CN109541212A

CN109541212A - PL2L60 albumen is preparing the application in early screening or diagnosing tumour kit

Info

Publication number: CN109541212A
Application number: CN201811391610.3A
Authority: CN
Inventors: 高建新; 李林凤; 陆虹旻
Original assignee: Shanghai Ke Ke Ke Biological Science And Technology Co Ltd
Current assignee: Shanghai Ke Ke Ke Biological Science And Technology Co Ltd
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2019-03-29
Also published as: CN112213488A

Abstract

The present invention relates to pharmaceutical technology fields, provide application of the PL2L60 albumen as diagnosing tumor marker, the application in early screening or diagnosing tumour kit is being prepared more particularly to PL2L60 albumen, the screening or diagnosing tumour reagent refers to the reagent of screening or diagnosing tumour stem cell or tumour progenitor cells, and the tumour refers to leukaemia, lymph cancer, breast cancer, cervical carcinoma, lung cancer, cancer of pancreas, colon cancer or liver cancer.Result of study shows, PL2L60 albumen has tumour-specific and tumour broad spectrum activity, it is especially specific expressed in Tumor Stem/progenitor cell surface, using PL2L60 as target, efficient antibody medicine, cell therapy technology and tumor vaccine can be not only researched and developed, and early diagnosis of tumor reagent can be developed, is made contributions for the progress of tumor diagnosis and therapy, be conducive to push clinical tumor immunization therapy, there is wide potential applicability in clinical practice.

Description

PL2L60 albumen is preparing the application in early screening or diagnosing tumour kit

Technical field

The invention belongs to field of biological medicine, are related to application of the PL2L60 albumen as tumor marker, specifically relate to And PL2L60 protein antibodies prepare the application in early screening or diagnosing tumour kit.

Background technique

Currently, immunotherapy is to explore the hot spot of oncotherapy new method.More and more evidences show Yao Zhiyu tumour Need the cooperation of immunotherapy.Immunotherapy mainly includes antibody-mediated passive immunization therapy and to activate tumour-specific to exempt from The active immunotherapy that epidemic disease cell is characterized.Active immunotherapy may include: (1) specific tumour vaccine, (2) immune biology Regulator (Immunomodulators) and (3) immune cell therapy.Currently, clinically immunotherapy be more successfully with PD-1 is the antibody medicine of target.Other therapies such as immune cell therapy etc. is unsatisfactory.It also can only be tumour even if CART therapy The transitional scheme of immunization therapy.

But there is significant limitation by the immunotherapy of target of PD-1.Start to think that anti-PD-1 antibody blocking is anti-swollen Immunologic test point in tumor immune response, can activate antineoplastic immune system.It was verified that this hypothesis is invalid.It is true It is that anti-PD-1 antibody is to activate ADCC access and direct killing tumour is thin by Direct Recognition tumor cell surface expression PD-1 Born of the same parents, rather than by blocking PD-1/PD-L1 immunosupress point to work.Since most tumors do not express PD-1, therefore anti-PD-1 is anti- The application of body medicine is very restricted.In addition, before unique one of the approval of FDA on April 29 in 2010 is used to treat advanced stage The face due to its clinical effectiveness difference tumor vaccine (actually immunocyte) the product PROVENGE (sipuleucel T) of column gland cancer Face unsuccessfully.In short, due to lacking tumour-specific ground broad-spectrum tumor antigen, no matter passive immunotherapy or active immunity treatment It all meets with and is difficult to the bottleneck gone beyond at present.

The bottleneck for overcoming current immunotherapy, needing to find existing tumour-specific again has the tumour of tumour broad spectrum activity raw Object marker (Tumor-specific antigen；TSA).Currently, the tumor targets that medicine enterprise is researching and developing both at home and abroad are generally not With tumour-specific and tumour broad spectrum activity, it is therefore difficult to there is landmark breakthrough in terms of immunotherapy of tumors.Currently, The breakthrough of immunotherapy of tumors needs to overcome following bottleneck:

(1) target (TSA) of tumour-specific and broad spectrum activity is needed, especially these targets can be expressed in Tumor Stem/ancestral Cell；

(2) corresponding high efficiency cell toxic antibodies (passive immunotherapy) are prepared；

(3) have not by MHC constraint and can be by the polypeptide sequence of the effective submission of antigen presenting cell (APC)；

(4) there is culture, amplification immune function normal (Immunocmpetent) and do not generate immune tolerance (Immunotolerant) technology of immunocyte, including Dendritic Cells (Dendritic cell；DC) and killing cell (Killer)。

If overcoming these bottlenecks, the epoch of immunotherapy of tumors just can really arrive.But it in the prior art, there is no document To can be specific expressed in kinds of tumors gene or albumen record.

