CN106701801A

CN106701801A - Detection marker and kit for B lymphoma and leukemia and application of detection marker and kit

Info

Publication number: CN106701801A
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: 2017-05-24
Anticipated expiration: 2037-02-17
Also published as: CN106701801B

Abstract

The invention belongs to the technical field of medicines, and in particular discloses a detection marker and kit for B lymphoma and leukemia and application of the detection marker and the kit. The invention provides a P4HA2 gene or protein and application of Carabin protein in preparation of a diagnosis kit or treating B lymphoma and leukemia. Study shows that the P4HA2 gene and protein are barely expressed or slightly expressed in normal people or reactive lymph nodes, and are remarkably highly expressed in diffuse large B cell lymphoma; Carabin protein is slightly expressed in the diffuse large B cell lymphoma. In addition, the expression of the P4HA2 gene in an acute myeloid leukemia blood sample is remarkably higher than that in normal people; the diffuse large B cell lymphoma of which expression of the P4HA2 gene is knocked down is slowly proliferated, and the tumorigenic ability of a nude mouse is remarkably degraded. Therefore, the P4HA2 gene can be used as a diagnosis marker and a treatment target of the hematopoiesis and lymphoma, and the Carabin protein can be also used as a diagnosis marker of such diseases. The invention further provides a corresponding detection method and kit.

Description

The detection label of B lymthomas and leukaemia, kit and its application

Technical field

The invention belongs to pharmaceutical technology field, and in particular to the detection label of B lymthomas or leukaemia, kit and its Prepare the purposes of antineoplastic.

Background technology

Be catalyzed proline residue in mammalian cell is prolyl -4- hydroxylases（P4H）, produce (2S, 4R) -4- hydroxyls Base proline.Prolyl -4- hydroxylases have extensive substrate, wherein before being most importantly catalyzed the collagen of new synthesis Body, it is crucial and necessary it to be folded and to form correct three-dimensional conformation.Prolyl -4- the hydroxylases of substrate mammal are one Individual α₂β₂The tetramer, its α subunit has substrate-binding domain and enzyme activity site.α subunits have 3 kinds of hypotypes, a type subunit (prolyl 4-hydroxylase, alpha polypeptide I, referred to as " P4HA1 ") is the enzyme for expressing wide spectrum the most. The type α subunits of prolyl -4- hydroxylases two (prolyl 4-hydroxylase, alpha polypeptide II, referred to as " P4HA2 "), it is made up of 535 amino acid, in the expression of some specific tissues.P4ha1 ^-/-Knock out mice embryonic death, butP4ha1 ^+/-OrP4ha2 ^-/-The mouse of gene knockout is not observed the phenotype of obvious exception, and knocks outP4ha1 ^+/-WithP4ha2 ^-/-Mouse cartilage depauperation.Recently research have indicated that, P4HA2 genes（Rather than P4HA1）Exception and the tight phase of disease Close, the mutation of the gene is along with non-syndrome high myopia disease, and of P4HA2 genes includes subregion in China It is lung cancer susceptibility loci in smoking population.With the exception of this, it is reported that P4HA1 and the P4HA2 height in breast cancer are expressed, and lead to The conformation for overregulating collagen participates in the pernicious transfer of breast cancer.But, have no P4HA1 and P4HA2 and the big B lymthomas of dispersivity Or the related report of leukaemia.

Carabin is the member of TBC1 protein families, and early-stage Study shows, Carabin is thin in spleen and periphery hemolymph Expressed in born of the same parents, and negative regulation T cell antigen acceptor（T cell antigen receptor TCR）Signal path.Further grind Study carefully the endogenous inhibiting factor for finding that Carabin is calcinerin Calcineurin.In addition, it also passes through its Ras GAP activity suppression Ras signal paths.Carabin is to the regulation and control of Calcineurin and Ras paths because by directly and Ras And Calcineurin interact and realize.In addition, Carabin can also be by same mechanism negative regulation B cell BCR signal paths, suppressing the expression of Carabin can accelerate the early stage response of B cell.Recently, the mouse of Carabin is being knocked out Middle discovery Carabin is relevant with myocardial hypertrophy, is also to be swashed by suppressing Calcineurin, Ras and Ca2+/Ca-dependent albumen The signal path of enzyme 2 prevents myocardial hypertrophy.But, have no expression of the reported in literature Carabin albumen in B lymthomas and its Correlation.

