, and this large group of diseases is known as hematopoietic and lymphoid tissue Tumors (Tumors). In the mortality from malignant tumors, leukemia is in the 6 th (male) and 8 th (female) position, and in children and adults under 35 years of age, in the l th position. The incidence of leukemia has been twice as high over the last 10 years as before. At present, more than 400 million leukemia patients exist in the whole country, 4 million leukemia patients are newly added every year, and half of the leukemia patients account for children. Leukemias can be classified into four major types, Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), and Chronic Myelogenous Leukemia (CML), as well as other less common classes. Clinically, acute leukemia is more common than chronic leukemia in China, acute leukemia must be treated immediately due to the sharp increase and spread of malignant cells, otherwise, patients die within months or even weeks. Among them, adults have the highest AML, while ALL is more common in children. Leukemia is the most common cancer in children. AML is a highly heterogeneous disease, and can be classified into different types according to cellular morphological and histochemical characteristics. At present, the treatment of AML still mainly adopts combined chemotherapy, the total complete remission rate of AML patients is only 50-70%, and the long-term disease-free survival rate is 25-30%. Therefore, the discovery and development of new leukemia markers is of great significance for the accurate typing, diagnosis, prognosis of leukemia, and selection of optimal treatment regimens.

Disclosure of Invention

The invention aims to provide a specific marker for detecting B lymphoma or leukemia, a diagnostic kit and application thereof in preparing anti-tumor drugs.

In particular, the present invention provides the use of a gene, mRNA, cDNA or protein of prolyl-4-hydroxylase type alpha subunit (P4 HA 2) and the use of TBC1 domain family member 10C protein as: (1) detecting a marker for B lymphoma or leukemia; (2) preparing a kit for detecting B lymphoma or leukemia; (3) treating the molecular target of B lymphoma and preparing the related anti-tumor drugs.

The marker provided by the invention can be used as a marker of B lymphoma or leukemia, and is a Gene (SEQ. ID. NO1 (Gene: P4HA2; CDS: 565.. 2172)) of prolyl-4-hydroxylase type alpha subunit (P4 HA 2), an mRNA (SEQ. ID. NO2 (Gene: P4HA2; mRNA: 1.. 2588)), a cDNA or a Protein (SEQ. ID. NO3 (Protein: P4HA2; Protein _ id = "NP-004190.1")), and a Gene (Carabin Gene) (SEQ. ID. NO4), mRNA (SEQ. ID. NO5), cDNA or a Protein (SEQ. ID. NO6)) of a TBC1 domain family member 10C. Research shows that the P4HA2 gene and protein are hardly expressed or low expressed in normal human or reactive hyperplasia lymph nodes, and are significantly highly expressed in diffuse large B lymphoma; while the Carabin protein is less expressed in diffuse large B lymphoma. In addition, the expression level of the P4HA2 gene in blood samples of acute myelogenous leukemia is obviously higher than that of normal people. The proliferation of the diffuse large B lymphoma cell strain for knocking down the expression of P4HA2 is slowed down, and the tumor forming capability of a nude mouse is obviously reduced; the proliferation of the diffuse large B lymphoma cell strain with the reduced Carabin is accelerated, and the tumor forming capability of a nude mouse is obviously improved. Therefore, P4HA2 can be used as a diagnostic marker and a therapeutic target of the hematopoietic and lymphoid tumors, and Carabin can also be used as a diagnostic marker of the diseases.

In a specific example of the present invention, 205 diffuse large B lymphomas and 20 reactive hyperplastic lymph nodes were examined by immunohistochemical methods using P4HA2 protein antibody. The P4HA2 is found to be hardly expressed or expressed at a low level in a tissue sample of a reactive hyperplastic lymph node, but is expressed in a plurality of diffuse large B lymphomas, and the expression of P4HA2 is obviously related to clinical indexes such as the typing and the International Prognostic Index (IPI) of the diffuse large B lymphomas. The self-made antibody is also used for detecting that the Carabin protein is low expressed in a B lymphoma sample and is high expressed in normal human B cells. Thus, P4HA2 and Carabin were considered as markers for B lymphoma. In another embodiment of the invention, 15 samples of acute myelogenous leukemia and 3 samples of peripheral blood mononuclear cells extracted from normal human blood were detected by Real-time fluorescent quantitative PCR (RT-PCR) method using P4HA2 specific primers. P4HA2 was found to be hardly expressed in 3 normal human samples and highly expressed in 12 (80%) acute myeloid leukemia human samples, and therefore P4HA2 was considered as a leukemia marker.

The invention also provides a reagent for detecting B lymphoma or leukemia. The reagent comprises: (a) an antibody against P4HA2; (b) a primer or primer pair for specifically amplifying P4HA2 mRNA or P4HA2 cDNA; (c) an antibody against Carabin.

The invention also provides a kit for detecting B lymphoma or leukemia. Namely, a kit for detecting B lymphoma or leukemia constructed by using the above-mentioned reagent or a composition thereof.

The kit for detecting B lymphoma or leukemia comprises:

a container (1) containing an antibody specific for P4HA2, a corresponding secondary antibody and paraffin sections of human normal or reactive hyperplastic lymph node tissue, together with a label or instructions; the label or the instruction indicates that the kit is used for immunohistochemistry or flow cytometry detection and analysis of the protein content of P4HA2 to judge the probability and prognosis of B lymphoma. Specifically, the P4HA2 antibody is incubated with paraffin sections of human normal or reactive hyperplastic lymph nodes or paraffin sections of abnormal lymph tissue of a patient at a certain concentration, and if the paraffin sections of the patient are stained more than the human normal or reactive hyperplastic lymph nodes, that is, P4HA2 is expressed in the abnormal lymph tissue of the patient, and is hardly expressed in the normal or reactive hyperplastic lymph tissue of the patient according to P4HA2, the patient can be judged to have the possibility of having B lymphoma. Alternatively, the P4HA2 antibody is incubated with PBMC sorted from normal human or patient blood at a certain concentration and combined with corresponding fluorescent secondary antibody, and the content of P4HA2 protein is analyzed by flow cytometry to judge the probability of B lymphoma.

A container (2) containing a pair of fluorescent quantitative PCR specific primers for specific amplification of P4HA2 mRNA or cDNA, together with a label or instructions; the label or the instruction indicates that the kit is used for judging the probability of having leukemia by quantitatively detecting the expression level of P4HA 2. Further, the container (2) contains specific primers for fluorescent quantitative PCR amplification of the P4HA2 gene, the sequences are SEQ.ID.NO7 and SEQ.ID.NO8, and specific primers for fluorescent quantitative PCR amplification of the internal reference gene GAPDH, the sequences are SEQ.ID.NO9 and SEQ.ID.NO10. After the mononuclear cells in the blood of the patient are extracted, whether the P4HA2 is expressed or not is detected by a fluorescence quantitative PCR method. If the expression of P4HA2 gene is detected in the blood mononuclear cells of the patient and is hardly expressed in the blood mononuclear cells of normal human based on P4HA2, the patient is judged to have the possibility of having leukemia.

