CN101041820A - Macrophagocyte transfer inhibition factor monoclonal antibody and method for making same - Google Patents

Abstract

The invention discloses a preparing method of monoclonal antibody (hMIF) to against human macrophage transferring inhibitor and getting method of amino acid sequence in biological medicine engineering technical domain, which comprises the following steps: augmenting out of hMIF gene from human breast cancer cell strain MDA-MB453 and human breast cancer tissue through reverse transcript polyose chain type reaction; constructing pET-11b/hMIF restructuring plasmid; expressing and purifying on bacillus coli BL21(DE3); immunizing Balb/c mouse; getting splenocyte of mouse and anti-hMIF monoclonal antibody 2D10 merged with SP2/0 cell through hybridomas technology; proceeding antibody subclass appraise and specificity test of antibody; making the secreting monoclonal antibody from 2D10 belong to IgG; setting the light chain as K type; setting the amino acid sequence of variable area of light chain as CRASGRNNAWYKGADGWYKGKAKRYAASSSGVSRSGSG SGTTTSSDATYYCHNDAVYSYGTKVKR; making the amino acid sequence of variable area as VWSGGSTVKG GRSCGYYASGTSTYSMGMNWVRAGKGWVSSSTSVDTYYYADSVKGRTSRDNAKNSYDY DVSRADTAVYYCARDGASSGWGVDATGSRVSCYWGGTVSSWMSAT. This anti-hMIF monoclonal antibody provides effective criterion for further transformation of human source, which can lay the groundwork for treating whole body inflammation in clinical therapeutics.

Description

A kind of Macrophagocyte transfer inhibition factor monoclonal antibody and preparation method thereof

Technical field

The present invention relates to bio-pharmaceutical engineer technology domain, is to comprise the method that relates to anti-Macrophagocyte transfer inhibition factor (MIF) Monoclonal Antibody and aminoacid sequence acquisition thereof.

Background technology

The treatment of traumatic shock is the clinical major issue that needs solution.Research in the past mostly traumatic shock owing to Hypovolemia, but many traumatic shock patients are after injured a couple of days because multiple organ dysfunction syndrome (multipleorgan dysfunction syndrome takes place, MODS) and dead, Q volume of blood is normal when dead.Think at present, severe trauma can cause serious systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS), and SIRS pathogenetic key link that is MODS, prevention will improve the survival rate of traumatic shock greatly with the generation of correcting SIRS, referring to Baue AE, Shock.1995,4 (1): 39-40; Lee CC etc., Ann Emerg Med, 2001,38 (2): 170-176.

SIRS is complex interactions result between the excessive inflammation that produces after the body wound, blood coagulation and the weakened fibrinolytic process, referring to Cunneen J etc., AACN Clin Issues.2004,15 (1): 18244; Riewald M etc., CritCare.2003,7 (2): 123-129.Wound activates the effector cell of body reticuloendothelial system, produces the various kinds of cell factor (as TNF-α, IL-1, IL-6, IL-8, IFN-γ).These cytokines promote that further more inflammatory mediators produce, and various media interact, and cause cascade reaction, strengthen inflammatory process.Constantly enhanced inflammation, coagulation process cause a large amount of inflammatory cells and endothelial cell apoptosis, thrombosis, microcirculation function destroys, simultaneously a large amount of secondary medium discharge, and as arachidonic acid metabolite, oxyradical, oxynitrides etc., directly cause physiologic derangement, cause serious clinical consequences, referring to Ely EW etc., AmJ Crit Care.2003,12 (20:120-133; Mao Yilei etc., the practical surgical magazine .2000 of China, 20 (12): 748-775. is because the various early stage cytokines that inspire inflammatory reaction are being brought into play not same-action in SIRS, in the research of the nineties in last century, effect in SIRS has proposed anti-cytokine therapies treatment SIRS to many scholars at these cytokines, as the TNF monoclonal antibody, soluble TNF acceptor, the IL-1 receptor antagonist, anti-IL-6 monoclonal antibody, anti-IL-8 monoclonal antibody, PAF antagonist TCV-309 and paf receptor antagonists BN52021, but because these target molecules do not have critical effect to the generation of SIRS, the result of clinical trial is disappointing, referring to Minneci P etc., Eur J Clin Microbiol InfectDis.2003,22 (1): 1-9; Bloom B etc., Science.1966 (1530:80-82.After entering this century, a kind of new thrombin inhibitors people activated protein C (Drotrecogin alfa) is treated the medicine of serious septicemia and is entered clinical as first by drugs approved by FDA.It can reduce the level of blood coagulation marker (D-dimers) and markers of inflammation thing (IL-6) in the blood plasma simultaneously, improves the survival rate of serious septic patient, and the crucial upstream molecule that medium discharges before the prompting blocking-up inflammation can be treated SIRS.Though Drotrecogin alfa determined curative effect, as a kind of natural anticoagulant, hemorrhage is its untoward reaction that is difficult to avoid, and makes it be difficult to widespread usage in the SIRS treatment, so medium retardance target spot more and more comes into one's own before seeking better inflammation.