B cell lymphoma is the entity tumor that B cell occurs, and is divided into Hodgkin lymphoma and NHL.Its In, the big B lymthomas of dispersivity are most commonly seen NHL lymthomas, account for adult's NHL case More than 1/3, its invasive ability is stronger, and grade malignancy is higher.The big B lymthomas of dispersivity are divided into GCB again（germinal centre B-cell）, ABC and PMBL types, 5 years overall survivals of GCB types are 76%, rather than 5 years overall survivals of GCB types are only 34%.At present, Clinically to B lymthomas especially the big B Lymphoma patients of dispersivity using CHOP or R-CHOP standard regimens.Although having Patient more than 50% can be cured by this therapeutic scheme, but still at least more than 1/3 patient is difficult to treat or recurs.It is right This kind of patient will use autologous hematopoietic stem cell transplantation, so that its chemotherapy medicament sensitive to standard regimens, but produce effects It is little.Therefore, find it is new be worth with B Lymphoma Diagnosis or Combining diagnosis, the gene or protein of special expression high have Important diagnosis and therapeutic potential.

Leukaemia belongs to the one kind in the middle of influence blood, marrow and lymphsystem tumor, this major class disease with lymthoma It is referred to as hematopoiesis and lymphoproliferative dissorders（Tumors of the hematopoietic and lymphoid tissues）. In mortality of malignant tumors, leukaemia occupies the 6th (male) and the 8th (women), is then occupied in adult in children and less than 35 years old L.Over nearest 10 years, the incidence of disease of leukaemia is twice before this.At present the whole nation leukaemic up to more than 400 ten thousand people, Increase 40,000 people newly every year, and children have accounted for half.Leukaemia can be divided into acute lymphatic leukemia（ALL）, acute myeloid Leukaemia（AML）, chronic lymphatic leukemia（CLL）And chronic myelogenous leukemia（CML）This four major types, and other one A little more uncommon species.Clinically, China's acute leukemia is more common than chronic leukemia, and acute leukemia is due to malignant cell Sharp increase and diffusion, it is necessary to treat immediately, otherwise patient is dead even in several weeks in the several months.Wherein it is grown up with AML at most, and youngster Relatively common in child is ALL.Leukaemia is most common childhood cancer.AML is a kind of disease with height heterogeneity, root Can be different types by AML points according to cytomorphology and Histochemical characteristics.The treatment for AML is still with combined chemotherapy at present Based on, the total complete remission rate of AML patient only 50%~70%, long-term disease-free survival rate is 25%~30%.Thus, it is found that and exploitation New leukaemia label has important meaning for the accurately typing of leukaemia, diagnosis, prognosis and the selection of therapeutic regimen Justice.

The content of the invention

It is an object of the invention to provide a kind of specific mark thing, diagnosis examination that can be used to detect B lymthomas or leukaemia Agent box and its purposes in antineoplastic is prepared.

Specifically, the invention provides the type α subunits of prolyl -4- hydroxylases two（P4HA2）Gene, mRNA, cDNA Or the purposes and the purposes of TBC1 domain family member 10C albumen of albumen, they are used as：（1）Detection B lymthomas or white blood The label of disease；（2）Prepare the kit reagent box of detection B lymthomas or leukaemia；（3）The molecular target of B lymthomas is treated, And prepare relevant antineoplastic.

What the present invention was provided can be the type α subunits of prolyl -4- hydroxylases two as B lymthomas or the label of leukaemia （P4HA2）Gene（SEQ.ID.NO1（ Gene: P4HA2; CDS: 565..2172））、mRNA（SEQ.ID.NO2（Gene: P4HA2; mRNA: 1..2588））, cDNA or albumen（SEQ.ID.NO3（ Protein: P4HA2; protein_id="NP_ 004190.1"））, and TBC1 domain family members 10C gene（Carabin genes）（SEQ.ID.NO4）、mRNA （SEQ.ID.NO5）, cDNA or albumen（SEQ.ID.NO6）.Research shows that P4HA2 genes and albumen increase in normal person or reaction Hardly expressed or low expression in raw lymph node, and the significantly high expression in the big B lymthomas of dispersivity；And Carabin albumen exists Low expression in the big B lymthomas of dispersivity.In addition, expression quantity of the P4HA2 genes in acute myeloid leukaemia blood sample is notable Higher than normal person.The big B lymphoma cell strains propagation of dispersivity for striking low P4HA2 expression slows down, and nude mice one-tenth knurl ability is significantly reduced； And the big B lymphoma cell strains propagation of dispersivity for striking low Carabin is accelerated, nude mice one-tenth knurl ability is significantly improved.Therefore, P4HA2 Can be used as this kind of hematopoiesis and the diagnostic marker and therapeutic targets of lympha tumour, and Carabin also can examining as such disease Disconnected label.

