CN101161283B - New use of CMTM1-v17 and its antagon - Google Patents
New use of CMTM1-v17 and its antagon Download PDFInfo
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- CN101161283B CN101161283B CN2006101136216A CN200610113621A CN101161283B CN 101161283 B CN101161283 B CN 101161283B CN 2006101136216 A CN2006101136216 A CN 2006101136216A CN 200610113621 A CN200610113621 A CN 200610113621A CN 101161283 B CN101161283 B CN 101161283B
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Abstract
The present invention relates to application of CMTM1-v17 as a new drone in diagnosing and/or treating cancer, application of antagonists of CMTM1-v17 in diagnosing and/or treating cancer medicine and application of reagent for detecting CMTM1-v17 expression in preparing composition for detecting cancer.
Description
Technical field
The present invention relates to new application and the antagonist thereof of CMTM1-v17; Specifically, the present invention relates to CMTM1-v17 as application, antagonist application, the diagnosis in the medicine that prepare diagnosis and/or treatment cancer of CMTM1-v17 and/or the application of treating the pharmaceutical composition of cancer and detecting the reagent of CMTM1-v17 expression of a new target in diagnosing tumour.
Background technology
Cancer is the general name of malignant tumor, has become the disease that is only second to cardiopathic another serious threat human life at present, and patient's quantity is the trend of rising always in the world wide, has caused enormous economic loss to society.It is generally acknowledged that it is infinite multiplication property that the tumor cell of vicious transformation is different from one of Normocellular key property.See that from apoptotic angle tumor is that the apoptosis mechanism of tumor cell is suppressed, can not normally carries out the result that cell death is removed, promptly because due to the apoptosis mechanism of tumor cell is interfered.Therefore, Apoptosis Mechanism capable of using comes tumor is treated.
Have been found that now a lot of genes have played important facilitation in the morbidity of cancer; They can suppress apoptosis through promoting the propagation of tumor cell, perhaps promote the mechanism such as generation of new vessels to play a role; Therefore; This genoid can be used as the target of treatment tumor, knocks out this genoid or reaches the purpose that suppresses growth of tumour cell with corresponding antagonist through corresponding braking measure, like VEGF, WT etc.
Chemotactic factor is a type cytokines that can make the motion of cell generation chemotactic, in processes such as inflammation, autoimmune and allergy, plays an important role.CMTM1-v17 (CKLF-like MARVELtransmembrane domain containing; Once be called CKLF-H1A) be the inventor with the nucleotide sequence (seeing Chinese patent 99107284.7 for details) of CKLF-1 serve as that the basis is found through analyzing EST (EST), and find that it can be used for treating disease of hematopoietic system and disease of immune system (Chinese patent ZL00121027.0).
Summary of the invention
An object of the present invention is to provide the application of antagonist in the medicine of preparation diagnosis and/or treatment cancer of CMTM1-v17.
Another object of the present invention provides a kind of polyclonal antibody.
Another object of the present invention provides the pharmaceutical composition of a kind of diagnosis and/or treatment cancer.
Another object of the present invention provides reagent that a kind of CMTM1-v17 of detection expresses is used for detecting the compositions of cancer in preparation application.
According to above-mentioned first aspect, CMTM1-v17 of the present invention might be as the target of a new oncotherapy.The inventor finds that in-vitro transfection CMTM1-v17 can resist the inductive apoptosis of TNF-α.After using siRNA to disturb the expression of CMTM1-v17, find that the apoptosis-induced ability of MDA-MB-231 cell resistance TNF-α reduces, thereby prove that fully CMTM1-v17 has the inductive effect of apoptosis of opposing TNF-α.
The inventor has proved that also in-vitro transfection CMTM1-v17 can promote the MDA-MB-231 cell in external propagation, so CMTM1-v17 of the present invention also has the effect that promotes cell proliferation when having the apoptosis-induced effect of opposing TNF-α.
In the present invention, CMTM1-v17 comprises CMTM1-v17 gene or CMTM1-v17 albumen etc.Said CMTM1-v17 albumen is the aminoacid sequence shown in SEQ ID NO:2, and it has 149 aminoacid.Said CMTM1-v17 gene is the polynucleotide sequence of coding SEQ ID NO:2 of the present invention; It can be amino acid whose coded sequence shown in the SEQ ID NO:2; Perhaps except the coded sequence of above-mentioned aminoacid sequence; Can also comprise non-coding sequence, for example intron, coded sequence 5 ' or the non-coding sequence of 3 ' end etc.Said polynucleotide sequence can be DNA or RNA, and wherein DNA comprises cDNA, genomic DNA and synthetic DNA.Wherein preferred gene is the polynucleotide sequence shown in the SEQ ID NO:1, and 603 nucleotide of this sequence total length, coded sequence are the 1st～450 nucleotide (CDS: the 1st～447 nucleotide).Perhaps, more preferably a kind of isolating nucleotide sequence, it comprises coding CMTM1-v17 protein sequence, the for example coded sequence shown in the SEQ ID NO:1: nucleotide 1～450.Those of ordinary skills are known, and the nucleotide sequence of CMTM1-v17 of the present invention can be entirely identical to the coded sequence shown in SEQ IDNO:1, also can not exclusively be equal to the coded sequence of above-mentioned nucleotide owing to the degeneracy of genetic code.
CMTM1-v17 polynucleotide sequence of the present invention can establishing criteria the pcr amplification technology with cDNA, mRNA or genomic DNA as template, and choose suitable oligonucleotide primers amplification and obtain.The nucleotide that obtains like this can be cloned in the suitable carriers, and carries out sequence description with the DNA analysis technology.Also can obtain through the standard DNA synthetic technology, for example, use can be synthetic on dna synthesizer by solid phase phosphorous acid amide triester method well known in the art.CMTM1-v17 albumen of the present invention can obtain through conventional method; For example through transformed host cell and after being grown into suitable cell density by transformed host cells; With appropriate method (inducing) evoked promoter, continue then to cultivate like temperature change or chemical speciality.After cultivating completion, available centrifuging collecting cell, and with any known method, like freeze-thaw method, supersound process method, lysozyme dissolution method or mechanical crushing method smudge cells.Can use various known methods from the host cell culture, to reclaim and purification CMTM1-v17 albumen of the present invention, these methods comprise ammonium sulfate or ethanol precipitation, acid extraction method, ultrafiltration, ion exchange chromatography, phosphoric acid fiber chromatography, hydrophobic interaction chromatography method, gel filtration, affinity chromatography and high pressure fluid column chromatography.