Summary of the invention

The present inventor has found and identifies tumour ancestral cells specific proteins by concentrating on studies for many years PL2L60, it is novel tumor stem cell markers.In addition, efficient anti-PL2L60 antibody is prepared for, from PL2L60 protein screening Confirm that PL2L60 is the important target of immunotherapy of tumors with more strongly immunogenic polypeptide, and in animal experiment.Base It can break through the bottleneck of current immunization therapy in the immunotherapy of tumors scheme of PL2L60, effectively solve the above problems.

PL2L60 is the germline stem cell gene PIWIL2 product PIWIL2 General Expression that alienation activates in tumour in life It grows in stem cell, during Sperm specific enzyme, cell nuclear reprogramming or chromatin remodeling is participated in by piwi-piRNA access (chromatin remodeling)；The activity of retrotransposon can be also adjusted by DNA methylation.But normal condition Under do not expressed in body cell.When body cell is exposed in carcinogenic bad border, DNA is impaired, and PIWIL2 gene will be activated, and participates in The reparation of DNA damage, still, being overexpressed PIWIL2 can inducing apoptosis of tumour cell.Therefore, PIWIL2 has tumor barrier gene (TBG) function.

But under carcinogenic environment, when PIWIL2 intragenic promoter is activated, the albumen of generation alienation activation is such as PL2L60.PL2L60 is not expressed in normal cell, is mainly expressed in transformed cells and tumour ancestral cells, and tumour is promoted Occurrence and development.Tumor stem cell is the seed cell of tumor development, inhibits tumor stem cell that can reach inhibition very To the purpose for curing tumour.

Tumor stem cell can originate stem cell (TIC) from normal cell turnover at tumour, by stem cell before cancer (pCSC) and cancer stem cell (CSC) development is cancer progenitor cells (CPC), and the latter becomes the main seed cell of tumor development. PL2L60 has an expression before cancer in stem cell, cancer stem cell, cancer progenitor cells and tumor cell line, expression and swollen Tumor cell proliferation ability is positively correlated.Importantly, PL2L60 expression in various neoplastic hematologic disorders and solid tumor, has tumour special Anisotropic and broad spectrum activity.Using PL2L60 as the Immunotherapy regimens of drone design, various leukemia and solid tumor can be effectively prevented.

PL2L60 high level expression in stem cell before cancer, is effective target of nature antineoplastic immune (NATI).It is anti- PL2L60 monoclonal antibody can identify the PL2L60 of people and mouse simultaneously, before monoclonal antibody (KAO2 and KAO3 are cloned) processing cancer of anti-PL2L60 Stem cell can effectively block its tumorigenesis ability in immunodeficient mouse, so, the result obtained from mouse is easy to People repeats.Recently, it has been found that except in cytoplasm, PL2L60 can also be identified in the surface expression of Partial tumors cell strain, The tumour cell that these cell surfaces express PL2L60 has the property of tumour ancestral cells, prompts monoclonal antibody KAO2 and KAO3 pairs The inhibiting effect of tumour can be worked by target tumor ancestral cells.

Stem cell can induce nature antineoplastic immune before cancer, inhibit the oncogenic function of stem cell itself before cancer.This function It can be that immune system is played a role by the PL2L60 that stem cell before targeting cancer is expressed, because with PL2L60 polypeptide sensitization tree Prominent shape cell (DC-PL2L tumor vaccine) can activate antineoplastic immune, obtain the antineoplastic immune induced with stem cell before cancer The same effect.

Therefore, PL2L60 albumen has the potentiality as tumor marker and tumor targets.

Application the purpose of the present invention is to provide PL2L60 albumen as the molecular marked compound of tumour, is characterized in particular in PL2L60 albumen is preparing the application in early screening or diagnosing tumour reagent or kit.

The first aspect of the present invention is that providing PL2L60 albumen is preparing early screening or diagnosing tumour reagent or reagent Application in box.