Before more than 40 years, in the process of research delayed type hypersensitivity, from the culture supernatant of activated T cells, find to have the protein factor that suppresses the effect that scavenger cell moves first, at that time with its called after macrophage migration inhibitory factor (macrophage migration inhibitor factor, MIF).This also is human first cytokine of finding owing to can not clone its encoding gene, to the research secular stagnation of its function not before.Along with development of molecular biology, successfully cloned mif gene in 1989 first, rediscover the many even more important function of MIF except that the inhibition scavenger cell moves in recent years: 1. MIF does not belong to any cytokine superfamily, all can find its homologous gene from blue-green algae, tick class insect, bird to mouse etc., constructive expression in immunocyte and endocrine cell (as pituicyte) not only, also can with epithelial cell that external environment directly contacts in abduction delivering, referring to Bernhagen J etc., Nature.1993 (365): 756-759.2. MIF can directly or indirectly promote the generation and the expression of the preceding medium (TNF, IL-1 β, IL-2, IL-6, IL-8, IFN-γ) of inflammation, macrophage inflammatory protein 2, NO, COX2 and arachidonic acid pathway product, referring to Thierry C etc., NATURE REVIEWSIMMUNOLOGY.2003, (3): 791-800.3. MIF not only can escape the immunosuppressive effect of glucocorticosteroid, and can reverse the restraining effect of glucocorticosteroid to other inflammatory mediators, serves as the physiology antagonist of glucocorticosteroid; Referring to Fingerle-RowsonG etc., Am.J.Pathol.2003 (162): 47-56; Beishuizen A etc., Clin Endocrino Meta.2001,86 (6): 1811-1817.4. MIF can keep the effect that it promotes inflammation by the macrophage apoptosis that suppress to rely on wild type p53, referring to Thierry C etc., and NATURE REVIEWS IMMUNOLOGY.2003, (3): 791-800.Calandra and Roger write articles on NATURE REVIEWS IMMUNOLOGY and pointed out in October, 2003, MIF is the indispensable composition of inflammatory reaction, being the regulatory factor of inherency immunity (innate immunity) maincenter, is that the host resists indispensable member in microorganism defence system and the stress reaction.Under the normal condition, when physical stress and severe infections, hypothalamic-pituitary-adrenal system (hpa axis) activates, and ACTH, level of adrenocortical hormone raise rapidly in the blood, plays the inhibition immune response, resists effect that stress, protect the host.Along with the hpa axis activation, MIF discharges (central release) from pituicyte, simultaneously after infection, extracellular toxin and cytokine induction mononuclear macrophage discharge a large amount of MIF (periphery release) in the bacterium that exists in the body, thereby induce scavenger cell and a large amount of cytokines of activated T emiocytosis, greatly promote inflammation and immune response.

Be stranded external calendar year 2001 and studied the patient that 17 examples continue SIRS, it is divided into survival group and dead group, detect blood MIF, TNF-α, IFN-γ and reflunomide level.The result shows, in the dead group of SIRS patient, MIF and TNF-alpha levels are apparently higher than SIRS survival group and control group, and MIF and TNF-alpha levels have obvious dependency, the poor prognosis that high MIF and TNF-alpha levels and SIRS patient have been described is closely related, and prompting MIF is a crucial medium among the SIRS, referring to Gando S etc., IntensiveCare Med.2001,27:1187-1193.By measuring the relation between the final prognosis of disease of reaching of MIF and HPA and other cytokine in the critical illness, disease severity, ARDS, studied the effect of the MIF in the critical illness in the neuroendocrine regulating networks, they find, the MIF level is lasting rising in the patients serum of Sepsis shock, parallel with concentration of cortisol, but with the horizontal negative correlation of ACTH.The non-survival patients serum MIF concentration of Sepsis shock is organized apparently higher than survival, Sepsis companion ARDS patient's serum MIF level also is significantly higher than no ARDS person, and multiple regression analysis shows that the MIF level raises and the ARDS existence is the independent prediction factor of its prognosis mala.Thierry etc. carry out intraperitoneal injection with intestinal bacteria in experiment, duplicate the animal model of bacterial peritonitis Sepsis shock, find that MIF concentration obviously raises in PE and blood.In addition, at TNF-α gene knockout type (TNF-α ^--) mouse model in, utilization caecum ligation puncture is hindered and is caused peritonitis, gives the mono-clonal neutralizing antibody of anti-MIF immediately, found that its survival rate obviously improves (rising to 62% by 0%), referring to Calandra T etc., Nat Med.2000 (6): 164-170.Marcelo etc. also find in experimental mouse with knocking out (MIF with the MIF effect or with mif gene in the neutrality MIF antibody ^--) can watch for animals, prevent fatefulue endotoxemia or streptococcus toxin shock, referring to Pollak T etc., Immun.2005; 73 (10): 6488-6492.More than research is all pointed out MIF not rely on other inflammatory mediators and is brought into play the effect that promotes inflammation, is neuroendocrine medium crucial in the systemic inflammatory response.Launched to utilize the clinical development strategy of MIF specific antibody treatment septic patient in the U.S..In sum, very crucial upstream molecule when MIF may be the inflammation generation, the effect of blocking-up MIF not only can suppress the expression of the preceding medium of its downstream inflammation, also can reach the purpose of correcting SIRS by the immunosuppressive action that improves glucocorticosteroid, referring to Al-Abed D etc., J Biol Chem.2005; 280 (44): 36541-36544.

The mucomembranous cell of leaf cell, scavenger cell, eosinophilic granulocyte, stomach and enteron aisle, chief cell, pancreatic cell and mouse adipocyte are all found the high level expression of MIF before the physiological status pendant, and prompting MIF has biological function widely.Bucala etc. just find that LPS can induce prepituitary gland cell expressing MIF when clone mouse source property MIF, after laboratory animal gives LPS, MIF in the serum obviously raises, and anterior pituitary resection and the comparative study of T cellular immunity deficiency mouse confirm that the MIF that raises in the serum derives from the prepituitary gland cell.Reorganization MIF can significantly improve the septic shock lethality rate of LPS inductive laboratory animal, and anti-MIF monoclonal anti physical efficiency obviously reduces the mortality ratio of animal, and the function of prompting blocking-up or inhibition MIF may have a good application prospect in anti-septic shock.