According to another aspect of the present invention, the present invention provides the application of antagonist in the medicine of preparation diagnosis and/or treatment cancer of CMTM1-V17.Because antagonist (for example albumen, nucleic acid, carbohydrate) can combine and suppress or seal the biological activity of CMTM1-v17 of the present invention with CMTM1-V17 of the present invention, so antagonist of the present invention can be used for diagnosis and/or treatment cancer.
In an embodiment of the invention, described antagonist can be anti-CMTM1-v17 or segmental specific monoclonal of its antigenicity or polyclonal antibody." specificity " described here is meant that antibody capable is incorporated into CMTM1-v17 gene outcome of the present invention or fragment.Preferred those can combine with the gene outcome of CMTM1-v17 of the present invention but nonrecognition and not with the bonded antibody of other irrelevant antigen molecule.Those of ordinary skills are known; Monoclonal antibody of the present invention or polyclonal antibody can carry out the immunity acquisition as antigen through total length or its antigenic fragment of CMTM1-v17 of the present invention, and said antigenicity fragment for example is positioned at the 118th～149 amino acids of the C end of SEQ IDNO:2.Said antigenicity fragment sequence is lacked, is easy to synthesize, and relatively good through bioinformatic analysis and its antigenicity of experiment proof.
In an embodiment of the invention, described antagonist can be the antisense RNA to CMTM1-v17 of the present invention.Antisense RNA can combine with the mRNA molecular specificity ground of CMTM1-v17 of the present invention is complementary, thereby suppresses expression and the function of CMTM1-v17.Usually antisense RNA is made up of 15～20 nucleotide, can and obtain with the expression vector dual mode by synthetic.The latter utilizes gene recombination technology, between suitable promoter and transcription terminator, oppositely inserts one section target gene, thereby obtains the RNA of antisense expression.
In yet another embodiment of the present invention, described antagonist can be siRNA (siRNA).So-called siRNA comprises the small fragment RNA of 19 bases exactly, behind transfered cell or tissue, can bring out target gene mRNA homologous with it specifically and degrade, and causes target gene expression to be suppressed, thus control and target gene function associated.Said siRNA can prepare through express expression in vivo methods such as framework such as chemosynthesis, in vitro transcription or with external preparation method such as RNaseIII digestion long segment double-stranded RNA or siRNA expression vector, siRNA, and utilizes the whole bag of tricks to improve the stability of RNA of the present invention.Method for preparing can be referring to the document of WO2005/007623 and institute's reference thereof with the method that improves stability.
In an embodiment of the invention, in 8 tumor tissues such as colon cancer, rectal cancer, the esophageal carcinoma, pulmonary carcinoma, renal carcinoma, hepatocarcinoma, bladder cancer and gastric cancer, all detect the expression of CMTM1-v17; While lot of statistics digital proof CMTM1-v17 albumen of the present invention high expressed in breast cancer tissue.And CMTM1-v17 of the present invention not only has the effect that promotes cell proliferation, also has the apoptosis-induced effect of opposing TNF-α.Therefore, CMTM1-v17 of the present invention can be used as a novel targets of treatment of cancer.For example suppress gene or the albumen of CMTM1-v17 of the present invention, can be used to treat tumor, for example cancers such as breast carcinoma, colon cancer, rectal cancer, the esophageal carcinoma, pulmonary carcinoma, renal carcinoma, hepatocarcinoma, bladder cancer or gastric cancer.CMTM1-v17 of the present invention can be used as a new index of cancer diagnosis simultaneously; But utilize aforementioned expression or expression, thereby new diagnosis basis is provided for diagnosing tumour to CMTM1-v17 in CMTM1-v17 or its segmental polyclonal antibody or the monoclonal antibody test sample.
Can utilize any method known in the art to detect the expression of CMTM1-v17 nucleotide of the present invention in nucleic acid level; For example through with the antisense RNA of labelled with radioisotope or the hybridization of the DNA sequence after dna probe and the amplification, can come the expression of qualitative or detection by quantitative CMTM1-v17 like this according to radioactive intensity that has that it's too late.Also can adopt non-radioactive marker's biological example element, digoxigenin, dinitrobenzal-dehyde, photobiotin, acetaminofluorene etc.When said method with after PCR (polymerase chain reaction) method combines, the sensitivity and the specificity of its detection significantly improve, and also can use to be derived from PCR method and to detect, for example reverse transcription PCR (RT-PCR).Reverse transcription PCR not only can detect DNA, also can analyze and study RNA.As selection, the tissue slice (fixing and/or freezing) of the patient tissue that can original position directly obtains from biopsy or excision detects the expression of CMTM1-v17 polynucleotide of the present invention, does not need purification of nucleic acid this moment.These analytical technologies comprise DNA or rna blot analysis, single-strand conformation polymorphism analysis, in situ hybridization analysis and polymerase chain reaction.These analyses both can disclose the situation of the quantitative aspect of CMTM1-v17 expression, also can disclose the situation of the qualitative aspect of CMTM1-v17 expression and/or compositions.For example can adopt the Southern hybridization analysis to detect the expression of polynucleotide of the present invention in nucleic acid level.Animal experimental model is arranged now, can carry out testing in the body, reach the effect of disturbing genes of interest with slow virus carrier.
Also can detect the expression of CMTM1-v17 of the present invention at protein level.Detection method also is known in the art; For example utilize the monoclonal antibody or the polyclonal antibody of CMTM1-v17 protein-specific of the present invention, utilize direct or indirect elisa, immunofluorescence, Western Blot, immunohistochemistry etc. to detect.In ELISA, enzyme commonly used be horseradish peroxidase (horserad-ishPeroxidase, HRP) and alkali phosphatase (alkaline phosphatease, AP).Also can use glucoseoxidase, beta-D-galactosidase and urase etc.Those of ordinary skills are known, to different enzymes, can use different substrates, and the substrate of HRP effect includes, but are not limited to o-phenylenediamine (OPD), tetramethyl benzidine (TMB) and ABTS etc.The immunofluorescence cell chemistry is the principle according to antigen antibody reaction; Earlier fluorescein on known antigen or the antibody labeling is processed fluorescent marker, this fluorescent antibody of reuse (or antigen) is as the corresponding antigens (or antibody) in the molecular probe inspection cell or tissue.Immunoblotting (Western Blot) is that existence is descended high-resolution PAGE electrophoresis that antigen is varied in size according to its molecular weight and effectively is divided into many zone of protein by SDS; Protein band after separating is transferred on a kind of solid support such as nitrocellulose membrane or the pvdf membrane through different approaches; Be probe then with antibody; With the target protein epitope generation specific reaction that is attached to solid support, the antibody available enzyme target two that is incorporated into solid support resists crosses detections such as the link coupled SA of fosterization thing enzyme like alkali phosphatase or Radix Cochleariae officinalis.Immunoblotting can identify in the mixture extremely molecular mass of agnoprotein.Immunohistochemistry is the antigen antibody reaction of in tissue slice, carrying out, and can detect content and the location of antigen in tissue.