The screening or diagnosing tumour reagent refers to the reagent of screening or diagnosing tumour stem cell or tumour progenitor cells.

It include the reagent of screening or diagnosing tumour stem cell or tumour progenitor cells in the kit.

The tumour refers to leukaemia, lymph cancer, breast cancer, cervical carcinoma, lung cancer, cancer of pancreas, colon cancer or liver cancer.

Further, early screening or diagnosis Brachyury positive tumor are being prepared in PL2L60 albumen provided by the invention In application in reagent or kit, the reagent include PL2L60 protein antibodies and to the expression quantity of PL2L60 albumen into The reagent that quantitatively or semi-quantitatively detects of row includes PL2L60 protein antibodies and the table to PL2L60 albumen in the kit The reagent quantitatively or semi-quantitatively detected up to amount.

Preferably, anti-PL2L60 protein antibodies are KAO3 monoclonal antibody, KAO3 monoclonal antibody sequences such as SEQ ID Shown in NO:1.

The action and effect of invention

The present invention confirms that PL2L60 albumen is not expressed in normal cell by many experiments, mainly in transformed cells and It is expressed in tumour ancestral cells, promotes the occurrence and development of tumour, and tumor stem cell or tumour progenitor cells are tumor developments Seed cell, pass through detection PL2L60 albumen expression quantity, so that it may can be realized the diagnosis to infantile tumour, pass through PL2L60 Protein antibodies inhibit tumor stem cell that can achieve the purpose that inhibit even to cure tumour.

In addition, result of study show PL2L60 albumen have tumour-specific and tumour broad spectrum activity, especially Tumor Stem/ Progenitor cell surface it is specific expressed, using PL2L60 as target, can not only research and develop efficient antibody medicine, cell therapy technology and swollen Tumor vaccine, and early diagnosis of tumor reagent can be developed, it makes contributions for the progress of tumor diagnosis and therapy, is conducive to push away Dynamic clinical tumor immunization therapy, has wide potential applicability in clinical practice.

Detailed description of the invention

Fig. 1 is that starting activation and its product diagram, (A) are the expression of PIWIL2 gene in PIWIL2 gene；It (B) is mRNA Expression；It (C) is the expression of protein；

Fig. 2 is PL2L60 albumen compared with PIWIL2 gene expression difference；

Fig. 3 is PL2L60 albumen high expression of results in the various leukaemia and tumor cell line of people and mouse；

Fig. 4 is expression of results of the PL2L60 albumen in tumour cell film surface；

Fig. 5 is that KAO3+ cell surface expresses PL2L60 result；

Fig. 6 is KAO3+ cell and KAO3- cell tumorigenesis ability comparison result；

Fig. 7 is PL2L60 protein overexpression and KAO3+ tumour cell quantitative relation figure；

Fig. 8 is that PL2L60 albumen improves tumor cell proliferation and transfer ability verification result；

Fig. 9 is that PL2L60 albumen improves tumour cell tumorigenesis proficiency testing figure；

Figure 10 is stem cell induction nature tumour immunity (Naturally occurring tumor immunity before cancer； NOTI) result；

Figure 11 is that induction NOTI is the distinctive functional result figure of stem cell before cancer；

Figure 12 be cancer before stem cell induce NOTI be non-tissue specificity result figure；

Figure 13 is immune for the vaccine-induced high efficiency anti-tumor of DC-PL2L, inhibits metastatic lung cancer result figure；

Figure 14 is that PL2L60 monoclonal antibody (KAO2) effectively inhibits tumour cell tumor formation and transfer result figure；

Figure 15 is that PL2L60 monoclonal antibody (KAO3) processing people and mouse tumor can effectively inhibit various tumor growth delay figures；

Figure 16 is KAO3 antibody induction apoptosis of tumor cells result figure；

Figure 17 is that KAO3 monoclonal antibody activates people's complement to kill people and mouse tumor cell result figure.

Specific embodiment

Below with reference to embodiment and attached drawing, the present invention will be described in detail.But the following example should not be regarded as to the present invention The limitation of range.

In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, or according to manufacturer Proposed condition.Unless otherwise stated, the percentage of substance and number are calculated by volume.

Material used in the present invention is as follows:

Reconstruction in Sever Combined Immunodeciency (SCID) mouse: using 8~12 weeks mouse, these mouse were raised harmless in animal Change in facility.

