CN101831434A

CN101831434A - Anti-human CD45RA rat immune globulin variable region gene and application

Info

Publication number: CN101831434A
Application number: CN200910246023A
Authority: CN
Inventors: 汤永民; 李思思
Original assignee: Zhejiang University ZJU
Current assignee: Hangzhou Yongshen Biotechnology Co ltd
Priority date: 2009-07-27
Filing date: 2009-11-20
Publication date: 2010-09-15
Anticipated expiration: 2029-11-20
Also published as: CN101831434B

Abstract

本发明提供一种抗人CD45RA鼠免疫球蛋白可变区基因，具有SEQ ID NO. 1和SEQ ID NO. 2的核苷酸序列，以及编码的SEQ ID NO. 3和SEQ ID NO. 4的氨基酸序列。研究表明，抗人CD45RA鼠免疫球蛋白可变区基因能够有效识别白血病病人及白血病细胞株CD34+CD38-CD123+的白血病干细胞及CD34+CD38+CD123+的子代白血病细胞，具有靶向治疗白血病的应用前景，可在制备靶向治疗血液系统恶性肿瘤药物中应用。The present invention provides an anti-human CD45RA mouse immunoglobulin variable region gene, which has the nucleotide sequences of SEQ ID NO. 1 and SEQ ID NO. 2, and the encoded SEQ ID NO. 3 and SEQ ID NO. 4 amino acid sequence. Studies have shown that the anti-human CD45RA mouse immunoglobulin variable region gene can effectively identify the leukemia stem cells of leukemia patients and leukemia cell lines CD34+CD38-CD123+ and the offspring leukemia cells of CD34+CD38+CD123+, and has the application of targeted therapy for leukemia Foreground, it can be used in the preparation of drugs for targeted treatment of hematological malignancies.

Description

Anti-human CD 45 RA rat immune globulin variable region gene and purposes

Technical field

The invention belongs to biotechnology, relate generally to anti-human CD 45 RA rat immune globulin ZCH-6-3A4 (being called for short 3A4) (monoclonal antibody or monoclonal antibody) variable region gene and the purposes in preparation treatment blood system malignant tumour targeted drug thereof.

Background technology

The blood system malignant tumour is the disease of serious harm human health, and before the seventies in 20th century, leukemia is considered to incurable disease, and its case fatality rate almost is 100%.Along with the further investigation of countries in the world to the leukemia diagnosis and treatment, the continuous appearance of new antitumor drug and the enforcement of hematopoietic stem cell transplantation, leukemic prognosis has had tangible improvement.But serious toxic side effect remains clinical success and treats leukemic major obstacle, and recurs the selection that suffers for want of medical supplies of drug-fast case, has brought great difficulty to treatment.

In recent years along with molecular biology and antibody engineering development of technology, killing tumor cell and not the leukemia targeted therapy of injuring normal cell obtained paying close attention to widely and developing, wherein the targeted therapy of monoclonal antibody (monoclonal antibody) guiding is the main direction in this field.Compare with chemotherapy, the monoclonal antibody targeted therapy has good selectivity and specificity, they only kill and wound the cell of expressing target molecule, and do not damage hemocyte composition and the histoorgan cell that no target molecule is expressed, thereby can improve the specificity of targeted therapy greatly, reduce the whole body toxic side effect of medicine.Effectively targeted therapy depends on good guide's molecule, series specific surfaces differentiation antigen monoclonal antibody is as guide's molecule, medicine or toxin are taken on the tumor cell membrane or in the cell specifically, or do in order to reach the purpose of killing the leukemia cell, to have the advantage that can not be substituted by complete antibody activating complement (CDC) or by antibody-mediated cell toxicant (ADCC).Rituximab (trade(brand)name: Mabthera) be first monoclonal antibody medicine that drugs approved by FDA is used for oncotherapy, it is a kind of non-binding type people mouse mosaic type CD20 monoclonal antibody, its action target spot is a CD20 antigen, mainly on pre B cell and mature B cell, express, 95% above B is high expression level on non-Hodgkin lymphoma (B-NHL) the case lymphoma cell, obtains gratifying curative effect in adult B is the ALL treatment of NHL and CD20+.In the research of targeted drug, in order to improve the selective killing of targeted drug, reduce toxic side effect to non-target tissue to target cell, the monoclonal antibody of determining a high specificity is a matter of utmost importance.