Clone total length MIF cDNA from the activated human T lymphocyte, sequential analysis can realize that in prokaryotic expression carrier pET-11b MIF efficiently expresses after confirming, the expression amount of MIF accounts for about 20% of bacterial protein amount.By hybridoma technology, the mouse monoclonal antibody of the anti-people MIF of acquisition has the significant protection therapeutic action to LPS septic shock animal.Because this monoclonal antibody is the mouse endogenous binding protein, can not carry out the transition to human body and use, can be widely used in the MIF content of vitro detection serum or tissue.

The research of monoclonal antibody medicine has in recent years obtained very great achievement, not only can implement humanization modified by genetic engineering technique to mouse source property monoclonal antibody, reduce the immunogenicity of mouse source property monoclonal antibody human, can also present the avidity that improves humanized antibody with the ribosomal display technology by phage surface, prepare the antibody drug that is fit to the human body application.In addition, also can utilize phage antibody library and transgenic animal research and development humanized antibody.Up to now, 9 kinds of treatments of U.S. FDA and European Community's approved antibody enters clinical use, and annual sales amount reaches 2,000,000,000 dollars.At present the whole world has 250 tame enterprises to be devoted to treat research with antibody, and product just under development has 700 kinds approximately, has wherein entered clinical study about 100 kinds, and monoclonal antibody medicine has accounted for 25% of biotechnology medicine in all exploitations.

Someone has implemented humanization modified to mouse-anti hepatoma monoclonal antibody HAb25, HAb25 antibody is light from analyzing, the heavy chain variable region gene structure is started with, the homology mould is built the three-dimensional structure of method prediction HAb25 antibody variable region, based on structured data, take single the most seemingly framework region method of substitution that it is carried out the humanization design, overlapping extension PCR method makes up the full gene of HAb25 Humanized single chain antibody (hscFv25), and recombinant expressed hscFv25 antibody has kept the specificity of parental antibody basically.Simultaneously, each amino acid of CDR3 district light, heavy chain of mouse monoclonal antibody is carried out random mutation, present the mutant that filters out high-affinity through phage surface again, affinity of antibody can have been improved tens of times with round pcr.

The control of septic shock is a thorny problem clinically, does not still have effective treatment means so far.The reorganization mif protein can aggravate the death of septic shock, and anti-MIF antibody then has significant protectiveness therapeutic action to the animal of septic shock.The mouse source property MIF monoclonal antibody that utilization acquires in LPS inductive septic shock animal model, shows clear and definite protectiveness therapeutic action.If in the past on the basis of mouse endogenous antibody monoclonal antibody human source transformation research, efficiently obtain the anti-MIF Humanized monoclonal antibodies of high-affinity by the biology recombinant technology, will have very significant clinical meaning to treating SIRS.

Because the generation development and the prognosis of diseases such as hMIF and struvite, autoimmune disease, tumour and wound are closely related, the clinical application of the especially anti-hMIF neutrality antibody of hMIF has become the focus of domestic and international research.Up-to-date studies confirm that, anti-MIF monoclonal antibody has significant provide protection in cecal ligation and perforation art (CLP) inductive mouse infection shock model He in anti-II Collagen Type VI antibody/LPS inductive mouse arthritis model; In addition, the anti-hMIF monoclonal antibody growth and the blood vessel that also can suppress mouse interior tumor had an effect.Thereby anti-hMIF monoclonal antibody is with a wide range of applications.

Mouse source property monoclonal antibody is owing to be easy to be subjected to the repulsion of human immune system, and the transformation period is short, and its Fc section human activin effect system effectively, so have only this strain mouse endogenous antibody of Muromonab-CD3 to use clinically at present.At present, chimeric antibody, humanized antibody, medicine lotus root len antibody become the main flow of therapeutic antibodies, and human antibody occurred along with the maturation of antibody library technology and transgenic mice technology in recent years.Till 2005, in the 18 strain antibody class medicines of FDA approval, still based on people-mouse chimeric antibody, humanized antibody, human antibody has only Adalimumab one strain.The present invention has successfully prepared 2D ₁₀Anti-hMIF monoclonal antibody is for anti-hMIF chimeric antibody, single-chain antibody, humanized antibody preparation and clinical application thereof are laid a good foundation.

Summary of the invention

Purpose of the present invention proposes a kind of mouse source property MIF MONOCLONAL ANTIBODIES SPECIFIC FOR method just in order to address the above problem, and obtains aminoacid sequence, is its further humanization modified laying the foundation.

Technical solution of the present invention:

A. the acquisition of recombinant human mif gene: people MIF (hMIF) the cDNA sequence of delivering according to GenBank, be CCGATGTTCATCGTAAACACCAACGTGCCCCGCGCCTCCGTGCCGGACGGGTTCCT CTCCGAGCTCACCCAGCAGCTGGCGCAGGCCACCGGCAAGCCCCCCCAGTACATCG CGGTGCACGTGGTCCCGGACCAGCTCATGGCCTTCGGCGGCTCCAGCGAGCCGTGC GCGCTCTGCAGCCTGCACAGCATCGGCAAGATCGGCGGCGCGCAGAACCGCTCCTA CAGCAAGCTGCTGTGCGGCCTGCTGGCCGAGCGCCTGCGCATCAGCCCGGACAGGG TCTACATCAACTATTACGACATGAACGCGGCCAATGTGGGCTGGAACAACTCCACC TTCGCCTAAGAGCCGCA, having designed upstream primer is 5 '-CGCGGATCCTGCGGCTCTTAGGCGAAGG-3 ' (containing the BamhI site); Downstream primer is 5 '-CGCCATATGCCGATGTTCATCGTAAACAC-3 ' (containing the NdeI site); upstream and downstream primer 5 ' end all has the protection base; from human breast cancer cell strain MDA-MB453, human breast carcinoma tissue amplification hMIF gene, make up the pET11b/hMIF recombinant plasmid through RT-PCR.