Human cell line: human breast cancer cell line MDA-MB-231, human lung cancer cell line A549 and Human cervical cancer cell lines HeLa is all obtained from American type culture collection (ATCC, Manassas, VA, USA).Cell is maintained at and is supplemented with 10% In the DMEM (Gibco) of fetal calf serum (Gibco) and 0.1mg/ml Pen .- Strep (Gibco).

Mouse lymphoma cell system 2C4 and 326T-4 is voluntarily prepared in the lab: cell is placed on containing 5%CO₂ (v/v) in humidified incubator, R10F is maintained at 37 DEG C, and (RPMI 1640 plus 10mmol/L fetal calf serum, are supplemented with 5mmol/L glutamine, 50mmol/L2- methyl acetophenone, 100U/mL penicillin and 100mg/ml streptomysin) in.

Anti- PL2L60 monoclonal antibody (KAO3mAb, isotype IgM)) exist according to the conventional preparation flow of monoclonal antibody It is voluntarily prepared in laboratory, sequence is as shown in SEQ ID NO:1.

Analysis method used in the present invention and experimental result are described in detail below.

(1) PL2L60 protein expression has tumour-specific and tumour broad spectrum activity

As shown in Figure 1, have multiple promoters in PIWIL2 gene, and it is usually silent in body cell, it can in tumour cell It is activated.Wherein PL2L60 albumen is the primary product that the alienation of PIWIL2 gene activates in tumour cell.

PL2L60 protein expression has tumour-specific as shown in Fig. 2, (A) shows PL2L60 monoclonal antibody specific Can identify PIWIL2 the and PL2L protein in testis (mouse) and tumour cell (people): KAO1 is mainly identified in testis PIWIL2；KAO2 and KAO3 can recognize the PL2L60 in PIWIL2 and PL2L40 and human tumor cells (SW480) in testis And PL2L50；PIWIL2 expression in tumour cell is very low.

(B) expression high level PL2L albumen (2,4) in primary breast cancer (1-2) and cervical carcinoma (3-4) tissue is shown, PIWIL2 only expresses (1,3 in a small number of apoptotic cells；Arrow is signified).

(C) show in breast tissue (1-2) and lymph node (3-4) metastatic carcinoma, PIWIL2 do not express (1, arrow institute Refer to) or only express in a small amount of apoptotic cell (3, arrow is signified), still, PL2L60 is almost expressed in all metastasis cancer cells In (2,4).

(D) show PL2L60+ cell while expressing NF-kB:(1) the mono- dye of PL2L60；(2) the mono- dye of NF-kB；(3) The bis- dyes of PL2L60 and NF-kB；(4) after for cell amplification in figure (3) square, arrow meaning is PL2L60 and NF-kB bis- positive thin Born of the same parents.

The tumour spectrum of PL2L60 protein expression is as shown in figure 3, Fig. 3 (A)~Fig. 3 (D) respectively illustrates PL2L60 egg The white expression of results in the various leukaemia and tumor cell line of people and mouse, these cell strains include leukaemia cell, leaching Bar tumor, breast cancer, cervical carcinoma, lung cancer, cancer of pancreas, colon cancer, liver cancer etc..HLMVEC-Sv and HLVEC-Pri is to turn early stage Change cell and is used as negative control.In all cell strains, the level of PIWIL2 is extremely low, prompts the only table in a small number of apoptotic cells It reaches, it is consistent with Fig. 2 result.As a result confirm that PL2L60 expression has tumour-specific and tumour broad spectrum activity.

(2) PL2L60 promotes tumor stem cell occurrence and development

The tumour cell of endoglin expression PL2L60 has the feature of stem cell.KAO3+ cell has stronger tumorigenesis energy Power is overexpressed the tumour cell of PL2L60, and KAO3+ cell increases, and proliferative capacity improves, and invasion, migration and orbicule generate energy Power enhancing, these features are related with tumor stem cell.The result shows that PL2L60+CD44+ tumour cell has the spy of stem cell Sign, it can promote tumor stem cell occurrence and development.Specific experiment verification result is as shown in Fig. 4~Fig. 9:

Fig. 4 shows PL2L60 albumen in the expression of results of tumour cell film surface.In general, PL2L60 thinks expression thin In endochylema or nucleus, but inventor is recently, it has been found that Partial tumors cell membrane surface also expresses PL2L60, and streaming is respectively adopted Cell analysis technology, immunofluorescence microscopy and immunoblot assay (WB) are verified: (A) Fluorescein activated cell sorter Display people (MBA-231, HeLa and A549) and mouse (2C4 and 326T-4) tumor cell surface also expresses PL2L60 (KAO3+)； (B-C) immunofluorescence microscopy confirms that tumor cell surface (Surface) expresses PL2L60.Note: intracellular KAO3+ cell (C) it is more than cell membrane KAO3+ cell (B)；(D-E) immunoblot assay (WB) proves mouse (D) and people (E) various tumour cells All express PL2L60；It (F-G) is the semi-quantitative analysis result of figure D&E.

Hereafter, KAO3+ and KAO3- breast cancer cell (MBA-MD231) is separated with Flow cytometry, separates cell Film carries out immunoblot assay analysis, as a result as shown in Figure 5, it was demonstrated that contains PL2L60 albumen in KAO3+ cell membrane, demonstrates KAO3+ cell surface expresses PL2L60.

Fig. 6 shows KAO3+ cell and KAO3- cell tumorigenesis ability comparison result.From mouse (A) tumour cell (B16: Melanoma；EL-4: lymthoma；LLC: lung cancer) and people (B) tumour cell (MDA-MB-231: breast cancer；A549: lung cancer； HeLa: cervical carcinoma) separation cell surface KAO3+ and KAO3- cell, it is normal to immune function to be inoculated into mouse tumor cell The nude mice of B6 mouse and human tumor cells to immunodeficiency.Routine observation and detection tumor size.The result shows that: KAO3+ The tumorigenesis ability of tumour cell is higher than the tumour cell of KAO3-.With experiment in vivo on the contrary, KAO3+ cell is cultivated in vitro is not easy It survives, prompts as stem cell, KAO3+ tumour cell needs suitable alcove (niche) that can just survive.

Fig. 7 is PL2L60 protein overexpression and KAO3+ tumour cell quantitative relation figure, and display PL2L60, which is overexpressed, to be increased KAO3+ tumour cell, promote cell Proliferation: PL2L60 gene (GTX-920) stabilization is transferred to human breast cancer cells by (A) In (MDA-MB-231 and MCF-7), control group is transferred to GFP gene.The result shows that being transferred in the cell of PL2L60 gene, have swollen The KAO3+ cell of tumor cells and characteristic of stem doubles.(B) by siRNA-E7 and siRNA-E21 transfection MDA-MB-231-GFP and MDA-MB-231-GTX920 cell knocks out PIWIL2 (E7) mRNA and PL2L60 (E21) mRNA respectively, the result shows that knocking out PIWIL2mRNA cell proliferation does not have inhibiting effect, it might even be possible to promote tumor cell proliferation；But knock out PL2L60mRNA Significantly inhibit cell Proliferation.Reconfirm that PIWIL2 expression has inhibiting effect to tumour, and PIWIL2 alienation activation can be by luring It leads tumor stem cell and promotes tumor development.

Fig. 8 shows that PL2L60 improves the proliferation and transfer ability of tumour cell.Real Time Image System detection shows: mammary gland Cancer cell MDA-MB-231 and MCF-7, which are overexpressed PL2L60 (GTX-920), causes cell Proliferation to accelerate (A, B) and invasion, migration Ability enhances (C, D).

Fig. 9 shows that PL2L60 improves the tumorigenesis ability of tumour cell: as shown in figure A and figure B, PL2L60 (GTX-920) Breast cancer cell (MDA-MB-231 and MCF-7) spherosome is promoted to be formed and grown；As shown in figure C and figure D, PL2L60 promotes swollen Tumor generates.