CD45 is a leukocyte common antigen (LCA), CD45 molecule three exon A, B, C or

claim

4,5,6 alternative splicing to produce multiple allosteric body, CD45 is in the leukocyte surface wide expression, and on other solid tissue cell, do not express, this has been avoided most of chemotherapeutics can cause multiple organ injury's side effect to greatest extent ^[1]Adopted at present the CD45 antibody of radioisotope labeling to be used for the preceding pre-treatment of hematopoietic stem cell transplantation in the world.Glatting G utilizes ¹¹¹8 bone marrow transplantation patients of anti-CD45 monoclonal antibodies YAML568 pre-treatment of In mark, biodistribution detects the concentration show in the Red bone marrow apparently higher than vital tissues such as spleen, liver, kidneys, when marrow was produced obviously inhibition, other important organ function of body did not have obviously impaired ^[2]John M finds in testing in the clinical I phase that 46 AML patients adopt in the pretreating scheme before HLA joins the type autologous peripheral blood stemcell transplant ¹³¹The I-BC8 huge legendary turtle is closed busulfan and endoxan, having reached comparatively ideal marrow removes, the result shows that patient's graft-rejection alleviates, transplanting the back survival rate improves, compare with using no radioimmunity mark busulfan and endoxan AML transplant patient in 509 pre-treatment, the former overall mortality rate is the latter's 0.65 ^[3]But, common CD45 antibody can not routine be used for general targeted therapy leukemia, because, the reactivity of itself and hemopoietic tissue is too extensive, particularly it all has expression at all T, B, granulocyte, monocyte, NK cell, DC cell surface, and conventional the application can produce serious cellular immunity deficiency or neutrophilic granulocyte shortage ^[4]

CD45RA is a kind of allosteric body of CD45 molecule, at T cells, B cell, part granulocyte, part monocyte surface expression, and does not express on activating T cell, mature erythrocyte and thrombocyte and body organa parenchymatosum histocyte.CD45RA only with the T cell subsets that did not stimulate (

T) reaction, and do not react living through antigenic stimulation and making body set up immunocompetent memory T cell subgroup (CD45RO+) cell to contacted antigen, therefore, adopt CD45RA as molecular targeted when killing and wounding or treating, should be unable to destroy the cellular immune function that body had been set up already, and be subjected to the CD45RA cleaning antibody Though the T cell can temporarily lose the immune response to new pathogenic agent (as virus), still, after the end to be treated, the normal hematopoiesis stem cell can produce reserve

It is low that the T cell can remedy this temporary cellular immune function.As for the CD45RA high expression level is arranged on the B cell, this and CD20 antibody class seemingly can remedy temporary transient humoral immune function defective by the infusion gamma-globulin.In addition, CD45RA does not express on red corpuscle and thrombocyte, can not produce anaemia and thrombopenia.Therefore, CD45RA should be a kind of comparatively ideal target killing leukemia cell's a target spot.But domestic and international at present needleless still is to the systematic study of this antigen targeted therapy.

In leukemic treatment, cytotoxic drug can kill most leukemia cell, and marrow routine even flow cytometer all can not detect the leukemia cell.But still may exist leukemic stem cells in the patient body this moment, and leukemic stem cells is a group cell of denier among the leukemia cell, often is in outside the cell cycle, can not be killed by the cytotoxic drug of routine, becomes the root of recurrence.Theoretically, if there is monoclonal antibody can discern leukemic stem cells, just can thoroughly cure leukemia by this antibody drug.CD34+CD38-CD123+ is considered to the sign of marrow series leukemia stem cell ^[5]

Summary of the invention

An object of the present invention is to provide a kind of anti-human CD 45 RA rat immune globulin (ZCH-6-3A4 monoclonal antibody) heavy chain and chain variable region gene, the heavy chain variable region gene of described monoclonal antibody has the aminoacid sequence of the SEQ ID NO 3 of the nucleotide sequence of SEQ ID NO 1 and its coding; The chain variable region gene of described monoclonal antibody has the aminoacid sequence of the SEQ ID NO 4 of the nucleotide sequence of SEQ ID NO 2 and its coding.

Another object of the present invention provides anti-human CD 45 RA rat immune globulin heavy chain and the application of chain variable region gene (ZCH-6-3A4 monoclonal antibody) in preparation targeted therapy blood system malignant tumor medicine.Studies show that, the anti-human CD 45 RA monoclonal antibody can effectively be discerned the filial generation leukemia cell of leukemic stem cells and the CD34+CD38+CD123+ of leukemia patient and leukemia cell line CD34+CD38-CD123+, has the leukemic application prospect of targeted therapy.

Adopting the primary condition of certain specific neoplastic hematologic disorder of monoclonal antibody targeted therapy is that antibody must be able to be discerned certain blood tumor cell membrane antigen.The ZCH-6-3A4 monoclonal antibody has good identification human leukocyte film CD45RA antigen and nonrecognition mature erythrocyte and thrombocyte and body organa parenchymatosum histocyte.Experiment in vitro shows that strain has good selective killing effect to the 3A4 immunotoxin to 3A4 male marrow series leukemia cell, and to the no effect of the cell of 3A4 feminine gender.Further studies show that, 3A4 with can internalization after the antigen of cell surface combines to cytoplasm, therefore, be that targeted molecular prepares immunotoxin with it, help increasing medicine and killing and wounding of target cell reduced toxic side effect non-target tissue's organ.