B. the recombinate prokaryotic expression of hMIF: recombinant plasmid pET11b/hMIF makes up transformed into escherichia coli BL21 (DE3), induces the proteic expression of hMIF by grouping action time of IPTG; Do not compare with inducing group behind the Tricine SDS-PAGE electrophoresis, IPTG induces the group bacterium should have a molecular weight to be about the differential protein band of 12.5kD, conforms to the proteic estimated molecular weight of hMIF; Write down the influence of induction time, and further obtain the phraseology of mif protein the expressing quantity height.

C. the recombinate purifying of hMIF: the BL21 bacterium (pET11b/hMIF) of recombinating is induced fermentation, carrying out ultrasonic bacteria breaking, carry out purifying through cationic exchange and hydrophobic chromatography, determine purifying process, and whether can carry out specific reaction with mouse-anti hMIF monoclonal antibody with the hMIF albumen of Western blotting purification Identification.

D. anti-hMIF MONOCLONAL ANTIBODIES SPECIFIC FOR: with the hMIF protein immunization Balb/c mouse of purifying,, utilize indirect elisa method and cloning to filter out hybridoma cell strain that can the anti-hMIF monoclonal antibody of stably excreting and detect tiring of its cells and supernatant through cytogamy repeatedly by hybridoma technology.Afterwards to the hybridoma karyotyping, clear and definite chromosome number confirms that hybridoma is that mouse boosting cell and SP2/0 fusion form really, carries out the specificity experiment of antibody subgroup identification and antibody again, reach albumen test, with the purpose of other albumen no cross reactions with hMIF.

E. the sequential analysis of anti-hMIF monoclonal antibody amino acid:

Through amino acid sequence analysis, 15 aminoacid sequences of anti-hMIF monoclonal antibody variable region of light chain aminoterminal are: MDMGGAWDVGDRVTT, 15 aminoacid sequences of variable region of heavy chain aminoterminal are: VWSGGSTVKGGRSCG, 3 aminoacid sequences of variable region of light chain carboxyl terminal are: VKR, 3 aminoacid sequences of variable region of heavy chain carboxyl terminal are: SAT.Through liquid matter logotype amino acid sequence analysis, the aminoacid sequence that obtains anti-hMIF monoclonal antibody variable region of light chain is: CRASGRNNAWYKGADGWYKGKAKRYAASSSGVSRSGSGSGTTTSSDATYYCHNDAV YSYGTKVKR, the aminoacid sequence of variable region of heavy chain is: VWSGGSTVKGGRSCGYYASGTSTYSMGMNWVRAGKGWVSSSTSVDTYYYADSVKGR TSRDNAKNSYDYDVSRADTAVYYCARDGASSGWGVDATGSRVSCYWGGTVSSWMSA T.

The present invention can significantly improve the output of anti-MIF monoclonal antibody by technique scheme, provides effective foundation for further humanization modified.

Description of drawings

The behave agarose gel electrophoresis interpretation of result figure of MIF RT-PCR amplified production of Fig. 1.

Wherein, 1, DNA marker; 2, template is from the PCR product of MDA-MB453 cell strain; 3, template is from the PCR product of breast cancer tissue.

Fig. 2 is that the double digestion of recombinant plasmid pET11b/hMIF is identified collection of illustrative plates.

Wherein, 1, DNA marker; 2, the pET11b/hMIF plasmid is through BamH I and Nde I double digestion collection of illustrative plates.

Fig. 3 is the mif gene sequencer map of clone from the MDA-MB453 cell.

Fig. 4 is the protein induced expression and purification SDS-PAGE of reorganization hMIF electrophorogram.

Wherein, 1, protein molecular weight marker; 2, pET11b/hMIF/BL21 is abduction delivering not; 3, the pET11b/hMIF/BL21IPTG abduction delivering; 4, the first purifying hMIF of SP Sepharose Fast Flow chromatography column protein graphical spectrum; 5, the final purifying hMIF of Hitrap butylFF chromatography column protein graphical spectrum.

Fig. 5 is the proteic HPLC collection of illustrative plates of the first purifying hMIF of SP Sepharose Fast Flow chromatography column.

Fig. 6 is the proteic HPLC collection of illustrative plates of the final purifying hMIF of Hitrap butyl FF chromatography column.

Fig. 7 is that the immunoblotting of purified product is identified collection of illustrative plates.

Wherein, 1, the hMIF albumen of purifying; 2, the GST-hMAM contrast.

Fig. 8 is hybridoma monoclonal cell strain 2D ₁₀1000 o'clock analysis collection of illustrative plates are amplified in nuclear staining body Giemsa dyeing back.

Fig. 9 is ascites monoclonal antibody 2D ₁₀Immunoblotting colour developing collection of illustrative plates.

Embodiment

The present invention clones hMIF cDNA from human breast cancer cell strain MDA-MB453, human breast carcinoma tissue, make up its prokaryotic expression carrier, the expression and purification mif protein, and preparation mouse-anti people MIF monoclonal antibody, obtain the tire hybridoma cell strain of high affinity antibody of number strain secretion high specific height, for the applied research of hMIF albumen and anti-hMIF antibody lays the first stone.