A container (3) containing an antibody specific for Carabin, together with a label or instructions; the label or the instruction indicates that the kit is used for judging the probability of suffering from B lymphoma by detecting the expression level of Carabin through protein.

The invention also comprises the gene, mRNA, cDNA or protein of P4HA2, and the gene, mRNA, cDNA or protein of Carabin, which are applied to the preparation of medicines for detecting and treating B lymphoma or leukemia.

The research of the invention shows that P4HA2 can be used as a molecular target for treating B lymphoma. In an embodiment of the invention, a large B lymphoma cell strain and a control for stably inhibiting P4HA2 expression are constructed by specifically aiming at short hairpin RNA (shRNA) of P4HA2 gene, and the slow proliferation rate of the large B lymphoma cell strain after the P4HA2 expression is knocked down is detected. In another example, the constructed stable cell line is inoculated subcutaneously in nude mice. After the expression of the P4HA2 gene is inhibited, the tumor forming capability of the nude mice in vivo is greatly reduced. In another example, the constructed stable cell line is inoculated subcutaneously in nude mice. After the expression of the Carabin gene is inhibited, the in-vivo tumorigenic capacity of the nude mice is greatly improved. Therefore, P4HA2 can be used as a molecular target for treating B lymphoma, and the screening of P4HA2 inhibitors HAs positive therapeutic significance for hematopoietic and lymphoid tissue tumors.

Thus, the invention also includes P4HA2 inhibitors as a medicament for the treatment of B lymphoma.

Specific implementations of the invention are further illustrated below:

(1) detecting whether there is an abnormality in P4HA2 protein expression in tissue samples of diffuse large B lymphoma and reactive hyperplastic lymph nodes (fig. 1, 2, examples 1, 2, 3 and 4);

A. preparing a test sample of a subject;

B. detecting the expression quantity of P4HA2 protein in a diffuse large B lymphoma tissue and a reactive hyperplasia lymph node tissue sample to be detected by using a specific anti-P4 HA2 antibody;

C. comparing the expression level of P4HA2 protein in the diffuse large B lymphoma tissue with that in the control reaction hyperplastic lymph node tissue, if the expression level is higher than the control value, the expression level is abnormal for P4HA2 in the detected diffuse large B lymphoma tissue. An expression level of P4HA2 higher than the control value indicates that the subject HAs diffuse large B lymphoma, or HAs a higher probability of developing diffuse large B lymphoma than that of the normal population (fig. 1 (a));

D. the expression level of P4HA2 protein in the diffuse large B lymphoma tissue is subjected to correlation analysis with various clinical indexes, and the high expression of P4HA2 is found to be correlated with the typing and IPI index of the diffuse large B lymphoma, so that the P4HA2 can be used as a marker and a prognostic index of the B lymphoma;

E. the expression level of P4HA2 protein in diffuse large B lymphoma tissue is correlated with the survival time of patients, and the high expression of P4HA2 is correlated with the median survival time of non-GCB type diffuse large B lymphoma patients (figure 1 (B)), so that P4HA2 can be used as a prognostic index of B lymphoma.

(2) Detecting whether the expression of P4HA2 gene is abnormal in acute myelogenous leukemia and normal human blood samples (FIG. 3, example 6);

A. preparing a test sample of a subject;

B. detecting the expression level of P4HA2 gene in blood samples of acute myelogenous leukemia and normal human by using RT-PCR primers of specific P4HA2;

C. comparing the detected expression level of P4HA2 with the control value of normal human, the expression level of P4HA2 higher than the control value indicates that the subject HAs acute myelogenous leukemia or HAs higher probability of acute myelogenous leukemia than the normal human (FIG. 3).

(3) The invention provides that P4HA2 can be used as a molecular target for treating B lymphoma (figure 4, figure 5, examples 8 and 9);

A. screening shRNA which aims at the P4HA2 gene and effectively inhibits the expression of P4HA2;

B. preparing shRNA for inhibiting P4HA2 expression and a control into a lentivirus particle;

C. respectively infecting the B lymphoma cell strains with the lentivirus particles prepared in the step B, and screening GFP expression positive cell strains;

D. detecting the proliferation rate of the cell strain screened from the C, finding that the proliferation rate of the diffuse large B lymphoma cell after the P4HA2 is knocked down is remarkably reduced (figure 4), and taking the P4HA2 as a therapeutic target;

E. the cell strain selected from C is inoculated in nude mice subcutaneously, the volume of transplanted tumor is reduced, the volume growth is slowed down and the weight is reduced after the expression of P4HA2 is inhibited (figure 5), and P4HA2 can be used as a target for treating diffuse large B lymphoma. At the same time, a P4HA2 inhibitor was obtained.

(4) The present invention provides that Carabin can be used as a molecular target for diagnosis of B lymphoma (fig. 6, example 10);

A. preparing a test sample of a subject;

B. detecting the expression quantity of the Carabin protein in blood samples of B lymphoma patients and normal persons by using a specific Carabin antibody;

C. comparing the detection result of the amount of Carabin protein expression with the control value of normal human, the expression amount of Carabin higher than the control value indicates that the probability of the subject suffering from B lymphoma is higher than that of the normal human (FIG. 6).

(5) The present invention provides that Carabin can be used as a molecular target for diagnosis of B lymphoma (fig. 7, example 11);

A. screening shRNA which aims at the Carabin gene and effectively inhibits the expression of Carabin;

B. preparing shRNA for inhibiting the expression of Carabin and a contrast into a lentivirus particle;

D. detecting the proliferation rate of the cell strain screened from C, and finding that the proliferation rate of the dispersed large B lymphoma cell after the Carabin is knocked down is remarkably increased (figure 7);

E. and C, inoculating the cell strain screened out from the C under the skin of a nude mouse, and inhibiting the volume increase and volume increase of the transplanted tumor after the expression of Carabin.

Drawings

FIG. 1, P4HA2 protein is highly expressed in diffuse large B lymphoma. Wherein A, P4HA2 protein is differentially expressed in diffuse large B lymphoma; B. the expression level of P4HA2 is related to median survival of non-GCB diffuse large B lymphoma patients.

FIG. 2, flow cytometry is used for detecting the expression level of P4HA2 protein in different cell lines.

FIG. 3, P4HA2 gene is highly expressed in blood sample of acute myelogenous leukemia.

FIG. 4 shows that the proliferation rate of B lymphoma cell lines inhibiting the expression of P4HA2 is slow. Wherein A, shRNA inhibits P4HA2 protein expression; B. the proliferation rate of B lymphoma cell strain inhibiting the expression of P4HA2 is slow.