In addition, leukemic stem cells is the root of leukemia refractory and recurrence, catches and thoroughly removes leukemic stem cells and the leukaemic is cured become possibility.3A4 can discern the leukemic stem cells of the CD34+CD38-CD123+ in leukemia cell line and the leukemia patient marrow, has very important meaning for the research and the treatment of target leukemic stem cells.

The major obstacle that mouse source property monoclonal antibody is used for human body therapy is to produce immune response between foreign protein and patient, thereby produce the toxic side effect that patient is difficult to tolerate, its basic reason is that the Fc section of mouse source property immunoglobulin (Ig) (antibody) has immunogenicity to human body, therefore, be necessary the mouse endogenous antibody is carried out humanization modified, and the heavy chain of antibody and chain variable region gene sequence be the development humanized antibody key.Anti-human CD 45 RA new clone rat immune globulin ZCH-6-3A4 monoclonal antibody heavy chain provided by the invention and chain variable region gene (abbreviate VH respectively as _3A4And VL _3A4) sequence, be as immunogen with children's's undifferentiated type acute lymphoblastic leukemia cell, the purebred small white mouse of sensitization Balb/C, the monoclonal antibody of the mouse-anti human CD 45 RA membrane antigen of succeeding in developing by classical mouse-murine hybridoma monoclonal antibody technology [international differentiation bunch (CD) called after CD45RA], this antibody have carried out comprehensive evaluation and have obtained differentiation bunch (CD) name in the 7th world human leukocyte differentiation antigen cooperative groups meeting (HLDA7).

Because mouse-anti human CD 45 RA antibody ZCH-6-3A4 and non-natural exist, carry out immune sensitized animal but need painstakingly use specific antigen, a series of loaded down with trivial details and the complicated technology process develops by cytogamy, screening, evaluation etc. then, the present invention has finished the clone of this anti-human CD 45 RA antibody (ZCH-6-3A4) heavy chain and chain variable region gene, order-checking and the external target killing acute myeloid leukemia cell of complete 3A4 antibody mediated immunity toxin strain KG1a cell.Especially 3A4 can discern the CD34+CD38-CD123+ cell, and having very for the research of leukemic stem cells, important use is worth.And may have important application to clinical blood system malignant tumour targeted therapy based on the humanized genetic engineering antibody that this gene order is developed.

Description of drawings

Fig. 1 .3A4 and KG1a cell strain reaction streaming figure, 3A4 is to anti-CD45RA monoclonal antibody (clone's name L48) the row retardance experiment streaming figure of U.S. Becton-Dickinson (BD) company.

Fig. 2. identify 3A4 albumen subclass streaming figure.

Fig. 3.

-T Easy/VH _3A4With

-T Easy/VL _3A4Enzyme is cut the evaluation electrophorogram.

Fig. 4. the variable region heavy chain gene (VH of anti-human CD 45 RA antigen monoclonal antibody ZCH-6-3A4 _3A4) sequence (two black arrow between sequence).

Fig. 5. the variable region heavy chain gene (VH of anti-human CD 45 RA antigen monoclonal antibody ZCH-6-3A4 _3A4) sequence (two black arrow between sequence).

Fig. 6. adopting flow cytometry, is reacting cells with the KG1a cell strain, detects the internalization degree of differing temps and incubation time 3A4.

Fig. 7 .3A4-Norcantharidin immunotoxin (3A4-NCTD) to the Nalm-6 leukemia cell's of the KG1a of CD45RA+ and CD45RA-targeting killing effect relatively.

Fig. 8 .3A4 monoclonal antibody identification CD34+CD38-CD123+KG1a cell strain (8A) and the first ability of sending out marrow series leukemia patient marrow CD34+CD38-CD123+ cell (8B).

Embodiment

The present invention is further described in conjunction with the accompanying drawings and embodiments.

Embodiment one

The nucleotide sequence and the aminoacid sequence of 2 genes of the present invention:

1, anti-human CD 45 RA rat immune globulin heavy chain variable region gene (monoclonal antibody ZCH-6-3A4VH _3A4) nucleotide sequence (SEQ ID NO 1) and aminoacid sequence (SEQ ID NO 3).

2, anti-human CD 45 RA rat immune globulin chain variable region gene (monoclonal antibody ZCH-6-3A4VL _3A4) nucleotide sequence (SEQ ID NO 2) and aminoacid sequence (SEQ ID NO 4).

Embodiment two

The mark method detects 3A4 identification antigenic capacity and detects its anti-CD45RA monoclonal antibody to BD company (clone's name L48) retardation (Fig. 1) between flow cytometer, 1B is 3A4 and KG1a cell strain reaction streaming detection figure among the figure, 1C is and the anti-CD45RA monoclonal antibody comparison streaming figure of BD company that 1D is the retardance experiment streaming figure of 3A4 antagonism CD45RA antibody.Found that 3A4 and KG1a cell response positive rate are 99.51%, MFI is 432.63, shows that the 3A4 antibody activity is good.Peak 1 negative contrast among Fig. 1 E, peak 2 is retardance back figure, peak 3 shows that for not blocking figure 3A4 can block the anti-CD45RA antibody of BD company and the reaction of KG1a fully, points out the same epitope that is of anti-CD45RA antibody and 3A4 identification.