In a particular embodiment of the present invention, by the method for SABC, P4HA2 protein antibodies are used, have detected 205 The big B lymthomas of dispersivity and 20 tissue samples of reaction hyperplasia lymph node.It was found that tissue samples of the P4HA2 in reaction hyperplasia lymph node In hardly expression or expression it is very low, and expressed in the big B lymthomas of most dispersivitys, and the expression of P4HA2 and more Dissipate the parting and International prognostic index of the big B lymthomas of property（International Prognostic Index, IPI）Deng clinical Index is significantly correlated.Also Carabin albumen low expression in B lymthoma samples with homemade antibody test, and in normal person B cell in expression high.Therefore, P4HA2 and Carabin are considered as the label of B lymthomas.Another embodiment of the present invention In, by real-time fluorescence quantitative PCR（Real-time PCR, RT-PCR）Method, use P4HA2 special primers, have detected 15 Example acute myeloid leukaemia and 3 PMNC samples of normal human blood extracting.It was found that P4HA2 is normal at 3 Hardly expressed in people's sample, and at 12（80%）Expression high in acute myeloid leukaemia people's sample, therefore, P4HA2 is considered as Leukaemia label.

The present invention also provides the reagent of detection B lymthomas or leukaemia.Described reagent is included：（a）Anti- P4HA2's is anti- Body；（b）The primer or primer pair of specific amplification P4HA2 mRNA or P4HA2 cDNA；（c）The antibody of anti-Carabin.

The present invention also provides the kit of detection B lymthomas or leukaemia.Even if being built with mentioned reagent or its composition Detection B lymthomas or leukaemia kit.

The kit of detection B lymthomas of the present invention or leukaemia, including：

Container（1）, wherein containing P4HA2 specific antibodies, corresponding secondary antibody and people be normal or reactive hyperplasia lymph node tissue Paraffin section, and label or specification；The label or specification are indicated, and the kit is used for SABC or fluidic cell The protein content of instrument detection and analysis P4HA2 is come the probability and the prognosis that judge to suffer from B lymthomas.Specifically, will with finite concentration P4HA2 antibody is normal with people respectively or reactive hyperplasia lymph node paraffin section or patient's exception lymphoid tissue paraffin section are incubated Educate, if the dyeing of patient's paraffin section is normal higher than people or reactive hyperplasia lymph node, i.e. P4HA2 are in the abnormal lymphoid tissue of patient Middle expression, normally or in reactive hyperplasia lymphoid tissue hardly expresses according to P4HA2 in people, then can determine whether that the patient has and suffer from The possibility of B lymthomas.Or, the PBMC that P4HA2 antibody is sorted with normal person or blood samples of patients respectively is incubated with finite concentration Corresponding fluorescence secondary antibody is educated and combined, the probability for suffering from B lymthomas is judged with the content of flow cytometry analysis P4HA2 albumen.

Container（2）, wherein the quantitative fluorescent PCR specific primer pair containing specific amplification P4HA2 mRNA or cDNA, And label or specification；The label or specification are indicated, the kit be used for by the expression quantity of quantitative determination P4HA2 come Judge the probability of cases with leukemia.Further, container（2）It is contained within P4HA2 gene by fluorescence quantitative PCR amplifying specific primers, sequence SEQ.ID.NO7 and SEQ.ID.NO8, and reference gene GAPDH fluorescent quantitative PCR special primers are classified as, sequence is SEQ.ID.NO9 and SEQ.ID.NO10.After extracting blood samples of patients monocyte, detect that P4HA2 is with the method for quantitative fluorescent PCR No expression.If detecting P4HA2 gene expressions in blood samples of patients monocyte, according to P4HA2 in normal human blood monocyte In hardly express, then can determine whether that the patient has the possibility of cases with leukemia.

Container（3）, wherein containing Carabin specific antibodies, and label or specification；The label or specification are noted Bright, the kit is used to judge to suffer from the probability of B lymthomas by the expression quantity of Protein Detection Carabin.

Gene, mRNA, cDNA or albumen present invention additionally comprises P4HA2, and the gene of Carabin, mRNA, cDNA or Albumen, applies in detection and treatment B lymthomas or leukemia medicament is prepared.

Present invention research shows that P4HA2 can be used as the molecular target for the treatment of B lymthomas.In one embodiment of the present invention In, by the short hairpin RNA for being specifically directed to P4HA2 genes（shot hairpin RNA, shRNA）, build stabilization checking The big B lymphoma cell strains of P4HA2 expression and control, detect the propagation of the big B lymphoma cell strains after striking low P4HA2 expression Speed slows down.In another embodiment, the stable cell line that will be built is inoculated with nude mice by subcutaneous.After suppressing P4HA2 gene expressions, One-tenth knurl ability declines to a great extent in nude mouse.In another embodiment, the stable cell line that will be built is inoculated with nude mice by subcutaneous.Suppress After Carabin gene expressions, one-tenth knurl ability is greatly improved in nude mouse.Therefore, can using P4HA2 as treatment B lymthomas point Sub- target, screening P4HA2 inhibitor has positive therapeutic potential to hematopoiesis and lymphoproliferative dissorders.

Therefore, present invention additionally comprises P4HA2 inhibitor, as the medicine for treating B lymthomas.