FIG. 5 shows that the B lymphoma cell line inhibiting the expression of P4HA2 HAs reduced in vivo tumorigenic ability. Wherein, the in vivo tumor forming capability of the B lymphoma cell strain A and B lymphoma cell strain inhibiting the expression of P4HA2 is compared; B. a nude mouse tumor volume growth curve; C. tumor body weight of nude mice.

FIG. 6, Carabin protein is less expressed in diffuse large B lymphoma.

FIG. 7 shows that the in vivo tumorigenic ability of B lymphoma cell lines inhibiting the expression of Carabin is enhanced. Wherein, the in vivo tumorigenic capacity of the B lymphoma cell strain inhibiting the expression of Carabin is compared; B. a nude mouse tumor volume growth curve; C. tumor body weight of nude mice.

Detailed Description

Example 1 immunohistochemical analysis expression of P4HA2 protein in diffuse large B lymphoma.

Detection material and preparation thereof: 205 diffuse large B lymphoma and 20 reactive hyperplastic lymph node tissue samples were collected and fixed in buffered formalin for 24 hours. After washing with running water for 1 hour, the sample was placed in 30% ethanol and 50% ethanol for 30 minutes each, and finally stored in 70% ethanol at 4 ℃. The fixed sample is dehydrated by gradient ethanol, transparent by xylene, embedded by paraffin at about 52 ℃, and sections with the thickness of 4-10 mu m and the diameter width of 0.1mm are respectively stuck on clean glass slides processed by polylysine in a point arrangement mode to prepare the tissue chip. Baking at 34 deg.C overnight, and storing at 4 deg.C under sealed condition.

The operation method comprises the following steps: taking the prepared tissue chip, firstly carrying out xylene dewaxing and gradient ethanol rehydration, and then adding 0.3% hydrogen peroxide for incubation for 20 minutes at 37 ℃; soaking the slices in citric acid buffer solution (pH 6.0), performing antigen retrieval for 15 min by microwave, and naturally cooling; PBS soak (5 min × 3 times); rabbit anti-human P4HA2 polyclonal antibody (Proteitech, 1:100 diluted in 5% BSA) was added, reacted at 37 ℃ for 1 hour, and incubated at 4 ℃ overnight; PBS soak (5 min × 3 times); adding HRP-labeled goat anti-rabbit ready-to-use secondary antibody (Dako corporation), and reacting at 37 ℃ for 1 hour; PBS soak (5 min × 3 times); DAB substrate (Dako Co.) color development, hematoxylin counterstain, ethanol dehydration, xylene clarity, neutral gum post-coverslipping scan (Leica SCN400 slide scanner). The expression intensity of P4HA2 in 204 diffuse large B lymphoma tissues is judged and scored, and a 0-3 grade system is adopted for judging the dyeing intensity: 0 is no staining (no brown positive signal), 1 is weak staining, 2 is moderate staining, 3 is strong staining.

As a result: as shown in fig. 1A, the left and right panels are representative pictures of P4HA2 expression in reactive hyperplastic tissue and diffuse large B lymphoma tissue, respectively. The figure shows that P4HA2 is hardly or poorly expressed in reactive hyperplastic tissue, but highly expressed in diffuse large B lymphoma tissue with significant difference (P < 0.01).

Example 2 expression intensity of P4HA2 protein is correlated with the typing and prognosis of diffuse large B lymphoma

According to the results of example 1, the correlation of the expression of P4HA2 protein in diffuse large B lymphoma with clinical pathological parameters was further analyzed. As shown in table 1, the expression of P4HA2 was not correlated with age and sex, but was significantly correlated with both the type and prognosis of diffuse large B lymphoma. P4HA2 expression was higher in patients with more malignant non-GCB type diffuse large B lymphoma. IPIs are scored by 5 prognostic-related pathological indicators, with higher scores representing poorer prognosis. The higher the expression of P4HA2, the higher the IPI score, and the worse the prognosis of diffuse large B lymphoma.

TABLE 1 correlation analysis of P4HA2 expression intensity with clinical pathology index

。

Example 3 expression intensity of P4HA2 protein and median survival analysis in patients with diffuse large B lymphoma

Based on the results of example 1, we also analyzed the association of P4HA2 with median survival in patients with diffuse large B lymphoma. The stronger the expression of P4HA2 was found to be, the shorter median survival in patients with non-GCB diffuse large B lymphoma. This shows that P4HA2 is closely related to the development of diffuse large B lymphoma, and can be used as a diagnostic or therapeutic prognostic marker for diffuse large B lymphoma.

Example 4 flow cytometry detection of P4HA2 protein expression

Detection material and preparation thereof:

A. p4HA2 positive cells 293T, and P4HA2 negative cells Jurkat were obtained. PBS was washed twice, each at 1000rpm for 5 minutes. Washing off the culture medium from the cells;

B. fixing liquid: putting methanol at-20 deg.C overnight in advance;

C. PBA 2% BSA in PBS was prepared.

The operation method comprises the following steps:

A. suspending 293T, Jurkat cells in 100 μ l PBS buffer, adding 1ml methanol pre-cooled at-20 deg.C dropwise (shaking while adding), standing at-20 deg.C for 10 min;

B. centrifugation was carried out at 1000rpm for 5 minutes, the supernatant was carefully removed, the cells were resuspended in 1ml of PBA solution and divided into two portions (the cells were gently blown in this and subsequent steps), and the supernatant was centrifuged at 1000rpm for 5 minutes;

C. IgG, P4HA2 antibody were diluted to 2. mu.g/ml, 200. mu.l each, with PBS, respectively;

D. the 293T, Jurkat two cells were resuspended with IgG, P4HA2 antibody, respectively, and numbered: 293T-IgG, 293T-P4HA2, Jurkat-IgG, Jurkat-P4HA 2. Standing at 4 ℃ for 2 hours;

E. cells were washed once with PBA (1000 rpm, 5 min);

F. adding a fluorescence labeled secondary antibody (Jackson) at the ratio of 1:1000, and standing for 30min at room temperature;

G. PBA cells were washed twice (1000 rpm, 5 min) and resuspended in 100. mu.l PBS;

H. detection was performed by flow cytometry. (BD Accuri C6).

As a result: as shown in FIG. 2, the P4HA2 test result was negative in Jurkat cells not expressing P4HA2, while the P4HA2 test result was positive in 293T cells expressing P4HA2, indicating that the specificity of the P4HA2 antibody used for the test was better. The method can be used for detecting the protein content of P4HA2 in peripheral blood mononuclear cells separated from blood of patients with B lymphoma or leukemia, and judging the probability of the patients suffering from hematopoietic and lymphoid tissue tumor diseases including acute myelogenous leukemia.

Example 5 kit for detecting diffuse Large B lymphoma

Preparing a kit for detecting diffuse large B lymphoma, the kit comprising: A. a label or instructions indicating that the kit is for use in the detection or diagnosis of diffuse large B lymphoma; B. an antibody specific for P4HA2 (available from Proteintech).