Step is as follows:

1, counting KG1a cell is adjusted concentration to 1 * 10 ⁷/ ml, each streaming pipe get 100 μ l cell suspensions; 4 reaction tubess are established in experiment, are followed successively by A. negative control pipe; B.3A4 discern the antigenic capacity detector tube; C. anti-CD45RA monoclonal antibody pipe; D.3A4 block anti-CD45RA monoclonal antibody pipe.

2, B pipe and D pipe add 3A4 culture supernatant 50 μ l, hatch 30min for 4 ℃;

3, added PBS 1000g centrifugal 10 minutes, the unconjugated 3A4 of flush away;

4, A pipe and B pipe add 2 μ l GAM-FITC respectively, and C pipe and D pipe add respectively and 2 μ lCD45RA-FITC;

5, added PBS 1000g centrifugal 10 minutes, the unconjugated antibody of flush away;

6, flow cytometer detects, analyzes.

Embodiment three

The evaluation of 3A4 immunoglobulin subclass: adopt mark method between flow cytometer, the KG1a cell strain is a reacting cells, with 3A4 is one anti-, the immunoglobulin subclass identification kit is two anti-, detect different immunoglobulin (Ig)s and be two positive rate when anti-and average fluorescent strengths, referring to Fig. 2, Fig. 2 is for adopting mark method between flow cytometry, with the KG1a cell strain is reacting cells, 3A4 is one anti-, with the sheep anti mouse immunoglobulin subclass antibody of FITC mark is two anti-, detects different two positive rate and average fluorescent strengths (MFI) when anti-.Found that IgG1 FITC is that two positive rates when anti-are 98.48%, MFI 332.85, and Kappa FITC is two when anti-, and positive rate is 99.02%, and MFI is 252.20, shows that 3A4 antibody belongs to IgG1 κ.

Step is as follows:

1, counting KG1a cell is adjusted concentration to 1 * 10 ⁷/ ml, each streaming pipe get 100 μ l cell suspensions;

2, every pipe adds 3A4 culture supernatant 50 μ l, hatches 30min for 4 ℃;

3, added PBS 1000g centrifugal 10 minutes, flush away is bonded 3A4;

4, add two in the immunoglobulin (Ig) test kit and resist, be respectively sheep anti-mouse igg 1, IgG2a, IgG2b, IgG3, IgM, Ig κ, the Ig λ of FITC mark, 4 ℃ of lucifuges are hatched 30min;

5, added PBS 1000g centrifugal 10 minutes, flush away unconjugated two is anti-;

6, flow cytometer detects, analyzes.

Embodiment four

2 anti-human CD 45 RA rat immune globulin variable region genes provided by the invention obtain through the following steps:

1, the development of ZCH-6-3A4 monoclonal antibody: press Koller﹠amp substantially; Mouse-murine hybridoma the classical way of Milstein report carries out ^[6], with children's acute undifferentiated type Lymphocytic leukemia (uALL) cell (its immunophenotype is HLA-DR+, CD19+, CD10-, CD33-, TdT+, CD7-is CD41a-) as immunogen, with 10 ⁷The leukemia cell makes abdominal injection for 8 ages in week female Balb/C mouse, once in a week, totally 4 times, injected the back the 3rd day in the 4th, mouse is killed in dislocation, and aseptic extracting spleen cell mixes by 6: 1 with the murine myeloma cell strain NS-1 cell (U.S. ATCC product) that is in logarithmic phase, with 50% polyoxyethylene glycol (PEG, U.S. Sigma company, molecular weight 3350 dalton) solution carries out cytogamy as merging media, carries out selectivity and cultivate in 96 orifice plates (U.S. Falcon company).In merging the back the 9th～20 day, the next day with immunogen cell culture supernatant is carried out indirect immunofluorescence (IIF) screening.Positive porocyte reaches positive through 3 time cloningizations and continuous 2 times 100% holes, has promptly set up the hybridoma cell line of energy continuous release 3A4 monoclonal antibody.Through the continuous passage cultivation and the multigelation of 8 wheat harvesting periods, the ability of its secretion 3A4 monoclonal antibody is stable.With its ascites (fluorescent method was tired 1: 3200) or culture supernatant (fluorescent method was tired 1: 16) source as the 3A4 monoclonal antibody.