According to another aspect of the present invention, the invention provides a kind of polyclonal antibody, it combines with the 118th～149 amino acids specificity of SEQ IDNO:2.Through experimental check of the present invention, the 118th～149 amino acids of SEQ IDNO:2 can effectively produce antibody.Above-mentioned antibody not only comprises complete monoclonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, like Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, United States Patent(USP) No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.Antibody of the present invention can prepare by known by one of ordinary skill in the art the whole bag of tricks.For example; The CMTM1-v17 albumen of the present invention of purification or its have antigenic fragment; Can be applied to animal (for example animals such as mice, Cavia porcellus, chicken, rabbit, goat, sheep, horse etc.) to induce the sero-fast generation that contains polyclonal antibody; When immunity, can add adjuvant in case of necessity, with the enhance immunity effect.In order to make experimental result true and reliable, need antagonistic Serum to carry out purification.The antiserum purification can use several kinds of diverse ways, and commonly used is protein A/G combined techniques and antigen affinity purification.A albumen or G albumen have special absorption to IgG antibody Fc arm, and this characteristic can be used for IgG purification antibody.To the IgG that different plant species produces, the proteic absorbability of A albumen and G has certain difference.The immunoaffinity purification method is the most popular method that is used for separating from former serum antigen-specific antibodies.This method can obtain specific Ig.Antigen at first is covalently bonded in the immobile phase on the detached dowel, has the antibody of special absorption to stay on the detached dowel owing to the absorption of synantigen to this antigen in the multi-resistance sample.Fail to combine the non-specific antibody will be at first by eluting from the detached dowel, further eluting will obtain specific antibody.The antibody that obtains with the immunoaffinity purification method has very high specificity.The present invention uses this two kinds of methods, the preparation and purification anti-CMTM1-v17 polyclonal antibody, the antibody behind the verified purification has good active and specificity.Prepare CMTM1-v17 of the present invention or its segmental monoclonal antibody method is methods known in the art; For example with albumen or its fragment as antigen-immunized animal (for example animals such as mice, Cavia porcellus, chicken, rabbit, goat, sheep, horse); The splenocyte of getting immune animal merges with the myeloma cell of syngeneic animal as the plasma cell (immunocyte) that can produce antibody.Screening can produce the hybridoma of purpose antibody, and carry out monoclonalization.
According to a further aspect in the invention, the present invention also provides the pharmaceutical composition of a kind of diagnosis and/or treatment cancer.This pharmaceutical composition comprises to antagonisies such as CMTM1-v17 or its segmental antibody, antisense RNA, siRNA, can also comprise one or more pharmaceutically acceptable carriers or excipient.Said carrier or excipient can comprise normal saline, etc. ooze the combination of glucose solution, BS, glycerol, ethanol and above-mentioned solution.Said pharmaceutical composition can also further comprise penetration enhancer, antioxidant and/or protease inhibitor etc.; Particularly when active component was siRNA, also preferred said pharmaceutical composition comprised the RNA enzyme inhibitor, or each composition of preferred pharmaceutical compositions does not contain the RNA enzyme.
Be therapeutic purposes, said pharmaceutical composition can adopt the appropriate formulations form and use through suitable route of administration.For dosage form, can said pharmaceutical composition be processed various dosage forms, for example solution, Liposomal formulation, polymer formulations, microcapsule and other slow releasing preparation etc.For route of administration,, can adopt the pharmaceutical composition of atomised form to realize through sucking for example for the administration of pulmonary carcinoma; For the administration of breast carcinoma etc., can adopt the said pharmaceutical composition of solution form to realize through injecting; For the administration of bladder cancer etc., can adopt the pharmaceutical composition of solution form to realize through cleaning target tissue.For the administration of skin carcinoma, can adopt forms such as Emulsion or gel to realize through topical (for example as liquid, Emulsion or gel).Pharmaceutical composition of the present invention can the whole body administration, for example through intravenous injection; Perhaps topical is for example to suffering from cancer position local injection.The righttest dosage will be according to patient's the state of an illness, age, sex or body weight, administering mode and required effect and decide, and those skilled in the art can need not creative work in its ken just can confirm best method of application.
As for pharmaceutical composition of the present invention in the application aspect the diagnosis; Can utilize said medicine that the sample from the experimenter is detected; Said sample is from the blood that breaks away from the experimenter, hair, tissue slice etc., especially from the tissue slice of tissues such as colon, rectum, esophagus, lung, kidney, liver, bladder, stomach, breast; The concrete grammar that detects is for example measured the expression of CMTM1-v17 albumen in target tissue through direct or indirect elisa, immunofluorescence, WesternBlot, immunohistochemistry etc.; Perhaps; For example through hybridizing with antisense RNA or the DNA sequence after dna probe and the amplification with radioactivity or non radioactive isotope labelling; Detect, when said method with after PCR (polymerase chain reaction) method combines, the sensitivity and the specificity of its detection significantly improve; Also can use and be derived from PCR method detection, for example reverse transcription PCR (RT-PCR).As selection, the tissue slice (fixing and/or freezing) of the patient tissue that can original position directly obtains from biopsy or excision detects the expression of CMTM1-v17 polynucleotide of the present invention, does not need purification of nucleic acid this moment.These analytical technologies comprise DNA or rna blot analysis, single-strand conformation polymorphism analysis, in situ hybridization analysis and polymerase chain reaction analysis.
The reagent that the present invention also provides the gene that detects CMTM1-v17 or proteic expression is used for detecting the application of the compositions of tumor in preparation.Said reagent can be preferably antibody, antisense RNA or siRNA (siRNA) for albumen, nucleic acid, carbohydrate etc.