The present invention utilizes the RT-PCR method successfully to be cloned into hMIFcDNA from human breast cancer cell strain MDA-MB453 and human breast carcinoma tissue, and has made up prokaryotic expression carrier pET11b, and it is in full accord that hMIF sequence that the order-checking confirmation is cloned into and Genebank report.Recombinant plasmid pET11b/hMIF successful expression in BL21 (DE3) goes out the hMIF of solubility, after the fermentation of reorganization bacterium, obtain highly purified reorganization hMIF albumen by cationic exchange and hydrophobic chromatography, it is 1.145g/l that the Lowry method is measured protein concentration, and the purity that HPLC measures finished product hMIF reaches 95.40%.It is 95.40% hMIF albumen that the wet bacterium of 1g can obtain 53mg purity, and yield is about 17.7%.The utilization hybridoma technology has successfully been set up 1 strain and has been secreted the hybridoma cell strain 2D of anti-hMIF monoclonal antibody ₁₀, its chromosome number is 84, and near mouse boosting cell karyomit(e) and SP2/0 karyomit(e) sum, confirmation is the syzygy of mouse boosting cell and SP2/0 cell, and the cell strain vitro culture still can stably excreting antibody more than 3 months.The fast qualitative test paper is measured 2D ₁₀Cell strain secrete monoclonal antibody subclass is IgG ₁, light chain is the κ type.Immunoblotting and ELISA result show: this strain monoclonal antibody high specificity, only with the hMIF specific combination, and survey the albumen no cross reaction with other.The present invention has also set up Tricine-SDS-PAGE electrophoresis method that detects small molecular weight protein and the indirect elisa method that detects the hMIF antibody titer, simultaneously, and 2D ₁₀The successful preparation of monoclonal antibody and CHARACTERISTICS IDENTIFICATION are laid a good foundation for the applied research of this antibody.

Material and source

Bacterial strain and plasmid: escherichia coli cloning bacterial strain DH5 α and escherichia coli expression bacterial strain BL21 (DE3) are the applicant laboratory and preserve, and pET-11b is available from Novagen company.

Enzyme and test kit: restriction enzyme BamH I and Nde IRT-PCR test kit and plasmid extraction test kit are available from Promega company, and sepharose reclaims test kit available from Qiagen company.

Other reagent: the HRP-goat anti-mouse igg is available from Beijing ancient cooking vessel state company, and GST-hMAM fusion rotein, anti-hMIF monoclonal antibody are applicant laboratory purification storage.

PCR primer: give birth to worker's biotechnology company limited by Shanghai and synthesize.Upstream primer is 5 '-CGCGGATCCTGCGGCTCTTAGGCGAAGG-3 '; Downstream primer is 5 '-CGCCATATGCCGATGTTCATCGTAAACAC-3 '.

Embodiment

1.RT-PCR method obtains the hMIF gene

(1) design of primers is with synthetic

According to the hMIF cDNA sequence of having delivered among the GenBank: CCGATGTTCATCGTAAACACCAACGTGCCCCGCGCCTCCGTGCCGGACGGGTTCCT CTCCGAGCTCACCCAGCAGCTGGCGCAGGCCACCGGCAAGCCCCCCCAGTACATCG CGGTGCACGTGGTCCCGGACCAGCTCATGGCCTTCGGCGGCTCCAGCGAGCCGTGC GCGCTCTGCAGCCTGCACAGCATCGGCAAGATCGGCGGCGCGCAGAACCGCTCCTA CAGCAAGCTGCTGTGCGGCCTGCTGGCCGAGCGCCTGCGCATCAGCCCGGACAGGG TCTACATCAACTATTACGACATGAACGCGGCCAATGTGGGCTGGAACAACTCCACC TTCGCCTAAGAGCCGCA, designed a pair of special primer. Upstream primer is 5 '-CGCGGATCCTGCGGCTCTTAGGCGAAGG-3 ' (containing the BamhI site); Downstream primer is 5 '-CGCCATATGCCGATGTTCATCGTAAACAC-3 ' (containing the NdeI site), and upstream and downstream primer 5 ' end all has the protection base, entrusts Shanghai to give birth to worker's biotechnology company limited above-mentioned primer and synthesizes.

(2) the MDA-MB453 cell total rna extracts

Mammary cancer MDA-MB453 cell frozen in the liquid nitrogen is taken out, put into 37 ℃ of water-baths rapidly, and rock frozen pipe frequently, dissolve fully up to frozen storing liquid, in containing the RPMI1640 substratum of 10% foetal calf serum, 37 ℃, 5% CO ₂After cultivating in the incubator, the centrifugal 800rpm 10min of level removes supernatant, the resuspended sedimentary cell of 1ml serum-free RPMI1640 substratum, and inverted microscope numeration down is with the centrifugal again collection of cell; Extract total RNA of breast carcinoma cell strain with Trizol reagent: with Trizol 1ml/5 * 10 by volume ⁶The ratio of cell joins in the cell precipitation, room temperature is placed 5min, makes its abundant cracking, the centrifugal 5min of 12000rpm, collect supernatant, add chloroform by 200 μ l chloroform/ml Trizol, room temperature is placed 15min, 4 ℃ of centrifugal 15min of 12000g behind the vibration mixing, draw the upper strata water, to another centrifuge tube, to press 0.5ml Virahol/ml Trizol and add the Virahol mixing, room temperature is placed 5-10min, 4 ℃ of centrifugal 10min of 12000g, abandon supernatant, RNA is sunken to pipe and adds 75% ethanol by 1ml 75% ethanol/ml Trizol at the end, gentle vibration centrifuge tube, precipitation suspends, 4 ℃ of centrifugal 5min of 8000g abandon supernatant as far as possible, and room temperature is dried or vacuum-drying 5-10min, dissolve the RNA sample with the sterile pure water that 50 μ l DEPC handle, 55-60 ℃, 5-10min surveys quantitative RNA concentration of OD value and purity.

(3) the total RNA of breast cancer tissue extracts

Mammary cancer sample source: human breast carcinoma tissue is southwest department of general surgery of hospital of a Third Military Medical University excision sample, and the pathology somatotype is the early infiltrate duct carcinoma; The collection of breast cancer cell: the mammary cancer sample is placed on the copper mesh, remove adipose connective tissue,, can obtain cell suspension while grind with the incomplete substratum flushing of RPMI1640 cell, with the centrifugal 800 * g 10min of cell suspension level, collecting cell precipitates again; Extract of the extraction of total RNA step with the MDA-MB453 cell total rna.