2, heavy, the light chain gene (VH of 3A4 _3A4, VL _3A4) amplification and clone

2.1 extracting and the processing of total RNA:

Collect 3A4 hybridoma 8 * 10 ⁶, with Yeast Nucleic Acid (RNA) extraction agent box (TRIZOL liquid) by specification step extracted total RNA.Be dissolved at last in 25 μ l DEPC (diethyl-pyrocarbonate, the diethylpyrocarbonate) water, and add

(ribonucleotide enzyme inhibitors) to final concentration is 1U/ μ l (unit/microlitre).Ultraviolet spectrophotometer is measured A260, A280 and ratio thereof, and 1% agarose electrophoresis is observed total RNA.It is preceding to carry out reverse transcription-polymerase chain reaction,PCR (RT-PCR), gets the total RNA of 2 μ l3A4, and the method according to RQ1 (the DNA enzyme reagent kit of no RNA enzyme) illustrates digests the genomic dna that pollutes among total RNA.

2.2 the extracting of total RNA:

(1) counting cells, totally 8 * 10 ⁶Individual viable cell;

(2) normal temperature following 1000 rev/mins (rpm) is centrifugal 15 minutes, abandons supernatant;

(3) add after the stroke-physiological saline solution under the 1000rpm condition centrifugal 15 minutes in the precipitation, repeated washing 2 times;

(4) in precipitation, add 8ml TRIZOL (1ml/10 ⁶Cell), lash shearing several minutes repeatedly with the aseptic syringe of 5ml immediately;

(5) above-mentioned TRIZOL solution is changed in 1.5ml little plastics with cover (eppendorf) pipe by the 1ml/ pipe, left standstill under the room temperature 5 minutes;

(6) every pipe adds the 0.2ml chloroform, acutely shakes 15 seconds, leaves standstill under the room temperature 10 minutes;

Centrifugal 15 minutes of (7) 4 ℃ of following 12000g change water in the eppendorf pipe of new 1.5ml, and every pipe adds the 0.5ml Virahol, shakes up immediately, leaves standstill under the room temperature 10 minutes;

Under (8) 4 ℃, centrifugal 10 minutes of 12000g;

(9) abandon supernatant, every pipe adds 1.5ml 75% ethanol, mixing, and under 4 ℃, centrifugal 5 minutes of 7500g abandons supernatant;

(10) dry in the Vacuumdrier, every pipe adds the DEPC water dissolution precipitation that 25 μ l do not have the RNA enzyme, and-80 ℃ of refrigerators are preserved.

2.3 the mensuration of total rna concentration:

Take out the new extractive total RNA of 2 μ l, add 98 μ l DEPC water mixings, ultraviolet spectrophotometer (GeneQuant II) is measured 260 light absorption values (A260) and 280 light absorption values (A280), and the RNA actual concentrations calculates with following formula:

2.4 total RNA gel electrophoresis:

Get new extractive total RNA3 μ l and add electrophoresis sample-loading buffer 5 μ l, brominated second pyridine (EB) concentration 0.5 μ g/ml in 1% sepharose, electrophoresis is 5 minutes under the 100V volts DS, and gel imaging system observed result and shooting are preserved.

2.5 the processing of total RNA: get DNA enzyme (RNase-freeDnase) (1U/ μ l) 10 μ l+RNasin (40U/ μ l) the 1 μ l that total RNA 1 μ l+ does not have the RNA enzyme, total amount reaches 12 μ l, through 37 ℃ * 1 hour, incubated in 90 ℃ * 5 minutes put immediately after the bath stand-by on ice.

2.6RT-PCR：

To specifications, RQ1 is not had total RNA that the DNA enzyme of RNA enzyme handled carry out reverse transcription (25 μ l system), and with DNA purification kit (QIAquick) purified mRNA/cDNA heteroduplex.The cDNA 2.5 μ l that get purifying carry out PCR.

2.6.1RT reaction system: get total RNA 13 μ l+ oligomerization deoxythymidines [Oligo (dT)] 1.5 μ l that RQ1 handled, total amount reaches 14.5 μ L, through 65 ℃ of pre-sex change 5 minutes, put immediately on ice, add following reagent: DEPC water 2.5 μ l+5 times damping fluids 5 μ l+25mM dNTP (deoxynucleoside mixture) 0.5 μ l+RNA enzyme inhibitors, 1.5 μ l+ reversed transcriptive enzymes (200U/ μ L) 1 μ l is to cumulative volume 25 μ l, through 37 ℃, incubated bath in 30 minutes with synthetic complementary DNA Nucleotide (cDNA), put 95 ℃ 5 minutes, it is standby to move to 4 ℃ of ice baths then.