Gene or the albumen of CMTM1-v17 of the present invention can be used as diagnosis index.Therefore; Can utilize restriction fragment length polymorphism analysis (RFLP), reverse transcription-polymerase chain reaction (RT-PCR), FISH method method or their combinations such as (FISH), the pathological state due to the CMTM1-v17 expression of the present invention of detection bodies endogenous cause of ill is not enough or excessive.Equally, also can use the proteic antibody of CMTM1-v17 of the present invention, reach identical purpose through radioimmunoassay, competitive combined techniques, Western engram analysis method or enzyme linked immunosorbent assay (ELISA).
Description of drawings
Fig. 1 is the structure sketch map of pcDNA3.1-CMTM1-v17 plasmid.
Fig. 2 is GST-CMTM1-v17 Expression of Fusion Protein and purification result.Swimming lane 1: before inducing; Swimming lane 2: after inducing; Swimming lane 3: the supernatant after the ultrasonication; Swimming lane 4: behind the affinity chromatograph; Swimming lane 5: the GST albumen that thrombin cutting back discharges; Swimming lane 6: the CMTM1-v17C end protein that thrombin cutting back discharges; Swimming lane M: low-molecular-weight labelling.
Fig. 3 is through SDS-PAGE electrophoresis detection purifying antibody result.Antibody behind the swimming lane 1:Protein G affinity chromatography column purification; Antibody behind swimming lane 2 and the swimming lane 3:CMTM1-v17C end protein affinitive layer purification.
Fig. 4 detects the specific result of CMTM1-v17 polyclonal antibody through immunoblotting.Swimming lane 1: the antiserum before the transfection pCDB empty carrier group, purification; Swimming lane 2: transfection pCDB/CMTM1-v17 group, the antiserum before the purification; Swimming lane 3: transfection pCDB/CMTM1-v17 group, the antibody behind the purification.
Fig. 5 detects the result of pCDB-CMTM1-v17 recombinant mammalian expressing vector transient transfection to the short proliferation function of MDA-MB-231 cell through cell counting.
Fig. 6 detects the result of pCDB-CMTM1-v17 recombinant mammalian expressing vector to the short proliferation function of MDA-MB-231 cell through mtt assay.
Fig. 7 A, Fig. 7 B and Fig. 7 C cause the result to the apoptosis-induced resistant function of TNF-α behind the CMTM1-v17 transfection MDA-MB-231 cell.Overexpression CMTM1-v17 can increase the MDA-MB-231 cell resistance apoptosis-induced to TNF-α; After endogenous CMTM1-v17 was suppressed by siRNA, the MDA-MB-231 cell reduced the apoptosis-induced resistivity of TNF-α.Fig. 7 A:24h result; Fig. 7 B:48h result; Fig. 7 C: bar diagram.
Fig. 8 detects the result that CMTM1-v17 expresses through RT-PCR in people's tumor tissues, said organizing from left to right is followed successively by: gastric cancer, hepatocarcinoma, pulmonary carcinoma, bladder cancer, renal carcinoma, the esophageal carcinoma, rectal cancer and colon cancer; With house-keeping gene G3PDH as experiment contrast.
Fig. 9 is the expression of results that detects CMTM1-v17 in the different cell lines through RT-PCR.Said from left to right cell line is: (1) HEK293; (2) HEK293T; (3) Jurkat; (4) Raji; (5) HL-60; (6) MDA; (7) MCF-7; (8) HepG2; (9) HT29; (10) A549; (11) Hela; (12) HelaS3; (13) Du145; (14) LNcap; With house-keeping gene G3PDH as experiment contrast.
Figure 10 detects the result of CMTM1-v17 in normal galactophore tissue and expression in breast through immunoblotting, and wherein N1-N17 is a normal galactophore tissue; T1-T20 is a breast cancer tissue.
Figure 11 detects the result that CMTM1-v17 expresses through immunohistochemical staining in organization chip, upper left: normal galactophore tissue (10 *); Upper right: normal galactophore tissue (40 *); Under the left side: breast cancer tissue (10 *); Bottom right: breast cancer tissue (40 *).
Figure 12 is that immunohistochemical staining detects ER-α and the expression of ca153 in the mammary gland tissue chip, and is upper left: the negative non-cancer milk glandular tissue (40 *) of ER-α; Upper right: the positive breast cancer tissue (40 *) of ER-α; Under the left side: the negative non-cancer milk glandular tissue (40 *) of ca153; The positive breast cancer tissue (40 *) of bottom right: ca153.
The specific embodiment
Below the reference implementation example is come the present invention is further set forth.
1. function motif prediction
Through Motif Scan software (
Http:// myhits.isb-sib.ch/cgi-bin/Motif-scan) prediction; Find that CMTM1-v17 has a LXXLL signal motif; The prompting CMTM1-v17 maybe with one of nuclear receptor superfamily member's who has the LXXLL motif equally steroid appearance hormone receptor (Estrogen receptor; ER) the p160 auxiliary adjustment factor has similar effect, promptly mediates interaction and transcriptional activation function between the hormone receptor and the auxiliary adjustment factor.Other member with CMTM family is the same; CMTM1-v17 contains MARVEL domain (MARVEL; MAL and related proteins for vesicle trafficking andmembrane link), and have the tetratransmembrane structure, aminoterminal and c-terminus are positioned at born of the same parents.The aminoacid sequence 47～53 of SEQ IDNO:2 is " LXXLL " domain.
2. characteristic analysis
Analyzed the bioinformatic analysis of the proteinic primary structure of CMTM1-v17 through DNAStar software (http://www.dnastar.com/).The result shows that CMTM1-v17 is made up of a large amount of hydrophobic amino acids, and 32 aminoacid of 118～149 have good pliability, surperficial accessibility and hydrophilic and antigens with higher index.
The preparation of embodiment 2, anti-CMTM1-v17 antibody
1. antigenic design
According to the result of above bioinformatic analysis and take all factors into consideration hydrophilic, antigenicity and surperficial accessibility, the sequence (hereinafter to be referred as the C end) that the inventor has chosen CMTM1-v17 (118～149 amino acids) this section c-terminus extracellular region prepares antibody.