(4)RT-PCR

RT-PCR is undertaken by the test kit explanation of Promega company: 1 μ g (2 μ l) MDA-MB453 cell total rna and 1 μ lOligo (dT) primer are joined in the water of 14.2 μ l DEPC processing, behind 70 ℃ of heating 5min, ice bath 5min, add 5 μ l 5 * reaction buffer, 1.25 μ l dNTP, 1 μ l M-MLV ThermoScript II, 0.55 μ lRNA enzyme inhibitors more successively, final volume is 25 μ l, 42 ℃ of 2h, 95 ℃ of sex change 5min ,-20 ℃ of preservations.Get above-mentioned RT product 1 μ l, 10 * reaction buffer, 5 μ l, each 1 μ l (final concentration is 50pmol) of upstream and downstream primer, dNTP 2 μ l, Taq enzyme 1 μ l (2U), 25mmol/L MgCl ₂2.5 μ l, ddH2O 36.5 μ l, cumulative volume are 50 μ l.94 ℃ of pre-sex change 4min, 94 ℃ of 1min then, 55 ℃ of 1min, 72 ℃ of 1min, 35 circulations, last 72 ℃ are extended 5min, get PCR product 10 μ l after reaction finishes and do 1.5% agarose gel electrophoresis, confirm to obtain size and are the specific fragment of 353bp (Fig. 1).

(5) recovery of hMIF goal gene

Ultraviolet lamp downcuts the adhesive tape that contains the specific PCR product down, putting into the EP pipe weighs, the Buffer QG that adds 3 times of volumes, 50 ℃ of water-bath 10min melt fully until adhesive tape, and check whether color is yellow, as be purple or orange, add 10 μ l 3mol/L sodium-acetates (pH5.0), mixing is until color restoration, 100% Virahol of adhesive tape volumes such as adding (adding 1 μ l) as 1mg glue, put upside down mixing, 1 MinElute post is put into the 2ml collection tube that provides, and be put on the shelf, whole samples are moved in the post, and centrifugal 1min pours out the liquid that flows through pillar, the MinElute post is put back in the same collection tube, add 500 μ l Buffer QG in the MinElute post, centrifugal 1min pours out the liquid that flows through pillar, the MinElute post is put back in the same collection tube, add 750 μ l Buffer PE and wash in the MinElute post, centrifugal 1min behind the placement 5min pours out the liquid that flows through pillar, centrifugal 1min, the MinElute post is put into 1 clean 1.5ml centrifuge tube, add the center membrane of 10 μ l Buffer EB on the MinElute post, eluted dna, centrifugal 1min behind the pillar placement 1min, the DNA of collection wash-out.

2.hMIF construction of recombinant plasmid

With the pET-11b carrier with BamH I and Nde I double digestion digestion 4h after with the PCR product T that reclaims ₄4 ℃ of connections of spending the night of dna ligase, get an aseptic centrifuge tube, add the competence DH5 α bacterium 200 μ l that prepared, ice bath, drawing 1 μ l connects in the product adding pipe, transform DH5 α bacterium, pat the tube wall mixing, ice bath 30 minutes, 42 ℃ of water-baths 90 seconds, take out centrifuge tube ice bath 2 minutes again, 2 * YT nutrient solution the mixing that adds 800 μ l room temperatures, 37 ℃ of shaking table 220rpm shaking culture 1h are respectively with 50 μ l, 200 μ l and remaining whole transformed bacteria liquid are applied on 32 * YT culture plates that contain the Pyocianil resistance, 37 ℃ of constant incubator incubated overnight, next day, the picking white colony was inoculated in 2 * YT substratum enlarged culturing.

3.hMIF the recombinant plasmid enzyme is cut evaluation

The precipitation thalline, 12000rpm, centrifugal 1min, abandon supernatant, blot as far as possible, with the solution I of 150 μ l precoolings that above-mentioned bacterial precipitation is resuspended, thermal agitation, freshly prepared solution II 300 μ l put upside down mixing 5 times, ice bath 3-5min gently, make its clarification, add the solution III 150 μ l of precooling, gently ice bath 10min behind the mixing, make protein be uniformly distributed in aqueous phase, the solution IV 150 μ l that add precooling, mixing gently, the centrifugal 10min of 12000rpm.The careful water (about 400 μ l) of drawing moves in another 1.5ml centrifuge tube, adds 2 μ l RNaseA (10 μ g/ml), 55 ℃ of water-bath 10min.Add 400 μ lTris-phenol and 400 μ l chloroforms again, the whirlpool mixing that shakes, the centrifugal 10min of 12000rpm.Get supernatant and be equipped with in the 1.5ml centrifuge tube of 600 μ l Virahols to another, put upside down back and forth several times, 4 ℃ of centrifugal 10min of 12000rpm abandon supernatant.Wash DNA 2 times with 70% ethanol 1ml, the centrifugal 3min of 12000rpm abandons supernatant, and drying at room temperature 10-20min adds TE 20 μ l dissolving plasmid DNA ,-20 ℃ of preservations.

Get and run the evaluation of 1.5% agarose gel electrophoresis after 10 μ l plasmids are used BamH I Nde I double digestion, the endonuclease reaction system is: PET11b/hMIF plasmid DNA 10 μ l, BamH I 1 μ l, Nde I 1 μ l, 10 * buffer K, 2 μ l, ddH ₂O6 μ l, centrifugal 30s, 37 ℃ of water-bath 4h get enzyme and cut capable 1.5% agarose gel electrophoresis of product, and a visible size of downcutting is about the specific fragment of 353bp, shows and has inserted the fragment (Fig. 2) that size is about 353bp in the pET11b plasmid.