2.6.2PCR system

The PCR reaction system of (1) 50 μ l: the 3A4 of 2.5 μ l purifying, each 1 μ l of the light chain of 10pmol or variable region of heavy chain upstream and downstream primer, the dNTP of 0.5 μ l 25mM, 5 μ l10 * high-fidelity PCR damping fluid, 0.2 μ l high-fidelity Taq polysaccharase (

Taq High Fidelity); Primer sequence is as follows:

Variable region of heavy chain 5 ' primer sequence (SEQ ID NO 5):

CAG?GTG?CAG?CTGAAG?CAG?TC

Variable region of heavy chain 3 ' primer sequence (SEQ ID NO 6):

CCA GGG GCC AGT GGA TAG ACAAGC TTG GGT GTC GTT TT variable region of light chain 5 ' primer sequence (SEQ ID NO 7):

CAAATT?GTT?CTC?ACC?CAG?TCT

Variable region of light chain 3 ' primer sequence (SEQ ID NO 8):

GGATAC?AGT?TGG?TGC?AGC?ATC

(2) reaction conditions: 94 ℃ of pre-sex change 2 minutes, 94 ℃ of sex change 30 seconds, 57 ℃ of annealing 30 seconds, 72 ℃ were extended totally 38 circulations 30 seconds;

(3) after last circulation is finished, add 1 μ l Taq DNA Polymerase (Taq archaeal dna polymerase) and extended 7 minutes for 72 ℃;

(4) get 5 μ l PCR products and carry out 1% agarose gel electrophoresis, add standard molecule amount mark simultaneously, identify PCR product segment size, respectively called after VH _3A4And VL _3A4Gene.The result can be observed the VH about 350 base pairs (bp) _3A4VL about band and 325bp _3A4Band.Aforementioned positive band is cut glue recovery purifying obtain VH _3A4And VL _3A4Gene.

3.VH _3A4, VL _3A4The clone of gene amplification product and evaluation are with the VH of purifying _3A4And VL _3A4Gene fragment by ligase enzyme respectively with cloning vector -T Easy connects, the connection product of acquisition

-T Easy/VH and

-T Easy/VL, by transforming DH5 α competence bacterium, obtain dozens of white colony and several blue colonies, 3 white colonies of picking carry out plasmid amplification respectively, carry out nucleic acid restriction endonuclease EcoRI enzyme after the extracting and cut evaluation, the purpose fragment (Fig. 3) about all visible 350bp of 1% agarose electrophoresis result and 325bp.Among Fig. 3, M: low ribonucleotide (DNA) standard molecular weight mark; 1～3: recombinant plasmid

-T Easy/VH _3A4The EcoRI enzyme cut; 4～6: recombinant plasmid

-T Easy/VL _3A4The EcoRI enzyme cut; 7～8: empty plasmid

The EcoRI enzyme of-T Easy is cut.

4.VH _3A4, VL _3A4Gene sequencing and analysis

To checking order VH behind the positive recombinant plasmid purifying _3A4(referring to Fig. 4) and VL _3A4(referring to Fig. 5) gene all meets mouse Ig variable region skeleton construction.VH _3A4Full length gene 351bp (seeing sequence SEQ ID NO 1), 117 amino acid (seeing sequence SEQ ID NO 3) of encoding, at the enterprising line retrieval of American National biotechnology information center (NCBI), the homology of finding this heavy chain variable region gene and mouse immuning ball protein heavy chain variable region gene reaches 95.83%, the homology of aminoacid sequence and mouse Ig heavy chain reaches 87%, belongs to the VH gene of mouse Ig.The amino acid sequence analysis result shows that variable region of heavy chain contains clear and definite 4 framework regions (FR) and 3 complementary districts of antigenic determinant (CDR), is characteristic Cys (halfcystine) at the 21st and the 96th.VH _3A4The aminoacid sequence structural framing is as follows:

1～25 FR1

26～33 CDR1

34～50 FR2

51～58 CDR2

59～96 FR3

97～10 5CDR3

106～117 FR4

VL _3A4Full length gene 327bp (seeing sequence SEQ ID NO 2), 109 amino acid (seeing sequence SEQ ID NO 4) of encoding, at the enterprising line retrieval of NCBI, the homology of finding this chain variable region gene and mouse Ig κ chain reaches 98.58%, the homology of aminoacid sequence and mouse Ig κ chain reaches 88%, belongs to the V kappa gene of mouse Ig.The amino acid sequence analysis result shows that variable region of light chain contains clear and definite 4 framework regions (FR) and 3 complementary districts of antigenic determinant (CDR), is characteristic halfcystine (Cys) at the 23rd and the 89th.VL _3A4The aminoacid sequence framework is as follows:

1～26 FR1

27～33 CDR1

34～30 FR2

51～53 CDR2

54～89 FR3

90～98 CDR3

99～109 FR4

Embodiment five

Papain (Papain) enzyme is cut in conjunction with flow cytometer and detected 3A4 internalization degree: papain is the enzyme that extracts in the pawpaw, it can excise the Fc section of immunoglobulin (Ig) from the Fab section, antibody is decomposed into 2 Fab sections and 1 Fc section totally three fragments after the papain effect ^[7]The Fab section of antibody combines with antigen, and FITC then is connected in the Fc section of antibody.In the antibody internalization detected, papain can excise the Fc section of the antibody that is combined in cell surface, and the FITC that is connected with the Fc section is cut thereupon, but can not excise the antibody Fc section of internalization to endochylema.Experimental procedure is as follows:

(1) counting KG1a cell is adjusted concentration to 5 * 10 ⁶/ ml, each reaction tubes get 100 μ l.