Of the invention implementing is further described below：

（1）Detect whether P4HA2 protein expressions have exception in the tissue sample of the big B lymthomas of dispersivity and reaction hyperplasia lymph node （Fig. 1,2, embodiment 1,2,3 and 4）；

A, preparation subject's test sample；

B, the antibody with specific anti-P4HA2, detect the big B lymphoma tissues of dispersivity to be measured and reaction hyperplasia lymph node tissue sample The expression quantity of P4HA2 albumen in this；

In C, the P4HA2 expressing quantities and control reaction hyperplasia lymph node tissue that will be measured in the big B lymphoma tissues of dispersivity Be compared, the expression quantity for such as measuring is higher than control value, then it represents that be detected P4HA2 in dispersivity big B lymphoma tissues Abnormal expression.The expression quantity of P4HA2 shows subject with the big B lymthomas of dispersivity higher than control value, or suffers from the big B pouring of dispersivity The probability of bar knurl is higher than normal population（Fig. 1（A））；

D, P4HA2 expressing quantities in the big B lymphoma tissues of dispersivity are associated analysis with every clinical indices, discovery The expression high of P4HA2 and parting and IPI the correlations of indices connection of the big B lymthomas of dispersivity, thus can be using P4HA2 as B lymthomas Label and prognostic indicator；

E, P4HA2 expressing quantities and patient in the big B lymphoma tissues of dispersivity are associated analysis life cycle, found The expression high of P4HA2 is associated with the median survival interval of the big B Lymphoma patients of non-GCB types dispersivity（Fig. 1（B））, thus can be by P4HA2 as B lymthomas prognostic indicator.

（2）Detect whether P4HA2 gene expressions have exception in acute myeloid leukaemia and normal human blood sample（Fig. 3, Embodiment 6）；

A, preparation subject's test sample；

B, P4HA2 genes in acute myeloid leukaemia and normal human blood sample are detected with the RT-PCR primer of specific P4HA2 Expression quantity；

C, P4HA2 expression quantity testing result and normal controls value are compared, the expression quantity of P4HA2 shows higher than control value Subject suffers from acute myeloid leukaemia, or the probability for suffering from acute myeloid leukaemia is higher than normal population（Fig. 3）.

（3）Can be used as the molecular target for the treatment of B lymthomas the invention provides P4HA2（Fig. 4, Fig. 5, embodiment 8,9）；

A, screening are for P4HA2 genes and the effectively shRNA of suppression P4HA2 expression；

B, the shRNA that will suppress P4HA2 expression and control prepare lentiviral particle；

C, the lentiviral particle that will be prepared in B infect B lymphoma cell strains, screening GFP expression positive cell strains respectively；

The multiplication rate of cell line is screened in D, detection C, it is found that P4HA2 strikes the big B lymphoma cells of the dispersivity after subtracting and increases Speed is grown significantly to slow down（Fig. 4）, can be using P4HA2 as therapeutic targets；

E, the cell line that will be screened in C are inoculated with nude mice by subcutaneous, suppress the reduction of transplantable tumor volume, volume after P4HA2 expression Growth slows down, and weight saving（Fig. 5）, can be using P4HA2 as the treatment big B lymthomas target of dispersivity.Meanwhile, obtain P4HA2 inhibitor.

（4）Can be used as the molecular target of diagnosis B lymthomas the invention provides Carabin（Fig. 6, embodiment 10）；

A, preparation subject's test sample；

B, the expression with Carabin albumen in special Carabin antibody test B Lymphoma patients and normal human blood sample Amount；

C, Carabin expressing quantities testing result and normal controls value are compared, the expression quantity of Carabin is higher than right Show that probability of the subject with B lymthomas is higher than normal population according to value（Fig. 6）.

（5）Can be used as the molecular target of diagnosis B lymthomas the invention provides Carabin（Fig. 7, embodiment 11）；

A, screening are for Carabin genes and the effectively shRNA of suppression Carabin expression；

B, the shRNA that will suppress Carabin expression and control prepare lentiviral particle；

The multiplication rate of cell line is screened in D, detection C, it is found that Carabin strikes the big B lymphoma cells of the dispersivity after subtracting Multiplication rate dramatically speeds up（Fig. 7）；

E, the cell line that will be screened in C are inoculated with nude mice by subcutaneous, suppress the increase of transplantable tumor volume, body after Carabin expression Product growth adds.

Brief description of the drawings

The expression high in the big B lymthomas of dispersivity of Fig. 1, P4HA2 albumen.Wherein, the big B lymphs of A, P4HA2 albumen dispersivity Differential expression in knurl；B, P4HA2 expression quantity are related to the big B Lymphoma patients median survival interval of non-GCB types dispersivity.

The expression quantity of Fig. 2, flow cytomery P4HA2 albumen in different cell lines.