4.hMIF recombinant plasmid inserts the segment order-checking

Two pET11b/hMIF recombinant plasmids are sent to Shanghai Bo Ya company order-checking, and use Blast software the hMIF cDNA of sequencing result and GenBank is carried out homology analysis.The result of order-checking shows that the insertion fragment length is 353bp, with the hMIF cDNA sequence of reporting among the GenBank (Fig. 3) in full accord.

5.hMIF induction expression of protein and evaluation

(1) transforms the BL21 bacterium

The reorganization pET11b/hMIF plasmid of getting last fetched transforms BL21 (DE3) bacterium, coat the LB solid medium that contains the Pyocianil resistance, 37 ℃ of incubator incubated overnight, next day, the picking white colony was inoculated in LB substratum enlarged culturing, use the alkaline lysis method of extracting plasmid, get plasmid with BamHI Nde I double digestion 4h, it is the same that enzyme is cut system, and get enzyme and cut capable 1.5% agarose gel electrophoresis of product 10 μ l and identify consistent with design load.

(2) hMIF induction expression of protein

BL21 bacterium with the commentaries on classics pET11b/hMIF recombinant plasmid of enlarged culturing, with LB solid medium screening mono-clonal bacterium colony, with the transformed bacteria enlarged culturing, survey that to add final concentration when bacterium OD value reaches 0.6-0.8 be that the IPTG of 1mmol/L induces, press time point 2,4,6,8,10,12h collection inductive bacterium.The result shows that the specific proteins band that molecular weight is about 12.5kD appears in IPTG inductive bacterium, conform to the molecular weight values of expection hMIF, and the hMIF expression amount is the highest during 6h, accounts for 30% of total bacterial protein.The BL21 reorganization bacterium of again IPTG being induced 6h is with splitting the broken bacterium of bacterium liquid, get cleer and peaceful precipitation electrophoresis after centrifugal respectively, inductive bacterium liquid and induce the bacterium liquid of 6h to do feminine gender and positive control respectively not, the result finds the differential protein band with the corresponding position of positive control hMIF protein band in supernatant, and precipitation does not have this protein band, confirms that hMIF albumen is solubility expression.

(3) the proteic evaluation of hMIF

Get induced product and do 15%Tricine-SDS PAGE electrophoresis detection, analyze the expression of bacterium hMIF.Each time point is induced the centrifugal 10min of bacterium 12000rpm, collecting precipitation, press resolving gel concentration 15%, concentrate glue 6% and join glue, the liquid of getting each collection tube mixes with 2 * sample loading buffer equal-volume, 100 ℃ are boiled 5-10min, press sample 20 μ l samples on the every hole of gel, compare with middle molecular weight protein matter standard simultaneously, press 120V constant voltage electrophoresis 2h, the capable vertical electrophoresis of 80V 30min, electrophoresis finishes tetrabromophenol sulfonphthalein plastic emitting, take off gel,, from staining fluid, take out gel with the 6h that dyes under the Coomassie brilliant blue R-250 dye liquor room temperature, destainer repeatedly decolours and observes to the colourless rearmounted deionized water of background, scanning gel and shooting.

(4) hMIF recombinant protein solubility is identified

Induce the bacterium liquid of 6h to join in the centrifuge tube 10mlIPTG, the centrifugal 10min of 5000rpm, collect bacterium, the resuspended bacterium of 1ml GTE liquid, the centrifugal 5min of 5000rpm, abandon supernatant, the resuspended bacterium of 1ml GTE adds 100 μ l N,O-Diacetylmuramidases (10mg/ml), mixing, ice bath 20min adds 20 μ l Sodium desoxycholates (40mg/ml), 10 μ l MgCl more successively ₂(1mol/L) with 5 μ l DNase I (1mg/ml), put upside down mixing under the room temperature and make solution by thick thinning, the centrifugal 10min of 12000rpm gets cleer and peaceful precipitation electrophoresis respectively, the inductive bacterium liquid and the bacterium liquid of inducing 6h are not observed electrophoresis result respectively as negative control and positive control.

6.hMIF the purifying of recombinant protein and immunoblotting are identified

(1) purifying of hMIF recombinant protein

Picking mono-clonal reorganization LB bacterium, with high expression level bacterial strain enlarged culturing and inducing in fermentor tank, with the expression bacterium that obtains with the PBS mixing of 20mmol/L pH 5.8 after carrying out ultrasonic bacteria breaking, 10000 * g is centrifugal, and 30min gets supernatant, just purifying is selected the cationic exchange coloum of filling SPSepharose Fast Flow, damping fluid (20m mol/L PBS, pH 5.8) after the balance with the supernatant upper prop, elutriant (1mol/L NaCl with 0～50% gradient, 20mmol/L PBS, pH 5.8) 10 column volumes of wash-out, 100% elutriant is 5 column volumes of wash-out again; The first pure product that obtain are splined on damping fluid (0.75mol/L (NH ₄) ₂SO ₄20mmol/L PBS, pH 5.8) Hitrap butyl FF hydrophobic chromatography post that balance is good, elutriant (the 20mmol/LPBS of 0～100% gradient, pH 5.8) 10 column volumes of wash-out, 100% elutriant is 10 column volumes of wash-out again, collect to obtain finished product albumen, sampling row Tricine SDS-PAGE electrophoresis respectively then, IPTG not inductive reorganization bacterium makes negative control, induces the positive contrast of reorganization bacterium of 6h, and purity reaches (Fig. 4) more than 95% behind the purifying.