201 cases of diffuse large B lymphoma and 20 cases of reactive hyperplasia lymph node tissue samples are detected by an immunohistochemical method by using the detection kit. The result shows that the staining intensity of 20 reactive hyperplastic lymph node tissue samples is 0 or 1, and basically no positive signals exist; when the positive threshold value is more than 1, 120 diffuse large B lymphoma samples are evaluated as P4HA2 positive, and the positive rate is about 59.7%.

Example 6 fluorescent quantitative RT-PCR method for detecting the expression of P4HA2 mRNA in blood samples of acute myeloid leukemia

Detection material and preparation thereof:

A. peripheral Blood Mononuclear Cells (PBMCs) were obtained. Blood samples of 12 acute myeloid leukemia and 3 normal persons, each with 3ml, were collected. PBMCs were separated using Ficoll lymphocyte isolate (GE) to separate PBMCs in a Ficoll ratio of blood 1: 2, slowly adding the blood into the Ficoll liquid, standing for 15 minutes at room temperature, and centrifuging for 20 minutes at 4000 g; taking the interface cells after centrifugation, resuspending the interface cells with Phosphate Buffered Saline (PBS) and centrifuging the interface cells for 5 minutes at 1500 g; removing supernatant, adding erythrocyte lysate (Shanghai Song Van bioscience) for treating for 3 min, adding PBS for resuspension, and centrifuging at 1500g for 5min to obtain PBMC in blood sample;

B. cDNA templates for PBMCs were prepared. Adding appropriate amount of Trizol (Invitrogen company) into PBMC, extracting total RNA of PBMC according to the method recommended by the instruction, and reverse transcribing (Takara company) to prepare cDNA template;

C. the mRNA abundance of P4HA2 was measured. Designing fluorescent quantitative PCR primers for synthesizing P4HA2 and an internal reference gene GAPDH;

p4HA2 upstream primer sequence: 5'-CGGTGTTGTGGATGGAGCAGGTGC-3' (SEQ. ID. NO7)

P4HA2 downstream primer sequence: 5'-CTGCTCCATCCACAACACCGTATG-3' (SEQ. ID. NO8)

GAPDH upstream primer sequence: 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ. ID. NO9)

GAPDH downstream primer sequence: 5'-GAAGATGGTGATGGGATTTC-3' (SEQ. ID. NO10).

The operation method comprises the following steps:

the PCR reaction system was configured in the following volumes:

the PCR reaction was carried out as follows:

98℃：1min

95℃：10s

60 ℃ below zero: 10s40 cycles

72℃：20s

72℃：2min。

As a result: as shown in FIG. 2, P4HA2 was expressed little or very low in red in 3 normal human blood PBMC samples and was detected in 15 acute myelogenous leukemia samples among the 15 blood PBMC samples tested. Thus, P4HA2 mRNA was significantly highly expressed in acute myelogenous leukemia humans compared to normal humans (P < 0.01).

Example 7 preparation of P4HA2 mRNA detection kit

As described in example 5, P4HA2 mRNA was not expressed in normal human blood PBMC, but was detected in an acute myelogenous leukemia blood PBMC sample, from which a P4HA2 mRNA assay kit was prepared.

The kit comprises: A. a label or instructions for use of the kit for determining the probability of developing leukemia by quantitatively detecting the expression level of P4HA2; B. upstream and downstream primers for fluorescent quantitative PCR detection of P4HA2, e.g., seq.id.no5 and seq.id.no 6; C. upstream and downstream primers for fluorescent quantitative PCR detection of internal reference gene GAPDH, such as SEQ.ID.NO7 and SEQ.ID.NO8.

Samples were prepared and tested as described in example 6, and the results of the experiments were analyzed, and if the ratio of the amount of P4HA2 mRNA in the test subject to the amount of P4HA2 mRNA in the general population was 1.5 or more (preferably 2.0 or more, more preferably 2.5 or more), the subject had a greater chance of developing hematopoietic and lymphoid tissue tumor diseases including acute myelogenous leukemia than in the normal population.

Example 8 inhibition of P4HA2 expression significantly slowed the proliferation rate of diffuse large B lymphoma cells

Detection material and preparation thereof:

A. a P4HA2 knockdown lentiviral vector was constructed. A psicor shRNA effective in knocking down P4HA2 was designed and validated with the following sequence: shP1: GCGGTACTTTGAGCAGTTA (SEQ. ID. NO11),

shP2:GCAGGTGCTAAAGCAGCTT（SEQ.ID.NO12）；

B. preparing lentivirus particles with the knockdown P4HA2, and infecting a diffuse large B lymphoma cell strain. Constructing shRNA of the knocked-down P4HA2 into a lentiviral vector pLVX-shRNA-GFP, then co-transfecting 293T cells with pMD2.G and psPAX2, harvesting supernatant after 48 hours, and filtering with a 0.45 mu M filter membrane. Infecting the filtered supernatant with virus particles with a diffuse large B lymphoma cell strain SU-DHL-4, and observing GFP green fluorescence expression after 72 hours;

C. positive cells that stably knocked down P4HA2 were sorted. As the expression of the shRNA lentiviral plasmid with the knockdown P4HA2 emits GFP green fluorescence, the shRNA lentiviral plasmid serves as a flow cytometry screening marker. After a period of incubation of infected SU-DHL-4, GFP expressing positive cells were screened by flow cytometry and the knockdown efficiency of P4HA2 was verified by Western Blot (FIG. 4 (A)).

The operation method comprises the following steps: and (3) inoculating the selected SU-DHL-4 cells with the knocked-down P4HA2 gene into a 96-well plate, wherein the density is 6000 cells/well, detecting the relative number of the cells by using an Alamar blue method respectively on

days

1, 2, 3, 4 and 5 after inoculation, and drawing a cell proliferation curve.

As a result: as shown in fig. 4(B), the proliferation rate of diffuse large B lymphoma cells was reduced after P4HA2 knockdown, with significant differences in the results.

Example 9 inhibition of P4HA2 expression results in a substantial decrease in the tumorigenic capacity of nude mice

Detection material and preparation thereof: nude mice were inoculated subcutaneously with cells. Female nude mice (slyke) of 4 weeks of age were selected and raised in advance for one week for acclimatization. Mixing 5 x 10⁶The individual cells were resuspended in 100. mu.l of PBS solution, mixed with 100. mu.l of Matrigel (BD Co.), and inoculated subcutaneously into nude mice.

The operation method comprises the following steps: after observing the growth of tumor at the inoculated cells, the tumor volume and the weight of the nude mice were measured every other day. When the nude mice grow 21 days with tumor, the tumor volume does not exceed 2cm³Nude mice were sacrificed by Shi-An. Stripping off tumor, weighing, and taking a picture for recording.

As a result: as shown in FIG. 5, after the expression of the knockdown P4HA2, the tumor volume is obviously reduced compared with the control group, the tumor volume is obviously increased and is obviously reduced, and the tumor weight is also lighter. It can be seen that P4HA2 is closely related to tumor development and development, and can be used as a therapeutic target for diffuse large B lymphoma.