(3) stem cell can induce wide spectrum nature tumour immunity (Naturally occurring tumor before cancer immunity；NOTI)

PL2L60 is as tumour-specific wide spectrum marker, if has immunogenicity, the meaning in tumour immunotherapy It is great.Stem cell expresses high level PL2L60 albumen before cancer, it is the early stage of tumor stem cell development, has benign and dislikes Property differentiation potential.The relationship between stem cell before cancer and natural tumour immunity is inquired into this part, and concrete outcome is as schemed Shown in 10~Figure 12:

Stem cell can induce nature tumour immunity (Naturally occurring tumor before Figure 10 shows cancer immunity；NOTI).Scheme in A and figure B, stem cell (2C4,3B5c and 3B6C) and lymphoma cell strain EL-4 connect respectively before cancer Kind of C57BL/6 mouse (n=6) is inoculated with the mouse of stem cell before the cancer still not bearing tumor after 200 days, but is inoculated with EL-4's Mouse the 7th day bearing tumor after inoculation；

Scheme in C and figure D, at 210 days, it is thin to be inoculated with EL-4 again for the mouse of stem cell (3B5C, 3B6C) before preparatory inoculation cancer Born of the same parents, the tumorigenesis ability of EL-4 are suppressed in these mouse.Wherein 50% or more mouse not bearing tumor (C), even if bearing tumor Mouse, tumour is also very small (D).The result shows that stem cell can induce NOTI before cancer, tumour progression is prevented.

Figure 11 display induction NOTI is the distinctive function of stem cell before cancer: (A) uses the living cells and cell of stem cell before cancer After lysate (5x 106) is inoculated with B6 mouse 8 weeks respectively, B16F10 melanoma cells (7x 105) is inoculated.It lives as the result is shown Cell and cell pyrolysis liquid can effectively induce NOTI.(B) with stem cell and lymphoma cell EL-4 before the cancer of various concentration Lysate be immunized B6C mouse, after 8 weeks be inoculated with B16F10 cell.Only have stem cell lysate before cancer can induce as the result is shown NOTI, EL-4 cell are without this ability.The cell quantity of lysate is related with the NOTI intensity that it is induced.

The NOTI that Figure 12 shows that stem cell induces before cancer is non-tissue specificity: (A) use stem cell before cancer (pCSC, Source lymthoma), B16F10 tumour cell is inoculated with after lysate Mice Inoculated 8 weeks of EL-4 and B16F10 tumour cell.As a result The NOTI that stem cell lysate induces before display only cancer is most strong, can be in the life of 60% or more mouse complete inhibition melanoma It is long；(B) tumor size of figure A experiment.The results show that the NOTI before cancer after the inoculation of stem cell lysate is wide spectrum, to black The rejection ability of plain tumor and lymthoma (Figure 11) is similar.

(4) PL2L60 albumen is the main component of stem cell induction NOTI before cancer

It is immune using the vaccine-induced high efficiency anti-tumor of DC-PL2L, inhibit metastatic lung cancer.With PL2L polypeptide Pa or Pb sensitization bone After marrow Dendritic Cells (DC), C57BL/6 mouse is inoculated, B16F10 is injected intravenously after 4 weeks, put to death mouse after two weeks and collect Lung tissue, paraffin section, branch on count tumour or inflammation stove (Counts/section) under microscope.As the result is shown such as Figure 13: Pb polypeptide ratio Pa polypeptid induction stronger NOTI (A) tumor incidence；(B) metastatic tumor and inflammation stove；(C) H&E lung tissue dyes The microphoto of different amplification afterwards.Upper row: inflammation stove；Lower row: metastatic tumor.

(5) PL2L60 target monoclonal antibody KAO2 and KAO3 can kill mouse and the tumour cell of people, effectively inhibit people and The one-tenth knurl ability of mouse tumor cell

Figure 14 shows that PL2L60 monoclonal antibody (KAO2) effectively inhibits tumour cell tumor formation and transfer.Stem cell before cancer (2C4G2) is with being seeded to the subcutaneous (n=of SCID mice groin after monoclonal antibody KAO2 culture supernatant (100 microlitres) or medium treatment 3/ group).The result shows that: the cell of KAO2 monoclonal antibody processing does not grow up to tumour (A&B), and still, growing up to for medium treatment is swollen Tumor (B&C).In order to assess effect of the KAO2 monoclonal antibody to metastatic tumor, after handling B16F10 melanoma cells with KAO2 culture supernatant It is injected intravenously B6 mouse.Mouse is put to death after two weeks, is collected lung tissue and is checked transfer stove.The result shows that KAO2 processing B16F10 Transfer ability is suppressed significantly (D), in the lung tissue of the mouse of the B16F10 cell of KA injection O2 processing, transfer stove under microscope Significantly lower than untreated fish group.