(2) every pipe adds 3A4 FITC 2 μ l, hatches 0.25h, 0.5h, 1h, 2h, 4h, 24h at 37 ℃ and 4 ℃ respectively.

(3) PBS 1000g stopped hatching in centrifugal 10 minutes.

(4) experimental group adds papain (final concentration 2mg/ml) enzyme and cut 15 minutes.

(5) PBS 1000g termination in centrifugal 10 minutes enzyme is cut.

(6) flow cytometer detects.

The result is referring to Fig. 6: when hatching for 37 ℃, the cell transmembrane protein transport is near physiological status, and the quick internalization of 3A4 is to cell, and when hatching for 4 ℃, the transmembrane protein transhipment is static, and 3A4 is internalization (6A) not.37 ℃ when hatching, the internalization degree of 3A4 prolongs with incubation time and increases, and hatches 2 hours, and antibody internalization amount is near maximum value, and its internalization rate is 70.8% (6B).

Embodiment six

For confirming the clinical treatment application prospect of 3A4 antibody, we are with the 3A4 mouse source property complete antibody and the Norcantharidin (Norcantharidin of purifying, abbreviation NCTD) immunotoxin (3A4-NCTD) of 3A4 antibody is made in coupling, observe it to the 3A4 positive (acute myeloid cellularity leukemia cell line, KG1a) and negative (acute B chronic myeloid leukemia cell strain, Nalm-6) cell carries out target killing research, found that, 3A4-NCTD has very strong selective killing effect to the KG1a cell, apparently higher than control group (adding the PBS group), and 3A4-NCTD does not but have lethal effect to the Nalm-6 cell of 3A4 feminine gender, illustrate that 3A4-NCTD has the effect of good target killing marrow series leukemia cell strain, the result is referring to Fig. 7: acting on 72 hours, is 25.8% to the inhibiting rate of KG1a, and is 0.63% to the Nalm-6 inhibiting rate; Act on 96 hours, the inhibiting rate of KG1a is reached 42.8%, and be 3.5% to the Nalm-6 inhibiting rate, wherein PBS is a phosphate buffered saline buffer.3A4-NCTD has significant inhibitory effect to KG1a propagation, compares with the PBS group, and the 96h inhibiting rate reaches 42.8% (7A), and cell counting shows that equally 3A4-NCTD has lethal effect for KG1a, and Nalm-6 is not had obvious lethal effect (7B).

Embodiment seven

For confirming that 3A4 can catch leukemic stem cells, flow cytometer detects the ability of CD34+CD38-CD123+ leukemic stem cells in the 3A4 identification leukemia cell line and the blood patient marrow that just turns white.Found that 3A4 positive rate on the CD34+CD38-CD123+KG1a cell strain is 99.05% (referring to Fig. 8 A), positive rate can be up to 90.3% (referring to Fig. 8 B) on the blood patient marrow CD34+CD38-CD123+ cell that just turns white.

Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the embodiment of front is interpreted as only illustrating, but not limits the scope of the invention by any way.

The reference that the present invention relates to

1.Holmes?N.CD45：all?is?not?yet?crystal?clear.Immunology.2006，117(2)：145-155.

2.Glatting?G，Müller?M，Koop?B，et?al.Anti-CD45?monoclonal?antibody?YAML568：Apromising?radioimmunoconjugate?for?targeted?therapy?of?acute?leukemia.J?Nucl?Med.2006，47(8)：1335-1341.

3.John?M，Pagel，Frederick?R，et?al.131I-anti-CD45?antibody?plus?busulfan?andcyclophosphamide?before?allogeneic?hematopoietic?cell?transplantation?for?treatment?of?acutemyeloid?leukemia?in?first?remission.Blood，2006，107(5)2184-2191.

4.Michelle?L.Hermistonl，Zheng?Xu，et?al.CD45：A?critical?regulator?of?signalingthresholds?in?immune?cells.Annu.Rev.Immunol.2003，21：107-137

5.Hauswirth?AW，Florian?S，Printz?D，et?al.Expression?ofthe?target?receptor?CD33?inCD34+/CD38-/CD123+AML?stem?cells.Eur?J?Clin?Invest.2007，Jan；37(1)：73-82

6. forestry face, the Zhang Ling chief editor. modern cell and molecular immunology. first version. Beijing: the .2000 of science tech publishing house, p489-521.

7.Pierre?Haquette，Barisa?Talbi，Sigolène?Canaguier，et?al.Functionalized?cationic(η6-arene)ruthenium(II)complexes?for?site-specific?and?covalent?anchoring?to?papain?frompapaya?latex.Synthesis，X-ray?structures?and?reactivity?studies.Tetrahedron?Letters，2008，49(31)：4670-467.