The expression high in acute myeloid leukaemia human blood sample of Fig. 3, P4HA2 gene.

Fig. 4, the B lymphoma cell strain multiplication rates of suppression P4HA2 expression slow down.Wherein, A, shRNA suppress P4HA2 eggs White expression；B, the B lymphoma cell strain multiplication rates of suppression P4HA2 expression slow down.

Tumorigenesis ability reduction in Fig. 5, the B lymphoma cell strain bodies of suppression P4HA2 expression.Wherein, A, suppression P4HA2 expression B lymphoma cell strain bodies in one-tenth knurl ability compare；B, nude mice tumor volume growth curve；C, nude mice tumor weight.

Fig. 6, Carabin albumen low expression in the big B lymthomas of dispersivity.

Tumorigenesis ability enhancing in Fig. 7, the B lymphoma cell strain bodies of suppression Carabin expression.Wherein, A, suppression Carabin One-tenth knurl ability compares in the B lymphoma cell strain bodies of expression；B, nude mice tumor volume growth curve；C, nude mice tumor weight.

Specific embodiment

The expression of embodiment 1, immunohistochemical analysis P4HA2 albumen in the big B lymthomas of dispersivity.

Detection material and its preparation：Collect 205 big B lymthomas of dispersivity and 20 reaction hyperplasia lymph node tissue samples This, buffered formalin fixes 24 hours.Flowing water is rinsed 1 hour, and sample is placed in into each 30 minutes, last 4 in 30%, 50% ethanol DEG C it is stored in 70% ethanol.The sample that will be fixed is first transparent through graded ethanol dehydration, dimethylbenzene, then in 52 DEG C or so bars Carry out FFPE under part, and a width of 0.1mm sections of thick 4-10 μm, diameter are affixed on poly-D-lysine with a spread pattern respectively On treated clean slide, organization chip is made.34 DEG C of roasting pieces overnight, 4 DEG C of sealing preserves.

Operating method：The organization chip for preparing is taken, dimethylbenzene dewaxing, graded ethanol rehydration is first carried out, is subsequently adding 0.3% 37 DEG C of hydrogen peroxide is incubated 20 minutes；Section is dipped in citrate buffer solution（PH6.0）In, microwave carries out antigen retrieval 15 Minute, natural cooling；PBS embathes（5 minutes × 3 times）；Add rabbit-anti people P4HA2 to resist more（Proteintech companies, 1:100 It is diluted in 5%BSA）, 37 DEG C reaction 1 hour after 4 DEG C of overnight incubations；PBS embathes（5 minutes × 3 times）；Add HRP marks Goat-anti rabbit instant secondary antibody（Dako companies）, 37 DEG C are reacted 1 hour；PBS embathes（5 minutes × 3 times）；DAB substrates（Dako is public Department）Colour developing, haematoxylin is redyed, and ethanol dehydration, dimethylbenzene is transparent, is scanned after neutral gum mounting（Leica SCN400 slide scanner）.Expression power to P4HA2 in 204 big B lymphoma tissues of dispersivity carries out interpretation and marking, to staining power Judgement use 0-3 grades of system：0 is dye-free（Without brown positive signal）, 1 is weak dyeing, and 2 dye for moderate, and 3 is strong dye Color.

As a result：As shown in figure 1, left figure and right figure are respectively that P4HA2 drenches in reactive hyperplasia tissue and the big B of dispersivity The representative picture expressed in bar tumor tissue.Shown in figure P4HA2 in reactive hyperplasia tissue hardly express or express compared with It is low, and the expression high in the big B lymphoma tissues of dispersivity, as a result with significant difference（p<0.01）.

The parting of embodiment 2, the expression intensity of P4HA2 albumen B lymthomas big to dispersivity and prognosis are related

According to the result of embodiment 1, further expression and clinical pathology of the analysis P4HA2 albumen in the big B lymthomas of dispersivity The correlation of parameter.As shown in table 1, expression and age, the sex of P4HA2 find no correlation to result, but and dispersivity The parting and prognosis two indices of big B lymthomas have significant correlation respectively.In grade malignancy non-GCB types dispersivity higher In big B Lymphomas, the expression of P4HA2 is higher.IPI is got by 5 pathological index marking related to prognosis, and score value is got over Height, represents prognosis poorer.P4HA2 expression is higher, and IPI score values are higher, and the big B lymthomas prognosis of dispersivity is poorer.

The correlation analysis of table 1.P4HA2 expression intensities and clinicopathological parameters

。

The median survival interval analysis of embodiment 3, the expression intensity of P4HA2 albumen and the big B Lymphoma patients of dispersivity

According to the result of embodiment 1, we also analyze associating for P4HA2 B Lymphoma patients median survival intervals big with dispersivity Property.It was found that P4HA2 expression is stronger, the non-big B Lymphoma patients median survival interval of GCB dispersivitys is shorter.This shows that P4HA2 is certain B lymthomas generation development big with dispersivity is closely related, can be used as the diagnosis of the big B lymthomas of dispersivity or treatment prognostic markers Thing.