2.pCDB-CMTM1-v17 construction of recombinant plasmid
With MCF-7 MDA-MB-231 (available from ATCC) cDNA is template, uses forward primer P1 and downstream primer P2 according to known CMTM1-v17 sequence (SEQ ID NO:1) design (to be respectively 5 '-ATGTTGAAGATCCTGAGACT-3 '; 5 '-GCACGTGTCTGTCGAATCGCT-3 '; SEQ ID NO:3 and 4, AudioCodes biotech firm), through utilizing PCR appearance (Applied Biosystems) amplification CMTM1-v17; Adopt sepharose electrophoresis to reclaim the PCR product, and be connected in the pGEM-Teasy carrier (Promega company); Connect product transformed into escherichia coli XL1-Blue with gained; Screening positive clone, the upgrading grain checks order with ABI PRISM Sequencer genetic analyzer (Applied Biosystems).After order-checking is confirmed; With restriction endonuclease EcoRI (TAKARA company) enzyme action pGEM-Teasy-CMTM1-v17 plasmid, will discharge fragment and be inserted into equally in pcDNA.3.1/myc-His (-) the B carrier (Invitrogen company) of EcoRI enzyme action and commercial CIAP (calf intestinal alkaline phosphatase, TAKARA company) processing; The picking positive colony; Through PCR and sequence verification, obtain the recombiant plasmid pCDB-CMTM1-v17 that forward inserts, also can be described as pcDNA.3.1-CMTM1-v17 (Fig. 1).
The structure of embodiment 3, recombiant plasmid pGST-CMTM1-v17C end
With the pCDB-CMTM1-v17 plasmid is template, and utilization (is respectively 5 '-GAAAAGATTCCTGGGAGTCG-3 ' according to the forward primer P3 and the downstream primer P2 of the consequence devised of bioinformatic analysis; 5 '-GCACGTGTCTGTCGAATCGCT-3 ', SEQ IDNO:5 and 4, AudioCodes biotech firm), the sequence of C end 118～149 amino acids of amplification coding CMTM1-v17, the PCR reaction condition is the same.Adopt above same procedure to clone, obtain the recombiant plasmid pGST-CMTM1-v17C end that forward inserts.
1.GST the extensive abduction delivering and the purification of fusion rotein
The pGST-CMTM1-v17C end recombinant plasmid transformed of expression GST (glutathione S-transferase) fusion rotein that structure is obtained is in E.coli BL21 (day be Time Technology company limited); 37 ℃ of shaken cultivation are during to OD600=1.0; Adding IPTG (isopropylthio-) is 0.1mmol/L to final concentration, continuation inducing culture 3 hours.Centrifugal collection antibacterial, with glutathion-Sepharose4B gel beads affinity chromatography purification gst fusion protein, the method that said purification adopts Pharmacia company description to provide is identified proteic purity with SDS-PAGE.
All samples all carries out the SDS-PAGE electrophoresis, then scanning after Coomassie brilliant blue dyeing.As shown in Figure 2, the pGST-CMTM1-v17-C end protein is solubility expression in the BL21 bacterium, and its molecular weight is about 30kD, conforms to expection albumen size; These soluble proteins are behind glutathion-Sepharose4B gel column affinity chromatograph, and the GST-CMTM1-v17C end is single protein band; With thrombin (TAKARA company) fusion rotein is carried out eluting behind the enzyme action, successively from eluent, obtain two kinds of different protein bands, be respectively the CMTM1-v17C end protein and the GST albumen of purification.CMTM1-v17C end protein behind the purification is through the order-checking of N terminal amino acid, and institute's calling sequence is consistent with the sequence of expection.Use the same method and obtain the albumen of total length.
2. Polyclonal Antibody Preparation and purification
Initial immunity: with the CMTM1-v17C end protein 100 μ g/1ml and Freund's complete adjuvant (Sigma company) the 1:1 mixing of purification; With each 0.25ml of gained mixed liquor rabbit (Beijing University's medical board animal center) two foots are carried out subcutaneous injection, all the other solution are at this rabbit back Intradermal multi-point injection.Afterwards, per 3 all booster immunizations once.Strengthened altogether 3 times, the 10th day heart extracting blood behind the last booster immunization, with 5 times of the antiserum dilutions for preparing, last appearance to commercial Protein G affinity column (by specification operation) carries out purification.In order to obtain more special anti-CMTM1-v17 antibody; The inventor is coupled to the CMTM1-v17C end pure protein of escherichia coli expression on the Sepharose4B of cyanogen bromide-activated, and the preparation affinity column is on appearance to said affinity chromatographic column on the above-mentioned antibody-solutions; Re-use pH2.7, concentration is the glycine eluent eluting of 0.2mol/L; Substep is collected eluent, and the dialysis that neutralizes as stated above immediately, and gained antibody is subsequent use in-70 ℃ of preservations.
The SDS-PAGE electrophoretic analysis shows, through behind Protein G and the CMTM1-v17C end protein affinity column two-step purifying, it is thus clear that heavy chain of antibody and light chain band (Fig. 3) clearly, the antibody purity of CMTM1-v17 can reach more than 85% behind the purification.
3. the immunoblotting of polyclonal antibody detects
In order to verify that anti-CMTM1-v17 antibody whether can be specifically and the protein binding of eukaryotic cell expression; The present invention is an object of study with the cell pyrolysis liquid of overexpression CMTM1-v17, use antiserum and the purification before the purification respectively after polyclonal antibody carry out the immunoblotting detection.
MCF-7 MDA-MB-231 is cultivated in DMEM culture medium (Invitrogen company), with cell inoculation in 12 orifice plates.Inoculation back 24h (hour) after; The DNA that with total amount is 1.0 μ gpCDB-CMTM-v17 mixes with 80 μ l DMEM culture medium; Again 2.5 μ l Lipofectamine2000 (Invitrogen) are mixed with 80 μ l DMEM culture medium; Above-mentioned two kinds of solution are mixed, placed 20 minutes, join in the cell then in room temperature.
After to MDA-MB-231 cell transfecting 24h, collecting cell adds the SDS sample loading buffer; Boiled 10 minutes, centrifugal after, get supernatant and carry out SDS-PAGE; Then on electrotransfer to the nitrocellulose filter (Amersham-Pharmacia biotech company), the skim milk sealing with 5% 2 hours is spent the night in 4 ℃ of reactions with the polyclonal antibody of the anti-people CMTM1-v17 of above-mentioned rabbit; Washed once totally 3 times afterwards in per 10 minutes with TBST; Add corresponding IRDye
TM800-conjugated two anti-(LI-COR BIOSCIENCE INC), lucifuge was placed 1 hour, washed film 3 times with TBST, and each 10 minutes, last machine testing.