Example 10 Western blot analysis of the expression of Carabin protein in diffuse Large B lymphoma

Detection material and preparation thereof: the B cells from 9 diffuse large B lymphoma tissue samples and 3 normal human PBMCs were collected, lysed, mixed with SDS loading buffer and treated in boiling water bath (980C) for 8 minutes.

The operation method comprises the following steps: and sequentially adding the prepared protein samples into sample adding holes of SDS-PAGE gel by using a micro sample loading needle, carrying out electrophoresis at a constant voltage of 80V for about 30min, adjusting the electrophoresis to a constant voltage of 110V after the sample marked by the bromophenol blue enters the separation gel, and continuing the electrophoresis until the bromophenol blue completely leaves the separation gel and enters the electrophoresis solution, thus finishing the electrophoresis. Proteins were transferred from SDS-PAGE gels onto nitrocellulose membranes (NC membranes) using a Bio-Rad wet transfer system, and transferred at a constant flow of 220mA for 1.5hr in an ice water bath. Placing the NC membrane printed with the western blot in a blocking buffer, placing the NC membrane in a shaking table, and incubating for 45min at room temperature. Diluting with antibody diluent, adding to NC membrane, placing in shaker, and incubating at room temperature for 2hr or 4 deg.C overnight. The membrane was washed 3 times with 1 XTSST elution buffer for 10min each time. The secondary antibody was diluted with the antibody diluent according to the recommended ratio, applied to the NC membrane, placed on a shaker, and incubated at room temperature for 1 hr. The membrane was washed 3 times with 1 XTSST elution buffer for 10min each time. And uniformly mixing the ECL color developing A/B solution according to the ratio of 1:1, uniformly adding the mixture on an NC film, reacting for 1min, sucking, tabletting in a dark room, developing and fixing. And scanning and storing the X-ray film after the X-ray film is dried.  

As a result: as shown in FIG. 6, Carabin was highly expressed in B cells sorted from normal human PBMCs, but was less expressed in diffuse large B lymphoma tissue samples.

Example 11 inhibition of Carabin expression greatly improves tumorigenic capacity in nude mice

Detection material and preparation thereof: b lymphoma cell lines stably knockdown for Carabin (knockdown sequence shC, GGCGGTACAAGAAGGTAAA, (seq. id. no 13)) were constructed as described in example 8, and nude mice were inoculated subcutaneously with cells. Female nude mice (slyke) of 4 weeks of age were selected and raised in advance for one week for acclimatization. Mixing 5 x 10⁶The individual cells were resuspended in 100. mu.l of PBS solution, mixed with 100. mu.l of Matrigel (BD Co.), and inoculated subcutaneously into nude mice.

As a result: as shown in FIG. 7, after the expression of Carabin is knocked down, the tumor body is obviously increased relative to the control group, the increase of the tumor body volume is obviously accelerated, and the tumor body weight is increased. Carabin can be seen to be closely related to the occurrence and development of B lymphoma.