The expression of embodiment 4, flow cytomery P4HA2 albumen

Detection material and its preparation：

A, acquisition P4HA2 positive cell 293T, and P4HA2 negative cells Jurkat.PBS is washed twice, each 1000rpm, 5 Minute.Wash away the culture medium in cell；

B, fixer：Methyl alcohol is put into -20 DEG C overnight in advance；

C、PBA:Prepare the PBS solution of 2% BSA.

Operating method：

A, 293T, Jurkat cell are resuspended in 100 μ l PBSs, are added dropwise over the 1ml -20 DEG C of methyl alcohol of precooling（Side Edged shakes）, -20 DEG C stand 10 minutes；

B, 1000rpm, are centrifuged for 5 minutes, carefully remove supernatant, with 1mlPBA solution that cell is resuspended and be divided into two parts（This step and Later step needs careful soft piping and druming cell）, 1000rpm, centrifugation in 5 minutes removes supernatant；

C, with PBS IgG, P4HA2 antibody are diluted to 2 μ g/ml, each 200 μ l respectively；

D, IgG, P4HA2 antibody two kinds of cells of resuspended 293T, Jurkat are used respectively, and number：293T-IgG、293T-P4HA2、 Jurkat-IgG、Jurkat-P4HA2.4 DEG C are placed 2 hours；

E, cell is washed one time with PBA（1000rpm, 5 minutes）；

F、1:1000 secondary antibodies for adding fluorescence labeling（Jackson）, room temperature placement 30min；

G, PBA wash cell twice（1000rpm, 5 minutes）, it is resuspended in 100 μ l PBS；

H, detected with flow cytometer.（BD Accuri C6）.

As a result：As shown in Fig. 2 in not expressing the Jurkat cell of P4HA2, the testing result of P4HA2 is negative, and expresses In the 293T cells of P4HA2, P4HA2 results are positive, and illustrate preferable for the P4HA2 antibody specificities of detection.And the method Can be used for the protein content of P4HA2 in the PMBC for detect B lymthomas or the separation of leukaemic's blood, and with this Judge that patient suffers from the probability including the hematopoiesis including acute myeloid leukaemia and lymphoproliferative dissorders disease.

The kit of embodiment 5, the detection big B lymthomas of dispersivity

The kit for detecting the big B lymthomas of dispersivity is prepared, the kit includes：A, label or specification, the mark Sign or specification indicates the kit for detecting or diagnosing the big B lymthomas of dispersivity；The antibody of B, specificity for P4HA2 （It is purchased from Proteintech companies）.

Above-mentioned detection kit is used, 201 big B lymthomas of dispersivity be have detected by the method for SABC and 20 anti- Answer hyperplasia lymph node tissue sample.Result shows, 20 reaction hyperplasia lymph node tissue sample dyeing intensity are 0 or 1, substantially without Positive signal；When positive threshold value takes more than 1,120 big B lymthomas samples of dispersivity of evaluation are P4HA2 positive, and positive rate is about It is 59.7%.

Embodiment 6, fluorescent quantitative RT-PCR method detects P4HA2 mRNA in acute myeloid leukaemia human blood sample Expression

Detection material and its preparation：

A, acquisition PMNC（PBMC）.Collect 12 acute myeloid leukaemias and 3 each 3ml blood samples of normal person This.Using Ficoll lymphocyte separation mediums（GE companies）PBMC is separated, with Ficoll than blood 1：2 volume ratio is slowly by blood Liquid is added in Ficoll liquid, and after being stored at room temperature 15 minutes, 4000g is centrifuged 20 minutes；Interphase cells are taken after centrifugation, it is slow with phosphoric acid Rush salting liquid（PBS）Resuspended, 1500g is centrifuged 5 minutes；Erythrocyte cracked liquid is added after removing supernatant（Shanghai Ge Fan biotech firms）Place Reason adds PBS resuspended again after 3 minutes, 1500g centrifugations obtain the PBMC in blood sample after 5 minutes；

B, the cDNA templates for preparing PBMC.By appropriate Trizol（Invitrogen companies）Add in PBMC, push away to specifications The method recommended extracts the total serum IgE of PBMC, reverse transcription（Takara companies）Prepare cDNA templates；

The mRNA abundance of C, detection P4HA2.Design synthesis P4HA2 and reference gene GAPDH fluorescence quantification PCR primers；

P4HA2 upstream primer sequences：5’-CGGTGTTGTGGATGGAGCAGGTGC-3’（SEQ.ID.NO7）

P4HA2 downstream primer sequences：5’-CTGCTCCATCCACAACACCGTATG-3’（SEQ.ID.NO8）

GAPDH upstream primer sequences：5’-GAAGGTGAAGGTCGGAGTC-3’（SEQ.ID.NO9）

GAPDH downstream primer sequences：5’-GAAGATGGTGATGGGATTTC-3’（SEQ.ID.NO10）.