As shown in Figure 4; Can detect many protein bands with not purified antiserum; Specificity is relatively poor, and the multi-resistance of process affinitive layer purification can react with eukaryotic protein, and specificity obviously increases; Only, prove that anti-CMTM1-v17 polyclonal antibody has excellent specificity approximately locating to see a tangible protein band about 40kD.In the bioinformatic analysis CMTM1-v17 protein sequence N glycosylation site is arranged, its molecular weight can increase general eukaryotic protein through glycosylation modified back, so infers that the increase of the detected specific band molecular weight of immunoblotting is owing to glycosylated reason.
The functional study of embodiment 4, CMTMI-v17
1. detect of the influence of CMTM1-v17 overexpression through cell counting to the MDA-MB-231 cell proliferation
In the present embodiment, use pcDNA3.1, pcDNA-CMTM1-v17 transfection MDA-MB-231 cell respectively after, got 0 hour, 24 hours, 48 hours and 72 hours 4 time points, in microscopically to not counted by the painted living cells of trypan blue.Because the cell membrane of dead cell raises to the permeability of trypan blue, can be dyed blueness, therefore, count the TCS of not dyed blueness down at optics inverted microscope (Olympus company) and get final product.As can be seen from Figure 5, cause the quickening of cell proliferation rate behind the pCDB-CMTM1-v17 transfection MDA-MB-231 cell.
2. detect the influence of CMTM1-v17 on cell proliferation through mtt assay
(3-(4 for MTT; 5-dimethyltiazol-2-yl)-2; 5-diphenyltetrazolium bromide; Invitrogen company) be faint yellow methyl tetrazolium, the mitochondrial dehydrogenase of living cells can produce blue color crystalloid Jia Za ， Jia Za with MTT can carry out colorimetric through dimethyl sulfoxide (DMSO) dissolving.The amount that the Jia Za generates is directly proportional with the metabolism power of living cells, and only has living cells that this reaction is arranged.The available ELIASA of result is measured, and can reflect the power of cell metabolic activity according to the depth of colour developing.In the present embodiment, use pCDB and pCDB-CMTM1-v17 transfection MDA-MB-231 cell respectively after, got 0 hour, 24 hours, 48 hours and 72 hours 4 time points, measure the propagation situation of cell with the MTT detection method.As can beappreciated from fig. 6, will after cultivating 72 hours, be compared with the empty carrier matched group faster than the cell of contrast transfection empty carrier group by the speed of growth of the MDA-MB-231 cell of pCDB-CMTM1-v17 transfection, transfection CMTM1-v17 group can cause 25.12% increase.
3. utilize CMTM1-v17 and CMTM1-v17-siRNA transfection to detect the influence of CMTM1-v17 pair cell apoptosis
With the MDA-MB-231 cell according to 2 * 10
5The density of individual cells/well is inoculated in the 12 porocyte culture plates; Utilize CMTM1-v17 and CMTM1-v17-siRNA transfection to carry out (transfection method is the same); Carry out trypsinization after 24 hours and 48 hours in transfection respectively; Operate by the explanation of FITC-Annexin-V (Bao Sai company) test kit, (FACSCalibur flow cytometer Becton-Dickinson) detects apoptosis with flow cytometer.10000 cells of each sample collection.
Phosphatidylserine (PS) is the important biochemical indicator that apoptosis takes place from the double-deck plasma membrane internal layer of cell to outer field displacement; Annexin-V can combine with the outer field PS of film specifically; Apoptotic generation can be easily judged in variation according to Annexin-V coupling intensity of fluorescence, simultaneously through judge the integrity of cell membrane with the impermeable propidium iodide of cell membrane (PI) dyeing.Shown in Fig. 7 A and Fig. 7 B; Abscissa is represented the fluorescence intensity of FITC-Annexin-V; Vertical coordinate is represented the fluorescence intensity of PI, and left lower area is represented normal cell, the incomplete dead cell of cell membrane that on behalf of the PI fluorescence intensity, top left region raise; The cell that on behalf of the FITC-Annexin-V fluorescence intensity, lower right area raise, the cell that on behalf of FITC-Annexin-V and PI fluorescence intensity, right regions all raise.Change liquid with pCDB and pCDB-CMTM1-v17 transfection MDA-MB-231 cell after 6 hours respectively; One group as contrast; Another group adds 20ng/ml TNF-α and comes apoptosis-induced; Respectively at 24 hours (Fig. 7 A) and 48 hours (Fig. 7 B) back collecting cell, with PI, the two situation of dying the back of annexinV through the facs analysis apoptosis.Can find out that from Fig. 7 A, Fig. 7 B and Fig. 7 C pCDB-CMTM1-v17 transfectional cell group apoptosis number and matched group be obviously difference not, but under TNF-α inductive condition, pCDB-CMTM1-v17 transfectional cell group apoptosis number obviously is less than matched group.In conjunction with cell counting and MTT experimental result, explain that CMTM1-v17 has brought into play effect in the apoptosis-induced process of cell resistance TNF-α, promptly CMTM1-v17 has the effect that opposing is dead, promote cell survival.In the MCF-7 cell, also obtained identical result (data are not given).
The double-stranded SiRNA of people CMTM1-v17 (19bp) synthesizes from Shanghai Ji Kai company, PAGE purification.The positive-sense strand sequence is 5 '-ACC ACU UGC UGA CCU AUU UdTdT (SEQ ID NO:6), and the antisense strand sequence is 5 '-AAA UAG GUC AGC AAG UGG UdTdT (SEQ ID NO:7).Non-silence (5 '-UUCUCC GAA CGU GUC ACG U-3 ', SEQ ID NO:8) in Shanghai Ji Kai company synthetic and through the PAGE purification.With its transfection MDA-MB-231 cell, change liquid after 6 hours, one group as contrast, another group add 20ng/ml TNF-α come apoptosis-induced, respectively at collecting cell after 24 hours and 48 hours, with PI, annexin V is two passes through facs analysis apoptosis situation after dying.Can find out from Fig. 7 A, Fig. 7 B and Fig. 7 C, compare with non-reticent group, transfection its apoptosis number of groups of cells of siRNA than matched group tangible increase has been arranged.After explaining that CMTM1-v17 is disturbed, the MDA-MB-231 cell descends to the apoptosis-induced resistance of TNF-α.Further confirmed the effect that CMTM1-v17 brings into play in the apoptosis-induced process of cell resistance TNF-α.In the MCF-7 cell, also obtained result's (data are not given) of basically identical.