Figure 15 shows that KAO3 monoclonal antibody processing people and mouse tumor can effectively inhibit various tumour growths.It is done before mouse cancer Cell 2C4 and cancer stem cell 326T-4 and human tumor cells (breast cancer: MDA-MB-231；Lung cancer: A549；Cervical carcinoma: HeLa) respectively with inoculation immunodeficient mouse (SCID) observation period tumour tumor formation after KAO3 monoclonal antibody culture supernatant or medium treatment Rate and tumor size.Meanwhile tumour is grown in diameter 1.0CM size KAO3 Antybody therapy, control group culture supernatant (Ctrl).As schemed shown in A- figure D, KAO3 antibody can effectively inhibit people and mouse tumor generates, also can be effective in treatment Ground inhibits tumour growth (figure E and figure F).

Figure 16 shows that KAO3 antibody can induce apoptosis of tumor cells.With the KAO3 Mab supernatant of various concentration (0,1,2, 4,8 microlitres/hole) it handles people and detects its influence to cell viability after mouse tumor cell 24 hours.(A) flow cytometer is examined Survey apoptotic cell example.(B) KAO3 handles the statistical result influenced on tumor cell activity；(C) various concentration is to Apoptosis The statistical result of influence and (D) cell are cultivating aerial microphoto.As the result is shown: KAO3 antibody handles people and mouse is swollen After oncocyte, all induce cell apoptosis to some extent.These apoptotic cells may be tumor stem cell.

Figure 17 shows that KAO3 monoclonal antibody can activate people's complement to kill people and mouse tumor cell.(A) tumour cell with KAO3 and people's complement with flow cytometer check apoptotic cell after being incubated for one hour.(B) tumour cell is incubated with KAO3 and people's complement After educating 3 hours, cell activity is measured, determines the percentage of apoptotic cell.As the result is shown: KAO3 can effectively induce tumour thin Born of the same parents' apoptosis.

The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, various changes and improvements may be made to the invention without departing from the spirit and scope of the present invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent defines.

Sequence table

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Leu Pro Gly Pro Pro Ala Leu Leu Ile Thr Leu Val Ser Ala Leu Gly