Operating method：

PCR reaction systems are configured by volumes below：

It is carried out as follows PCR reactions：

98℃：1min

95℃：10s

60℃：10s40 circulation

72℃：20s

72℃：2min.

As a result：As shown in Fig. 2 in 15 blood PBMC samples of detection, P4HA2 is in 3 normal human blood PBMC samples This red hardly expression expresses very low, and is detected in 15 acute myeloid leukaemia samples.Therefore, P4HA2 MRNA is in acute myeloid leukaemia people than the significantly high expression of normal person（p<0.01）.

The preparation of embodiment 7, P4HA2 mRNA detection kits

As described in Example 5, P4HA2 mRNA are not expressed in normal human blood PBMC, and in acute myeloid leukaemia blood Expression is detected in PBMC samples, P4HA2 mRNA detection kits can have been prepared accordingly.

The kit contains：A, label or specification, the label or specification indicate the kit for by calmly The expression quantity of detection P4HA2 is measured to judge the probability of cases with leukemia；The upstream and downstream primer of B, P4HA2 fluorescence quantitative PCR detection, example Such as SEQ.ID.NO5 and SEQ.ID.NO6；The upstream and downstream primer of C, reference gene GAPDH fluorescence quantitative PCR detections, for example SEQ.ID.NO7 and SEQ.ID.NO8.

Sample is prepared according to embodiment 6 and is detected, analyze experimental result, if the P4HA2 mRNA of detection object The ratio between amount of P4HA2 mRNA >=1.5 in amount and population（Preferably >=2.0, more preferably >=2.5）, then the object hair The raw probability including the hematopoiesis including acute myeloid leukaemia and lymphoproliferative dissorders disease is more than normal population.

Embodiment 8, suppression P4HA2 expression make the big B lymphoma cells multiplication rate of dispersivity significantly slow down

Detection material and its preparation：

A, structure strike the slow virus carrier for subtracting P4HA2.Design and verify and effectively strike the psicor shRNA for subtracting P4HA2, sequence is such as Under：shP1:GCGGTACTTTGAGCAGTTA（SEQ.ID.NO11）,

shP2:GCAGGTGCTAAAGCAGCTT（SEQ.ID.NO12）；

The lentiviral particle for subtracting P4HA2 is struck in B, preparation, infects the big B lymphoma cell strains of dispersivity.The shRNA for subtracting P4HA2 will be struck Slow virus carrier pLVX-shRNA-GFP is built into, then with pMD2.G, psPAX2 cotransfection 293T cells, is harvested after 48 hours Supernatant, 0.45 μM of membrane filtration.The big B lymphoma cell strains SU- of the Supernatant infection dispersivity with virion after by filtering DHL-4, observes GFP green fluorescence expressions after 72 hours；

C, sorting stabilization strike the positive cell for subtracting P4HA2.Because GFP can be sent out when striking the shRNA slow virus plasmid expressions for subtracting P4HA2 Green fluorescence, in this, as flow cytometer selection markers.After SU-DHL-4 culture a period of times of infection, by fluidic cell The positive cell of instrument screening GFP expression, and verify that P4HA2's strikes decreasing effect rate with Western Blot（Fig. 4 (A)）.

Operating method：To screen and strike the SU-DHL-4 cells for subtracting P4HA2 genes and be inoculated in 96 orifice plates, density is 6000 Individual/hole, detects cell relative populations in the 1st, 2,3,4,5 days after inoculation with Alamar blue methods respectively, draws cell propagation bent Line.

As a result：As shown in Fig. 4 (B), the big B lymphoma cells multiplication rate reduction of the dispersivity after subtracting P4HA2 is struck, as a result had There is significant difference.

Tumorigenesis ability declines to a great extent in nude mouse after embodiment 9, the expression of suppression P4HA2

Detection material and its preparation：Nude mice by subcutaneous inoculating cell.Choose the Female nude mice of 4 week old（This Leco Corp.）, raise in advance Support one week and adapt to environment.By 5*10⁶Individual cell is resuspended in 100 μ l PBS solutions, with 100 μ l Matrigel（BD companies）It is mixed Nude mice by subcutaneous is inoculated in after conjunction.

Operating method：After growing knurl body at observation inoculating cell, tumor volume and nude mice weight are every other day measured.When naked Mouse band knurl grows 21 days, and tumor volume is no more than 2cm³When euthanasia nude mice.Knurl body is peeled off, is weighed, Taking Pictures recording.