The expression of embodiment 5, CMTM1-v17
1. analyze the express spectra of CMTM1-v17 through RT-PCR
1.1. the cultivation of each cell line and express spectra detect
Human cell line HEK293, HEK293T, HL-60, MDA-MB-231, MCF-7, HepG2, HT29, A549, Hela, HelaS3 and Du145 are incubated in the DMEM culture medium; Lncap, Jurkat and Raji (all from ATCC) then are incubated in the RPMI1640 culture medium (Invitrogen company), all contain 10% hyclone, 100U/ml penicillin and 100 μ g/ml streptomycins in all culture medium.The gained cell is used for following expression analysis and research.
1.2. through the synthesizing single-stranded cDNA of reverse transcription
1) the total RNA with each cell of collecting prepares reaction system as follows respectively:
| Composition | Volume (μ l) |
| Cell total rna Oligo (dT) 20(50μM)10mM?dNTP?Mix | 4.50.51.0 |
2) hatched 5 minutes in 65 ℃, make RNA template and primer degeneration, place then on ice;
3) preparation reaction system as follows adds respectively in the above-mentioned system;
| Solution | Volume (μ l) |
| 5 * cDNA synthesizes buffer (Synthesis buffer) 0.1M DTT (dithiothreitol, DTT) RNaseOUT TMWater Thermoscript through the DEPC processing TMRT | 2.00.50.50.50.5 |
4) hatched 50 minutes in 50 ℃;
5) hatched cessation reaction 5 minutes in 85 ℃;
6) hatched the DNase in the deactivation system 15 minutes in 90 ℃.
1.3.RT-PCR
Utilize Invitrogen Corporation's Super ScriptII test kit (Invitrogen company), with the above synthesizing single-stranded cDNA of total RNA of 2 μ g.PCR reaction the primer sequence is: CMTM1-v17 (forward primer P5:5 '-ATG TTG AAG ATC CTG AGA CT-3 ', SEQ ID NO:9, downstream primer P6:5 '-GCACGT GTC TGT CGA ATC GCT-3 ', SEQ ID NO:10); With G3PDH as system's confidential reference items (forward primer P7:5 '-CTA GCT AGC TGG GAT TAC AGG CGT GAG-3 '; SEQ ID NO:11; Downstream primer P8:5 '-CTA GCT AGC TTA TAT ACA GCG GTT TC-3 ', SEQ IDNO:12).Reclaim the PCR product through sepharose electrophoresis, gained is reclaimed product be connected in the pGEM-Teasy carrier; Connect product transformed into escherichia coli XL1-Blue; Screening positive clone extracts plasmid, carries out sequence verification with the ABIPRISM3100DNA sequenator.Result's proof all can detect CMTM1-v17 expression (Fig. 9) in various degree in different cells system.
Utilize similar approach, in 8 tumor tissues (the farsighted star in Shanghai company) such as gastric cancer, hepatocarcinoma, pulmonary carcinoma, bladder cancer, renal carcinoma, the esophageal carcinoma, rectal cancer and colon cancer, all detect the obvious expression (Fig. 8) of CMTM1-v17.
2. detect the expression of CMTM1-v17 through the Western trace
20 routine breast cancer tissue BIAO and BEN and 17 routine normal galactophore tissues are provided by the 9th (Beijing Shijitan Hospital) Pathology Deparment of clinical medicine institute of Peking University and School of Clinical Oncology, Peking University BIAO and BEN storehouse.All tissue specimens are frozen immediately in liquid nitrogen after obtaining.
In liquid nitrogen,, add the RIPA lysate, be positioned over then and treat its thawing on ice, be drawn in the eppendorf pipe the tissue specimen grind into powder; Placed 30 minutes in 4 ℃, in 4 ℃ with 18, centrifugal 20 minutes of 000g; After getting supernatant and carrying out quantitative equilibrium, albumen boiled heated 10 minutes, be placed on cooled on ice then immediately with BCA (U.S. Pierce Company products) method; In 4 ℃ with 18,000g gets supernatant after centrifugal 10 minutes, carries out the Western trace and detects (see figure 10).In the 17 routine normal galactophore tissues that the inventor detects, 2 examples are positive for CMTM1-v17 expresses, and account for 11.76%, and 15 examples are negative, account for 88.24%; In 20 routine breast cancer tissues, 13 examples are that CMTM1-v17 is positive, account for 65.00%, and 7 examples are negative, account for 35.00%.There are significant difference (p < 0.01) (seeing table 1) in X 2 test statistics prompting, the expression of CMTM1-v17 between normal breast and breast cancer tissue.
Table 1. is through the result of Western engram analysis
| Negative | Positive | The P value | |
| Breast cancer tissue of normal galactophore tissue (n=17) (n=20) | 15(88.24％)7(35.00％) | 2(11.76％)13(65.00％) | <0.01<0.01 |
3. immunohistochemical staining detects the expression of CMTM1-v17 in the organization chip
In order to have analyzed the expression of CMTM1-v17 in various cancers especially breast carcinoma, the inventor has bought organization chip (the ultra English in Shaanxi company, article No. is respectively CC08-02-004, CC08-11-001 and CC08-11-002).Processing method is the same.In room temperature sealing 20 minutes, add anti-CMTM1-v17 polyclonal antibody (through the PBS dilution) with the normal sheep serum working solution,, wash 5 minutes * 3 times, rock frequently with PBS in room temperature reaction 16h, with anti-ca153 antibody (company of China fir Golden Bridge in Beijing) as positive control.Use ChemMate according to DAKO company description
TMDAKO EnVision
TMSystem carries out subsequent experimental.Behind the haematoxylin redyeing nucleus, use the neutral gum mounting, observe and the record result in microscopically.