SEQUENCE LISTING

<110> university of Compound Dan

<120> B lymphoma and leukemia detection marker, kit and application thereof

<130> 001

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 1608

<212> DNA

<213>

<400> 1

atgaaactct gggtgtctgc attgctgatg gcctggtttg gtgtcctgag ctgtgtgcag 60

gccgaattct tcacctctat tgggcacatg actgacctga tttatgcaga gaaagagctg 120

gtgcagtctc tgaaagagta catccttgtg gaggaagcca agctttccaa gattaagagc 180

tgggccaaca aaatggaagc cttgactagc aagtcagctg ctgatgctga gggctacctg 240

gctcaccctg tgaatgccta caaactggtg aagcggctaa acacagactg gcctgcgctg 300

gaggaccttg tcctgcagga ctcagctgca ggttttatcg ccaacctctc tgtgcagcgg 360

cagttcttcc ccactgatga ggacgagata ggagctgcca aagccctgat gagacttcag 420

gacacataca ggctggaccc aggcacaatt tccagagggg aacttccagg aaccaagtac 480

caggcaatgc tgagtgtgga tgactgcttt gggatgggcc gctcggccta caatgaaggg 540

gactattatc atacggtgtt gtggatggag caggtgctaa agcagcttga tgccggggag 600

gaggccacca caaccaagtc acaggtgctg gactacctca gctatgctgt cttccagttg 660

ggtgatctgc accgtgccct ggagctcacc cgccgcctgc tctcccttga cccaagccac 720

gaacgagctg gagggaatct gcggtacttt gagcagttat tggaggaaga gagagaaaaa 780

acgttaacaa atcagacaga agctgagcta gcaaccccag aaggcatcta tgagaggcct 840

gtggactacc tgcctgagag ggatgtttac gagagcctct gtcgtgggga gggtgtcaaa 900

ctgacacccc gtagacagaa gaggcttttc tgtaggtacc accatggcaa cagggcccca 960

cagctgctca ttgccccctt caaagaggag gacgagtggg acagcccgca catcgtcagg 1020

tactacgatg tcatgtctga tgaggaaatc gagaggatca aggagatcgc aaaacctaaa 1080

cttgcacgag ccaccgttcg tgatcccaag acaggagtcc tcactgtcgc cagctaccgg 1140

gtttccaaaa gctcctggct agaggaagat gatgaccctg ttgtggcccg agtaaatcgt 1200

cggatgcagc atatcacagg gttaacagta aagactgcag aattgttaca ggttgcaaat 1260

tatggagtgg gaggacagta tgaaccgcac ttcgacttct ctaggaatga tgagcgagat 1320

actttcaagc atttagggac ggggaatcgt gtggctactt tcttaaacta catgagtgat 1380

gtagaagctg gtggtgccac cgtcttccct gatctggggg ctgcaatttg gcctaagaag 1440

ggtacagctg tgttctggta caacctcttg cggagcgggg aaggtgacta ccgaacaaga 1500

catgctgcct gccctgtgct tgtgggctgc aagtgggtct ccaataagtg gttccatgaa 1560

cgaggacagg agttcttgag accttgtgga tcaacagaag ttgactga 1608

<210> 2

<211> 2588

<212> DNA

<213>

<400> 2

agcgttgttt ttccttggca gctgcggaga cccgtgataa ttcgttaact aattcaacaa 60

acgggaccct tctgtgtgcc agaaaccgca agcagttgct aacccagtgg gacaggcgga 120

ttggaagagc gggaaggtcc tggcccagag cagtgtggtg agcgctgtgc tggaagggaa 180

tgcgggcagt gggtacttgg tagagcactg actgcctccg gccagaggac ttcccggagg 240

aggtgaccca tgagctggag tggtcagagg aaggctggca aaagggcatc gtggacagag 300

gaacagccta tgtgagtggg agcagagacc ttggccaatg ccattcctta tggccttgta 360

gtggaagcaa ggtgatgggg aaggaacact gtaggggata gctgtccacg gacgctgtct 420

acaagaccct ggagtgagat aacgtgcctg gtactgtgcc ctgcatgtgt aagatgccca 480

gttgaccttc gcagcaggag cctggatcag ggcacttcct gcctcaggta ttgctggaca 540

gcccagacac ttccctctgt gaccatgaaa ctctgggtgt ctgcattgct gatggcctgg 600

tttggtgtcc tgagctgtgt gcaggccgaa ttcttcacct ctattgggca catgactgac 660

ctgatttatg cagagaaaga gctggtgcag tctctgaaag agtacatcct tgtggaggaa 720

gccaagcttt ccaagattaa gagctgggcc aacaaaatgg aagccttgac tagcaagtca 780

gctgctgatg ctgagggcta cctggctcac cctgtgaatg cctacaaact ggtgaagcgg 840

ctaaacacag actggcctgc gctggaggac cttgtcctgc aggactcagc tgcaggtttt 900

atcgccaacc tctctgtgca gcggcagttc ttccccactg atgaggacga gataggagct 960

gccaaagccc tgatgagact tcaggacaca tacaggctgg acccaggcac aatttccaga 1020

ggggaacttc caggaaccaa gtaccaggca atgctgagtg tggatgactg ctttgggatg 1080

ggccgctcgg cctacaatga aggggactat tatcatacgg tgttgtggat ggagcaggtg 1140

ctaaagcagc ttgatgccgg ggaggaggcc accacaacca agtcacaggt gctggactac 1200

ctcagctatg ctgtcttcca gttgggtgat ctgcaccgtg ccctggagct cacccgccgc 1260

ctgctctccc ttgacccaag ccacgaacga gctggaggga atctgcggta ctttgagcag 1320

ttattggagg aagagagaga aaaaacgtta acaaatcaga cagaagctga gctagcaacc 1380

ccagaaggca tctatgagag gcctgtggac tacctgcctg agagggatgt ttacgagagc 1440

ctctgtcgtg gggagggtgt caaactgaca ccccgtagac agaagaggct tttctgtagg 1500

taccaccatg gcaacagggc cccacagctg ctcattgccc ccttcaaaga ggaggacgag 1560

tgggacagcc cgcacatcgt caggtactac gatgtcatgt ctgatgagga aatcgagagg 1620

atcaaggaga tcgcaaaacc taaacttgca cgagccaccg ttcgtgatcc caagacagga 1680

gtcctcactg tcgccagcta ccgggtttcc aaaagctcct ggctagagga agatgatgac 1740

cctgttgtgg cccgagtaaa tcgtcggatg cagcatatca cagggttaac agtaaagact 1800

gcagaattgt tacaggttgc aaattatgga gtgggaggac agtatgaacc gcacttcgac 1860

ttctctagga atgatgagcg agatactttc aagcatttag ggacggggaa tcgtgtggct 1920

actttcttaa actacatgag tgatgtagaa gctggtggtg ccaccgtctt ccctgatctg 1980

ggggctgcaa tttggcctaa gaagggtaca gctgtgttct ggtacaacct cttgcggagc 2040

ggggaaggtg actaccgaac aagacatgct gcctgccctg tgcttgtggg ctgcaagtgg 2100

gtctccaata agtggttcca tgaacgagga caggagttct tgagaccttg tggatcaaca 2160

gaagttgact gacatccttt tctgtccttc cccttcctgg tccttcagcc catgtcaacg 2220

tgacagacac ctttgtatgt tcctttgtat gttcctatca ggctgatttt tggagaaatg 2280

aatgtttgtc tggagcagag ggagaccata ctagggcgac tcctgtgtga ctgaagtccc 2340

agcccttcca ttcagcctgt gccatccctg gccccaaggc taggatcaaa gtggctgcag 2400

cagagttagc tgtctagcgc ctagcaaggt gcctttgtac ctcaggtgtt ttaggtgtga 2460

gatgtttcag tgaaccaaag ttctgatacc ttgtttacat gtttgttttt atggcatttc 2520

tatctattgt ggctttacca aaaaataaaa tgtccctacc agaagcctta aaaaaaaaaa 2580