As a result：As shown in figure 5, after striking and subtracting P4HA2 expression, knurl body phase is substantially reduced to control group, and tumor volume increases aobvious Work slows down, and tumor weight is also lighter.It can be seen that P4HA2 is closely related with tumor development, can be controlled as the big B lymthomas of dispersivity Treat target.

The expression of embodiment 10, Western blot analysis Carabin albumen in the big B lymthomas of dispersivity

Detection material and its preparation：Collect the B that 9 big B lymphoma tissues samples of dispersivity and 3 normal pbmcs are sorted out Cell, is mixed after cracking with SDS sample-loading buffers, boiling water bath treatment（980C）8 minutes.

Operating method：The protein sample that will be prepared using micro loading pin is sequentially added the well of SDS-PAGE glue In, electrophoresis after the sample of bromophenol blue mark enters separation gel, is adjusted to 110V permanent by 80V constant pressure electrophoresis 30min or so Pressure continues electrophoresis, until bromophenol blue is left after separation gel enters electrophoresis liquid completely terminates electrophoresis.Shifted using Bio-Rad wet methods From SDS-PAGE glue be transferred on nitrocellulose filter (NC films) albumen by system, 220mA constant currents transfer in ice-water bath 1.5hr.The NC films that Western blot will be printed on are positioned in Block buffer, are positioned over shaking table, are incubated at room temperature 45min.With anti- Body dilution is diluted, and is added on NC films, is positioned over shaking table, and 2hr or 4 DEG C of incubation at room temperature is overnight.Use 1xTBST Elution buffer washes film 3 times, each 10min.Recommendation ratio is diluted secondary antibody with antibody diluent to specifications, plus Onto NC films, shaking table is positioned over, is incubated at room temperature 1hr.Film is washed with 1xTBST elution buffers 3 times, each 10min. ECL develops the color A/B liquid according to 1:1 ratio is well mixed, and is uniformly added on NC films, is sucked after reaction 1min, dark place pressure Piece, develops and is fixed.X mating plates are scanned after drying and deposit piece.

As a result：As shown in Figure 6, Carabin expression high in the B cell that normal pbmc is sorted out, and in the big B of dispersivity Low expression in lymphoma tissue sample.

Tumorigenesis ability is greatly improved in nude mouse after embodiment 11, the expression of suppression Carabin

Detection material and its preparation：Stabilization is built as described in embodiment 8 and is struck and subtract Carabin（Decreasing order row shC is struck, GGCGGTACAAGAAGGTAAA,(SEQ.ID.NO13)）B lymphoma cell strains, nude mice by subcutaneous inoculating cell.Choose 4 week old Female nude mice（This Leco Corp.）, raise in advance one week and adapt to environment.By 5*10⁶Individual cell is resuspended in 100 μ l PBS solutions In, with 100 μ l Matrigel（BD companies）Nude mice by subcutaneous is inoculated in after mixing.

As a result：As shown in fig. 7, strike subtract Carabin expression after, knurl body phase is significantly increased to control group, tumor volume increase Dramatically speed up, tumor weight weightening.It can be seen that the generation development of Carabin and B lymthomas is closely related.

SEQUENCE LISTING

<110>Fudan University

<120>The detection label of B lymthomas and leukaemia, kit and its application

<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

1. the label of B lymthomas or leukaemia is detected, it is characterised in that one is gene, mRNA, cDNA or the albumen of P4HA2； Two is gene, mRNA, cDNA or the albumen of Carabin；Here, P4HA2 is the type α subunits of prolyl -4- hydroxylases two, Carabin is TBC1 domain family members 10C.

2. the reagent of B lymthomas or leukaemia is detected, it is characterised in that included：（a）The antibody of P4HA2；（b）Specific amplification The primer or primer pair of the mRNA or cDNA of P4HA2；（c）The antibody of Carabin albumen；Here, P4HA2 is prolyl -4- The type α subunits of hydroxylase two, Carabin is TBC1 domain family members 10C.

3. the kit of B lymthomas or leukaemia is detected, it is characterised in that as reagent described in claim 2 or its composition structure Build and obtain.

4. the kit of detection B lymthomas according to claim 3 or leukaemia, it is characterised in that the primer includes： P4HA2 gene by fluorescence quantitative PCR amplifying specific primers, sequence is SEQ.ID.NO7 and SEQ.ID.NO8；Reference gene GAPDH is glimmering Fluorescent Quantitative PCR amplifying specific primer, sequence is SEQ.ID.NO9 and SEQ.ID.NO10.

5. the type α subunits of prolyl -4- hydroxylases two are used as the molecular target for treating B lymthomas.

6. the inhibitor of the type α subunits of a kind of prolyl -4- hydroxylases two.

7. the type α subunit genes of prolyl -4- hydroxylases two, mRNA, cDNA or albumen, prepare detection and treatment B lymthomas or Applied in leukemia medicament.