Through organization chip is carried out immunohistochemical staining, analyze the expression (result sees Figure 11) of CMTM1-v17 in the mammary gland tissue.Carry out in one group of experiment the inventor, three chips amount to and comprise 103 routine breast carcinoma and 129 routine non-cancer tissues (wherein containing 100 normal structures and 29 other mastopathy tissues of example).The result shows that most of normal breast BIAO and BEN (82 examples in 100 examples) show negative findings, account for 82.0% of sum; The result of other mastopathy tissue is (account for 26 examples in 29 examples, percentage ratio is 89.66%) similarly.And in 103 routine tumor tissues, 70 examples (67.96%) demonstrate tangible CMTM1-v17 positive expression, remaining 33 routine negative expression.Like this, in 129 routine non-cancer tissues, amount to the expression that only has 21 examples to detect CMTM1-v17, account for 16.28% of sum; And in 103 routine breast carcinoma BIAO and BEN, have 70 examples to demonstrate the expression of CMTM1-v17, account for 67.96% of sum.Statistics shows, there is significant difference (p < 0.01) in the expression of CMTM1-v17 between normal breast and breast cancer tissue.Meanwhile, the inventor has also detected the expression (Figure 12) of ca153 in the same BIAO and BEN.The result shows, the expression (p < 0.01) of ca153 is arranged in 68 examples (40.96%) tumor specimen and the non-cancer BIAO and BEN of 7 examples (12.96%).Explain that CMTM1-v17 and the ca153 expression in breast cancer tissue has good dependency.
Ca153 is a tumor related antigen, is positioned at tumor cell surface, and when cell carcinogenesis, the activity of protease and sialidase increases on the cell membrane, and cytoskeleton destroys, thereby causes cell surface antigen to wither and fall, and serum ca153 content is raise.Ca153 is present in the multiple adenocarcinoma, like breast carcinoma, pulmonary carcinoma, ovarian cancer and cancer of pancreas, is considered to the comparatively sensitive tumor markers of diagnosing mammary cancer, in clinical position, is widely used.CMTM1-v17 high expressed in breast carcinoma is not only found in this research; And and ca153 between have high correlation; Prompting CMTM1-v17 has the probability that becomes the breast tumor diagnosis marker, is applied to the judgement of auxiliary diagnosis, curative effect monitoring and the transfer and relapse of tumor.
Another category that the present invention carries out like experiment in, the inventor has proved that further there is significant difference (p < 0.01) in the expression of CMTM1-v17 between normal breast and breast cancer tissue; CMTM1-v17 and the ca153 expression in breast cancer tissue has good dependency (result sees table 2, table 3 and table 4).On this basis, the inventor has further inquired into the relation between ER and the CMTM1-v17.The expression of ER-α in the mammary gland tissue chip seen Figure 12 and table 2.
There are some researches show that part breast carcinoma is a kind of estrogen-dependent tumor, (Estroge n receptor ER) has the mechanism of action that promotes the breast cancer cell growth, participates in regulation and control and the relevant expression of gene of breast carcinoma progress for estrogen and estrogen receptor.Show that through bioinformatic analysis CMTM1-v17 comprises a nuclear receptor box motif (nuclear receptor box motif)---LXXLL, this structure can mediate interaction and the transcriptional activation function with nuclear receptor.Estrogen receptor (Estrogen receptor, ER) one of nuclear receptor superfamily member just.And the positive and high expressed of most CMTM1-v17 is found in the breast cancer tissue of ER-alpha expression in statistical result; And in the mammary gland non-cancer tissue of ER-alpha expression, only have half CMTM1-v17 positive and be low the expression.This results suggest CMTM1-v17 possibly play a role in the breast cancer disease process that ER-α participates in.
Table 2.CMTM1-v17, ca153 and the expression of ER in organization chip
The diversity of table 3.CMTM1-v17, ca153 expression in the breast carcinoma BIAO and BEN
The diversity of table 4.CMTM1-v17, ca153 expression in non-cancer milk gland BIAO and BEN
Sequence table
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< 120>new application and the antagonist thereof of CMTM1-v17
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Claims (4)
1. be directed against the application of antagonist in the medicine of preparation treatment cancer of the polynucleotide sequence of the aminoacid sequence shown in the SEQ ID NO:2 or this sequence of encoding; Wherein said antagonist is double-stranded siRNA, and shown in SEQ ID NO:6 and 7, described cancer is a breast carcinoma respectively for the positive-sense strand of said siRNA and antisense strand.
2. application as claimed in claim 1, wherein said polynucleotide sequence are the 1st～447 nucleotide of sequence shown in sequence shown in the SEQ ID NO:1 or the SEQ ID NO:1.
3. pharmaceutical composition of treating cancer, this pharmaceutical composition comprises the antagonist to the polynucleotide sequence of the aminoacid sequence shown in the SEQ ID NO:2 or this sequence of encoding, and one or more pharmaceutically acceptable carriers or excipient; Wherein said antagonist is double-stranded siRNA, and shown in SEQ ID NO:6 and 7, described cancer is a breast carcinoma respectively for the positive-sense strand of said siRNA and antisense strand.
4. pharmaceutical composition as claimed in claim 3, wherein said polynucleotide sequence are the 1st～447 nucleotide of sequence shown in sequence shown in the SEQ ID NO:1 or the SEQ ID NO:1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5854202A (en) * | 1995-01-24 | 1998-12-29 | Dedhar; Shoukat | Peptide fragments of calreticulin, peptide mimetics thereof, and pharmaceutical compostions comprising same |
| CN1244584A (en) * | 1999-05-14 | 2000-02-16 | 北京医科大学 | Chemotarix factor with immunocyte chemotaxis and hemopoinesis stimulating activity |
| CN1283693A (en) * | 2000-07-13 | 2001-02-14 | 卫生部医学免疫学重点实验室 | Cell factor CKLF-HIA with functions of hematopoietic stimulation and immunoregulation and its variant CKLF-HIB |
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| US5854202A (en) * | 1995-01-24 | 1998-12-29 | Dedhar; Shoukat | Peptide fragments of calreticulin, peptide mimetics thereof, and pharmaceutical compostions comprising same |
| CN1244584A (en) * | 1999-05-14 | 2000-02-16 | 北京医科大学 | Chemotarix factor with immunocyte chemotaxis and hemopoinesis stimulating activity |
| CN1283693A (en) * | 2000-07-13 | 2001-02-14 | 卫生部医学免疫学重点实验室 | Cell factor CKLF-HIA with functions of hematopoietic stimulation and immunoregulation and its variant CKLF-HIB |
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