aaaaaaaa 2588

<210> 3

<211> 535

<212> PRT

<213>

<400> 3

Met Lys Leu Trp Val Ser Ala Leu Leu Met Ala Trp Phe Gly Val Leu

1 5 10 15

Ser Cys Val Gln Ala Glu Phe Phe Thr Ser Ile Gly His Met Thr Asp

20 25 30

Leu Ile Tyr Ala Glu Lys Glu Leu Val Gln Ser Leu Lys Glu Tyr Ile

35 40 45

Leu Val Glu Glu Ala Lys Leu Ser Lys Ile Lys Ser Trp Ala Asn Lys

50 55 60

Met Glu Ala Leu Thr Ser Lys Ser Ala Ala Asp Ala Glu Gly Tyr Leu

65 70 75 80

Ala His Pro Val Asn Ala Tyr Lys Leu Val Lys Arg Leu Asn Thr Asp

85 90 95

Trp Pro Ala Leu Glu Asp Leu Val Leu Gln Asp Ser Ala Ala Gly Phe

100 105 110

Ile Ala Asn Leu Ser Val Gln Arg Gln Phe Phe Pro Thr Asp Glu Asp

115 120 125

Glu Ile Gly Ala Ala Lys Ala Leu Met Arg Leu Gln Asp Thr Tyr Arg

130 135 140

Leu Asp Pro Gly Thr Ile Ser Arg Gly Glu Leu Pro Gly Thr Lys Tyr

145 150 155 160

Gln Ala Met Leu Ser Val Asp Asp Cys Phe Gly Met Gly Arg Ser Ala

165 170 175

Tyr Asn Glu Gly Asp Tyr Tyr His Thr Val Leu Trp Met Glu Gln Val

180 185 190

Leu Lys Gln Leu Asp Ala Gly Glu Glu Ala Thr Thr Thr Lys Ser Gln

195 200 205

Val Leu Asp Tyr Leu Ser Tyr Ala Val Phe Gln Leu Gly Asp Leu His

210 215 220

Arg Ala Leu Glu Leu Thr Arg Arg Leu Leu Ser Leu Asp Pro Ser His

225 230 235 240

Glu Arg Ala Gly Gly Asn Leu Arg Tyr Phe Glu Gln Leu Leu Glu Glu

245 250 255

Glu Arg Glu Lys Thr Leu Thr Asn Gln Thr Glu Ala Glu Leu Ala Thr

260 265 270

Pro Glu Gly Ile Tyr Glu Arg Pro Val Asp Tyr Leu Pro Glu Arg Asp

275 280 285

Val Tyr Glu Ser Leu Cys Arg Gly Glu Gly Val Lys Leu Thr Pro Arg

290 295 300

Arg Gln Lys Arg Leu Phe Cys Arg Tyr His His Gly Asn Arg Ala Pro

305 310 315 320

Gln Leu Leu Ile Ala Pro Phe Lys Glu Glu Asp Glu Trp Asp Ser Pro

325 330 335

His Ile Val Arg Tyr Tyr Asp Val Met Ser Asp Glu Glu Ile Glu Arg

340 345 350

Ile Lys Glu Ile Ala Lys Pro Lys Leu Ala Arg Ala Thr Val Arg Asp

355 360 365

Pro Lys Thr Gly Val Leu Thr Val Ala Ser Tyr Arg Val Ser Lys Ser

370 375 380

Ser Trp Leu Glu Glu Asp Asp Asp Pro Val Val Ala Arg Val Asn Arg

385 390 395 400

Arg Met Gln His Ile Thr Gly Leu Thr Val Lys Thr Ala Glu Leu Leu

405 410 415

Gln Val Ala Asn Tyr Gly Val Gly Gly Gln Tyr Glu Pro His Phe Asp

420 425 430

Phe Ser Arg Asn Asp Glu Arg Asp Thr Phe Lys His Leu Gly Thr Gly

435 440 445

Asn Arg Val Ala Thr Phe Leu Asn Tyr Met Ser Asp Val Glu Ala Gly

450 455 460

Gly Ala Thr Val Phe Pro Asp Leu Gly Ala Ala Ile Trp Pro Lys Lys

465 470 475 480

Gly Thr Ala Val Phe Trp Tyr Asn Leu Leu Arg Ser Gly Glu Gly Asp

485 490 495

Tyr Arg Thr Arg His Ala Ala Cys Pro Val Leu Val Gly Cys Lys Trp

500 505 510

Val Ser Asn Lys Trp Phe His Glu Arg Gly Gln Glu Phe Leu Arg Pro

515 520 525

Cys Gly Ser Thr Glu Val Asp

530 535

<210> 4

<211> 1341

<212> DNA

<213>

<400> 4

atggcccagg ccctggggga ggacctggtg cagcctcccg agctgcagga tgactccagc 60

tccttggggt ccgactcaga gctcagcggg cctggcccat atcgccaggc cgaccgctat 120

ggattcattg ggggcagctc agcagagcca gggccgggcc acccacctgc agacctcatc 180

cgccaacggg agatgaagtg ggtggagatg acctcgcact gggagaaaac catgtcccgg 240

cggtacaaga aggtaaagat gcagtgccgg aaaggcatcc cgtctgccct gcgcgcccga 300

tgctggcccc tgttgtgtgg ggcccatgtg tgccagaaga acagccctgg cacctatcag 360

gagctggcag aggcccctgg agacccacag tggatggaga ccattggcag ggacctgcac 420

cgtcaattcc ctctgcacga gatgtttgtg tcgcctcagg gccacgggca gcaggggctc 480

ctgcaggtgc tcaaggccta caccctgtat cgaccggagc agggctactg ccaggcccag 540

gggcccgtgg ctgctgtgct gctcatgcac ctgcccccag aggaggcctt ctggtgcctg 600

gtgcagatct gtgaggtcta cctccctggg tactacgggc cccacatgga ggctgtgcgg 660

ctggacgccg aggtgttcat ggccctgctg cggcggctgc ttccgcacgt gcacaagcac 720

ctgcagcagg tgggcgtcgg acccctgctg tacctgcccg agtggttcct gtgcctcttc 780

gcccgctccc tgcccttccc cacagtgctg cgtgtctggg atgccttcct cagtgagggt 840

gccagagtac tgttccgtgt ggggctgaca ctggtgcgcc tggcgctggg cactgcagag 900

cagcgagggg cctgccctgg cctcctggag acactgggag cccttcgagc catccccccc 960

gcgcagctgc aggaggaggc cttcatgtca caggtgcaca gcgtggtgct gtcagagcgg 1020

gacctgcagc gggagatcaa ggcccagctg gcccagctgc ccgattccgc gccgggaccc 1080

ccgccccggc cacaggtccg cctcgccggg gcccaagcca tctttgaggc ccagcagctg 1140

gcaggagtgc gacgaggcgc caagcctgag gtgcctcgga ttgtggtgca gcccccggag 1200

gagcccagac caccgcggcg gaaaccccag acccgcggca agactttcca tgggctcctg 1260

actcgggccc ggggcccccc catcgagggg ccccccaggc cccaacgagg ctccacctcc 1320

ttcctggaca cccgcttctg a 1341

<210> 5

<211> 1752

<212> DNA

<213>

<400> 5

gaggaggagg aggaagtgag aggaggaggt gaggtgctgc gggaggcccc gggcaccatg 60

gcccaggccc tgggggagga cctggtgcag cctcccgagc tgcaggatga ctccagctcc 120

ttggggtccg actcagagct cagcgggcct ggcccatatc gccaggccga ccgctatgga 180

ttcattgggg gcagctcagc agagccaggg ccgggccacc cacctgcaga cctcatccgc 240

caacgggaga tgaagtgggt ggagatgacc tcgcactggg agaaaaccat gtcccggcgg 300

tacaagaagg taaagatgca gtgccggaaa ggcatcccgt ctgccctgcg cgcccgatgc 360

tggcccctgt tgtgtggggc ccatgtgtgc cagaagaaca gccctggcac ctatcaggag 420

ctggcagagg cccctggaga cccacagtgg atggagacca ttggcaggga cctgcaccgt 480

caattccctc tgcacgagat gtttgtgtcg cctcagggcc acgggcagca ggggctcctg 540

caggtgctca aggcctacac cctgtatcga ccggagcagg gctactgcca ggcccagggg 600

cccgtggctg ctgtgctgct catgcacctg cccccagagg aggccttctg gtgcctggtg 660

cagatctgtg aggtctacct ccctgggtac tacgggcccc acatggaggc tgtgcggctg 720