The sequence that the present invention relates to

＜120〉anti-human CD 45 RA rat immune globulin variable region gene and purposes

<160>8

<170>Patent?In?Version?2.1

<210>1

<211>351

＜213〉artificial sequence

<220>

<223>94a?88c?91g?78t

<400>1

1 CAGGTGCAGC?TGAAGCAGTC?TGGGGCTGAA?CTGGCAAAAC?CTGGGGCCTC?AGTGAAGATG

61 TCCTGCAAGG?CTTCTGGCTC?CACCTTTACT?ACCTACTGGA?TACACTGGGT?AAAACAGAGG

121?CCTGGACAGG?GTCTGGAATG?GATTGGATAC?ATTAATCCTA?ACACTGGTTA?TACTGAGTAC

181?AATCAGAAGT?TCAAGGCCAA?GGCCACATTG?ACTGCAGACA?AATCCTCCAG?CACAGCCTAC

241?ATGCAACTGA?GCAGCCTGAC?ATCTGAGGAC?TCTGCAGTCT?ATTACTGTGT?AAGATTTATT

301?ACGGTAGTAG?GGGGGTGGGG?CCAAGGCACC?ACTCTCACAG?TCTCCTCAGC?C

<210>2

<211>327

<212>DNA

＜213〉artificial sequence

<220>

<223>75a?99c?75g?78t

<400>2

1 CAAATTGTTC?TCACCCAGTC?TCCAGCAATC?ATGTCTGCAT?ATCTAGGGGA?ACGGGTCACC

61 ATGACCTGCA?CTGCCAGTTC?AAGTGTAAGT?TCCAGTCACT?TGCACTGGTA?CCAGCAGAAG

121?CCAGGATCCT?CCCCCAAACT?CTGGATTTAT?AGCACATCCA?ACCTGGCTTC?TGGAGTCCCA

181?GCTCGCTTCA?GTGGCAGTGG?GTCTGGGACC?TCTTACTCTC?TCACAATCAG?CAGCATGGAG

241?ACTGAAGATG?CTGCCACTTA?TTACTGCCAC?CAGTATCATC?GTTCCCCGCT?CACGTTCGGT

301?GCTGGGACCA?AGCTGGAGCT?GAAACGG

<210>3

<211>117

<212>

＜213〉artificial sequence

<220>

<400>3

1 QVQLKQSGAE?LAKPGASVKM?SCKASGSTFT?TYWIHWVKQR?PGQGLEWIGY

61 INPNTGYTEY?NQKFKAKATL?TADKSSSTAY?MQLSSLTSED?SAVYYCVRFI

101?TVVGGWGQGT?TLTVSSA

<210>4

<211>109

<212>

＜213〉artificial sequence

<220>

<221>

<400>4

1 QIVLTQSPAI?MSAYLGERVT?MTCTASSSVS?SSHLHWYQQK?PGSSPKLWIY

51 STSNLASGVP?ARFSGSGSGT?SYSLTISSME?TEDAATYYCH?QYHRSPLTFG

101?AGTKLELKR

<210>5

<211>20

<212>

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain 5 ' primer sequence

<400>5

CAG?GTG?CAG?CTG?AAG?CAG?TC

<210>6

<211>38

<212>

＜213〉artificial sequence

<220>

＜223〉variable region of heavy chain 3 ' primer sequence

<400>6

CCA?GGG?GCC?AGT?GGA?TAG?ACA?AGC?TTG?GGT?GTC?GTT?TT

<210>7

<211>21

<212>

＜213〉artificial sequence

<220>

＜223〉variable region of light chain 5 ' primer sequence

<400>7

CAA?ATT?GTT?CTC?ACC?CAG?TCT

<210>8

<211>21

<212>

＜213〉artificial sequence

<220>

＜223〉variable region of light chain 3 ' primer sequence

<400>8

GGA?TAC?AGT?TGG?TGC?AGC?ATC

Claims

1. anti-human CD 45 RA rat immune globulin variable region gene has the nucleotide sequence of SEQ ID NO 1 and SEQID NO 2.

2. want 1 described a kind of anti-human CD 45 RA rat immune globulin variable region gene according to right, its amino acid sequence coded is SEQ ID NO 3 and SEQ ID NO 4.

3. want the application of 1 or 2 described a kind of anti-human CD 45 RA rat immune globulin variable region genes in preparation targeted therapy blood system malignant tumor medicine according to right.

4. according to want 3 described application according to right, it is characterized in that the application in preparation effective identification leukemic stem cells and filial generation leukemia cell medicine.

5. according to want 3 described application according to right, it is characterized in that, with described anti-human CD 45 RA rat immune globulin variable region gene is that targeted molecular prepares immunotoxin, effectively kills and wounds target cell and reduces the application in the medicine of the toxic side effect of non-target tissue's organ in preparation.