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785 790 795 800

Gly Gly Gly Thr Ser Val Thr Val Ser Ser Ala Ile Val Met Thr Gly

805 810 815

Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Gly Thr Val Thr Ile Thr

820 825 830

Cys Ala Ala Ser Gly Ala Gly Ala Ile His Ala Thr Leu Ala Thr Thr

835 840 845

Gly Gly Leu Gly Gly Leu Ser Pro Gly Leu Leu Val Thr Ala Ala Leu

850 855 860

Thr Leu Ala Ala Gly Val Pro Ser Ala Pro Ser Gly Ser Gly Ser Gly

865 870 875 880

Thr Gly Thr Ser Leu Leu Ile Ala Ser Leu Gly Pro Gly Ala Pro Gly

885 890 895

Ser Thr Thr Cys Gly His Pro Thr Ser Thr Pro Thr Thr Pro Gly Gly

900 905 910

Gly Thr Leu Leu Gly Ile Leu Gly Val Thr Leu Leu Gly Cys Gly Pro

915 920 925

Gly Ile Leu Leu Pro Ser Gly Thr Leu Ser Leu Thr Cys Ser Pro Ser

930 935 940

Gly Pro Ser Leu Ser Thr Ser Gly Met Gly Val Gly Thr Ile Ala Gly

945 950 955 960

Pro Ser Gly Leu Gly Leu Gly Thr Leu Ala His Ile Thr Thr Ala Ala

965 970 975

Ala Leu Thr Thr Ala Pro Ser Leu Leu Thr Thr Ala Pro Ser Leu Leu

980 985 990

Ser Gly Leu Thr Ile Ser Leu Ala Thr Ser Ala Ala Gly Val Pro Leu

995 1000 1005

Leu Ile Thr Ser Val Ala Thr Ala Ala Thr Thr Thr Cys Ala Ala Ala

1010 1015 1020

Gly Ala Thr Ala Thr Ala Met Ala Thr Thr Gly Gly Gly Thr Ser Val

1025 1030 1035 1040

Thr Val Ser Ser Ala Ile Val Met Thr Gly Ser Pro Ala Ser Leu Ala

1045 1050 1055

Val Ser Leu Gly Gly Ala Ala Thr Ile Ser Thr Ala Ala Ser Leu Ser

1060 1065 1070

Val Ser Thr Ser Gly Thr Ser Thr Met His Thr Ala Gly Gly Leu Pro

1075 1080 1085

Gly Gly Pro Pro Pro Leu Leu Ile Thr Leu Val Ser Ala Leu Gly Ser

1090 1095 1100

Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr

1105 1110 1115 1120

Leu Ala Ile His Pro Val Gly Gly Gly Ala Ala Ala Thr Thr Thr Cys

1125 1130 1135

Gly His Ile Ala Gly Leu Thr Ala Ser Gly Gly Gly Pro Ser Thr Leu

1140 1145 1150

Ala Gly Val Leu Leu Val Cys Gly Pro Gly Ile Leu Leu Pro Ser Gly

1155 1160 1165

Thr Leu Ser Leu Thr Cys Ser Pro Ser Gly Pro Ser Leu Ser Thr Ser

1170 1175 1180

Gly Met Gly Val Gly Thr Ile Ala Gly Pro Ser Gly Leu Gly Leu Gly

1185 1190 1195 1200

Thr Leu Ala His Ile Thr Thr Ala Ala Ala Leu Thr Thr Ala Pro Ser

1205 1210 1215

Leu Leu Gly Leu Thr Ile Ser Leu Ala Thr Ser Ala Ala Gly Val Pro

1220 1225 1230

Leu Leu Ile Thr Ser Val Ala Thr Ala Ala Thr Ala Thr Thr Cys Ala

1235 1240 1245

Ala Ala Gly Ala Thr Ala Thr Ala Met Ala Thr Thr Gly Gly Gly Thr

1250 1255 1260

Ser Val Thr Val Ser Ser Ala Ile Val Met Thr Gly Ser Pro Ala Ser

1265 1270 1275 1280

Leu Ala Val Ser Leu Gly Gly Ala Ala Thr Ile Ser Thr Ala Ser Leu

1285 1290 1295

Ser Val Ser Thr Ser Gly Thr Ser Thr Met His Thr Ala Gly Gly Leu

1300 1305 1310

Pro Gly Gly Leu Pro Gly Gly Pro Pro Pro Leu Leu Ile Thr Leu Val

1315 1320 1325

Ser Ala Leu Gly Ser Gly Val Pro Ala Ala Pro Ser Gly Ser Gly Ser

1330 1335 1340

Gly Thr Ala Pro Thr Leu Ala Ile His Pro Val Gly Gly Gly Ala Ala

1345 1350 1355 1360

Ala Thr Thr Thr Cys Gly His Ile Ala Gly Leu Thr Ala Ser Gly Gly

1365 1370 1375

Gly Pro Ser Thr Leu Ala

1380

Claims

Application of the 1.PL2L60 albumen as the molecular marked compound of tumour.
2.PL2L60 albumen is preparing the application in early screening or diagnosing tumour reagent or kit.
3. PL2L60 albumen according to claim 2 is preparing answering in early screening or diagnosing tumour reagent or kit With, which is characterized in that the screening or diagnosing tumour reagent refers to screening or diagnosing tumour stem cell or tumour progenitor cells Reagent.
4. PL2L60 albumen according to claim 2 is preparing answering in early screening or diagnosing tumour reagent or kit With, which is characterized in that it include the reagent of screening or diagnosing tumour stem cell or tumour progenitor cells in the kit.
5. PL2L60 albumen according to claim 2 is preparing answering in early screening or diagnosing tumour reagent or kit With, which is characterized in that the tumour refer to leukaemia, lymph cancer, breast cancer, cervical carcinoma, lung cancer, cancer of pancreas, colon cancer or Liver cancer.
6. preparing early screening or the diagnosis Brachyury positive is swollen according to any PL2L60 albumen of claim 2 to 5 Application in tumor reagent or kit, which is characterized in that the reagent includes PL2L60 protein antibodies and to PL2L60 egg The reagent that white expression quantity is quantitatively or semi-quantitatively detected includes PL2L60 protein antibodies in the kit and right The reagent that the expression quantity of PL2L60 albumen is quantitatively or semi-quantitatively detected.
7. PL2L60 albumen according to claim 6 is preparing answering in early screening or diagnosing tumour reagent or kit With, which is characterized in that the anti-PL2L60 protein antibodies are KAO3 monoclonal antibody, and the KAO3 monoclonal antibody sequences are such as Shown in SEQ ID NO:1.