gacgccgagg tgttcatggc cctgctgcgg cggctgcttc cgcacgtgca caagcacctg 780

cagcaggtgg gcgtcggacc cctgctgtac ctgcccgagt ggttcctgtg cctcttcgcc 840

cgctccctgc ccttccccac agtgctgcgt gtctgggatg ccttcctcag tgagggtgcc 900

agagtactgt tccgtgtggg gctgacactg gtgcgcctgg cgctgggcac tgcagagcag 960

cgaggggcct gccctggcct cctggagaca ctgggagccc ttcgagccat cccccccgcg 1020

cagctgcagg aggaggcctt catgtcacag gtgcacagcg tggtgctgtc agagcgggac 1080

ctgcagcggg agatcaaggc ccagctggcc cagctgcccg attccgcgcc gggacccccg 1140

ccccggccac aggtccgcct cgccggggcc caagccatct ttgaggccca gcagctggca 1200

ggagtgcgac gaggcgccaa gcctgaggtg cctcggattg tggtgcagcc cccggaggag 1260

cccagaccac cgcggcggaa accccagacc cgcggcaaga ctttccatgg gctcctgact 1320

cgggcccggg gcccccccat cgaggggccc cccaggcccc aacgaggctc cacctccttc 1380

ctggacaccc gcttctgaga ggaccatgga cttagtgtcc cccagtctca attgcctgat 1440

ggctgatgcc agcccggcaa ataggcaccg cactttactc ttgggactcg gggacttggc 1500

ttccttcctg gcaaggacca ggcagtgggg aaggaggagg tcctccgtgg tacatactgg 1560

gtcaggcact agcatggagg agggtcacag agtggggcac gtgaggaccc atggaaccgt 1620

cctggtgccc aggccctcac aagtaccaaa gccagcacca aaggagtcag ggaaggggtt 1680

ggctgagtca agggacccca gagggcacca ggaataaaat cttcttgaac agataaaaaa 1740

aaaaaaaaaa aa 1752

<210> 6

<211> 446

<212> PRT

<213>

<400> 6

Met Ala Gln Ala Leu Gly Glu Asp Leu Val Gln Pro Pro Glu Leu Gln

1 5 10 15

Asp Asp Ser Ser Ser Leu Gly Ser Asp Ser Glu Leu Ser Gly Pro Gly

20 25 30

Pro Tyr Arg Gln Ala Asp Arg Tyr Gly Phe Ile Gly Gly Ser Ser Ala

35 40 45

Glu Pro Gly Pro Gly His Pro Pro Ala Asp Leu Ile Arg Gln Arg Glu

50 55 60

Met Lys Trp Val Glu Met Thr Ser His Trp Glu Lys Thr Met Ser Arg

65 70 75 80

Arg Tyr Lys Lys Val Lys Met Gln Cys Arg Lys Gly Ile Pro Ser Ala

85 90 95

Leu Arg Ala Arg Cys Trp Pro Leu Leu Cys Gly Ala His Val Cys Gln

100 105 110

Lys Asn Ser Pro Gly Thr Tyr Gln Glu Leu Ala Glu Ala Pro Gly Asp

115 120 125

Pro Gln Trp Met Glu Thr Ile Gly Arg Asp Leu His Arg Gln Phe Pro

130 135 140

Leu His Glu Met Phe Val Ser Pro Gln Gly His Gly Gln Gln Gly Leu

145 150 155 160

Leu Gln Val Leu Lys Ala Tyr Thr Leu Tyr Arg Pro Glu Gln Gly Tyr

165 170 175

Cys Gln Ala Gln Gly Pro Val Ala Ala Val Leu Leu Met His Leu Pro

180 185 190

Pro Glu Glu Ala Phe Trp Cys Leu Val Gln Ile Cys Glu Val Tyr Leu

195 200 205

Pro Gly Tyr Tyr Gly Pro His Met Glu Ala Val Arg Leu Asp Ala Glu

210 215 220

Val Phe Met Ala Leu Leu Arg Arg Leu Leu Pro His Val His Lys His

225 230 235 240

Leu Gln Gln Val Gly Val Gly Pro Leu Leu Tyr Leu Pro Glu Trp Phe

245 250 255

Leu Cys Leu Phe Ala Arg Ser Leu Pro Phe Pro Thr Val Leu Arg Val

260 265 270

Trp Asp Ala Phe Leu Ser Glu Gly Ala Arg Val Leu Phe Arg Val Gly

275 280 285

Leu Thr Leu Val Arg Leu Ala Leu Gly Thr Ala Glu Gln Arg Gly Ala

290 295 300

Cys Pro Gly Leu Leu Glu Thr Leu Gly Ala Leu Arg Ala Ile Pro Pro

305 310 315 320

Ala Gln Leu Gln Glu Glu Ala Phe Met Ser Gln Val His Ser Val Val

325 330 335

Leu Ser Glu Arg Asp Leu Gln Arg Glu Ile Lys Ala Gln Leu Ala Gln

340 345 350

Leu Pro Asp Ser Ala Pro Gly Pro Pro Pro Arg Pro Gln Val Arg Leu

355 360 365

Ala Gly Ala Gln Ala Ile Phe Glu Ala Gln Gln Leu Ala Gly Val Arg

370 375 380

Arg Gly Ala Lys Pro Glu Val Pro Arg Ile Val Val Gln Pro Pro Glu

385 390 395 400

Glu Pro Arg Pro Pro Arg Arg Lys Pro Gln Thr Arg Gly Lys Thr Phe

405 410 415

His Gly Leu Leu Thr Arg Ala Arg Gly Pro Pro Ile Glu Gly Pro Pro

420 425 430

Arg Pro Gln Arg Gly Ser Thr Ser Phe Leu Asp Thr Arg Phe

435 440 445

<210> 7

<211> 24

<212> DNA

<213>

<400> 7

cggtgttgtg gatggagcag gtgc 24

<210> 8

<211> 24

<212> DNA

<213>

<400> 8

ctgctccatc cacaacaccg tatg 24

<210> 9

<211> 19

<212> DNA

<213>

<400> 9

gaaggtgaag gtcggagtc 19

<210> 10

<211> 20

<212> DNA

<213>

<400> 10

gaagatggtg atgggatttc 20

<210> 11

<211> 19

<212> DNA

<213>

<400> 11

gcggtacttt gagcagtta 19

<210> 12

<211> 19

<212> DNA

<213>

<400> 12

gcaggtgcta aagcagctt 19

<210> 13

<211> 19

<212> DNA

<213>

<400> 13

ggcggtacaa gaaggtaaa 19

Claims

The application of the gene of P4HA2 in preparing reagent for detecting diffuse large B lymphoma and acute myelogenous leukemia; here, P4HA2 is prolyl-4-hydroxylase type II alpha subunit.
Application of Carabin gene in preparation of a reagent for detecting diffuse large B lymphoma; here, Carabin is TBC1 domain family member 10C.
3. The kit for detecting the diffuse large B lymphoma or the acute myelogenous leukemia is characterized by being constructed by (a) an antibody of P4HA2, (B) a primer or a primer pair for specifically amplifying mRNA or cDNA of P4HA2, and (c) an antibody of Carabin protein; here, P4HA2 is prolyl-4-hydroxylase type alpha subunit, and Carabin is TBC1 domain family member 10C.
4. The kit for detecting diffuse large B lymphoma or acute myeloid leukemia according to claim 3, wherein said primers comprise: specific primers for fluorescent quantitative PCR amplification of the P4HA2 gene have sequences of SEQ.ID.NO7 and SEQ.ID.NO8; specific primers for fluorescent quantitative PCR amplification of an internal reference gene GAPDH have sequences of SEQ ID.NO9 and SEQ ID.NO10.
5. The application of the gene of P4HA2 in preparing medicine for treating diffuse large B lymphoma; here, P4HA2 is prolyl-4-hydroxylase type II alpha subunit.