(2) immunoblotting of hMIF recombinant protein is identified

Preparation 8%Tricine SDS-PAGE separation gel and 6% concentrates glue.Electrophoresis 80V 30min, 120V 1h.After electrophoresis finishes, take off gel, one with Coomassie brilliant blue R-250 dye liquor rapid dyeing, another piece places transfer printing damping fluid balance 20min-60min, cut the pvdf membrane and the thick filter paper of gel size, place the transfer printing damping fluid to soak 5min with filter paper pvdf membrane soaked 1min in 100% methyl alcohol after, 22V constant voltage electricity transfer printing 1h takes off pvdf membrane, Ponceau S room temperature dyeing 5min, observed result, distillation washing film 5min * 3 times, TBST washing 10min * 3 time, 5% skim-milk closing membrane spends the night under the room temperature, TBST washing 10min * 3 time, adding is diluted to 1: 1000 mouse-anti people MIF monoclonal antibody with 3%BSA/TBS, hatches 4h for 37 ℃, TBST washing 10min * 3 time, add 3%BSA/TBS and be diluted to 1: 5000 sheep anti-mouse igg, hatch 2h for 37 ℃, TBST washing 10min * 3 time, preparation substrate buffer solution, 5mg DAB is dissolved among the 10ml TBS, adds 10 μ l 30%H again ₂O ₂Mixing, film are soaked in the damping fluid, shake gently until colour developing, and the distilled water termination reaction is dried the film taking-up, and scanning is filed.The result shows that the albumen of BL21 transformed bacteria expression and purification can react with anti-hMIF antibody, negative control GST-hMAM fusion rotein then with above-mentioned antibody reactionless (Fig. 7)

7.hMIF recombinant protein concentration, purity are identified

(1) Lowry method (Folin phenol method) is measured the hMIF protein content

The preparation standard curve:

	Blank pipe	Standard pipe	1	Standard pipe 2	Standard pipe 3	Standard pipe 4	Standard pipe 5
								Standard protein liquid (ml) distilled water (ml)	0 1.0	0.2 0.8	0.4 0.6	0.6 0.4	0.8 0.2	1.0 0
Reagent first (ml) reagent second (ml)	(5.0 room temperature is placed 10min) 0.5 (mixing reacts 30min rapidly)

Under the 650nm wavelength, serve as the contrast zeroing with blank pipe, measure the absorbancy of each pipe respectively, be X-coordinate with the protein concentration, absorbancy is an ordinate zou, only makes typical curve.

After the testing protein dilution, ultraviolet spectrophotometer is measured A ₂₆₀The value and, A ₂₈₀Value.According to formula, protein concentration C=(1.45 * A ₂₈₀-0.75 * A ₂₆₀) * extension rate, calculate the rough concentration of testing protein, then with protein sample with distilled water diluting to 25-250 μ g scope, schedule of operation reaction according to last table, determine the 650nm absorbance, on typical curve, find corresponding concentration then, count the concentration of testing protein multiply by extension rate, the multitube calculating mean value, recording concentration is 1.145g/ml.

(2) purified product carries out purity testing and the calculating of hMIF yield with high performance liquid chromatography (HPLC).

The albumen of purifying is analyzed its purity with HPLC, the proteic purity of hMIF reaches (Fig. 5) more than 92.62% behind the first purifying of SP Sepharose Fast Flow cationic exchange coloum as can be known, and hMIF purity can reach 95.40% (Fig. 6) behind the Hitrap butyl FF hydrophobic chromatography purifying once more.As calculated, it is 95.40% hMIF albumen that the wet bacterium of 1g can obtain 53mg purity, and yield is about 17.7%.

8.hMIF immune Balb/c mouse

The hMIF albumen of purifying is taken out from-80 ℃ of refrigerators, the dissolving after-filtration, the Lawry method is measured protein content, and the 20mmol/LPBS of pH5.8 is diluted to 0.5mg/ml with it.Choose 15 6 week age, the about 20g of body weight female Balb/c mouse, be divided into A, B, three groups of immunity of C.The mutual pushing manipulation of double syringe is selected in antigen emulsification for use.During first immunisation, hMIF is mixed with isopyknic Freund's complete adjuvant emulsification, every mouse adds abdominal injection by the amount intracutaneous multiple spot of 100 μ g hMIF.Carried out the second time of immunity for the third time on the the 14th and the 28th day respectively, adjuvant is used incomplete Freund's adjuvant instead, and antigen amount, volume injected and approach are constant, and the 3rd time immunity back indirect elisa method is measured and tired.Merge and carried out booster immunization in preceding 3 days, every mouse peritoneal injection does not add 100 μ g hMIF of adjuvant, cytogamy after 3 days.

9. by the Balb/c mice serum titration of hMIF immunity

The 3rd immunity got blood examination from mouse tail vein in back 10 days and surveyed serum antibody titer.To newly purchase enzyme plate distilled water soaked overnight, standby after drying; Is best effort concentration 5 μ g/ml with coating buffer with antigen diluent, and every hole adds 100 μ l antigen liquids, and 37 ℃ of incubations are after 1 hour, with tape seal, spend the night in 4 ℃, liquid in the plate hole to the greatest extent blots residual reaction liquid in the hole, filling it up with washings crosses once, fill with washings again and slowly rock 2min, incline, five times repeatedly, at last Sptting plate is upside down on the thieving paper, makes that washings flows to end in the hole.With tape seal, this is the enzyme plate of known antigens bag quilt after the seasoning, adds confining liquid 300 μ l, hatches 1-1.5 hour for 37 ℃, washs 5 times; Blood sampling and dilute serum: pinch the mouse tail, use scissors at tail vein scissors one breach behind 75% alcohol disinfecting, get blood 20 μ l, the centrifugal 30min of 2000rpm gets supernatant 1 μ l and adds 999 μ l antibody diluent mixings, and carries out doubling dilution, from 1: 100 to 1: 3200, the every hole of tested serum of dilution is added 100 μ l, get the dilution in 1: 100 of the preceding serum of mouse immune simultaneously and do negative control, antibody diluent is done blank.Hatched 1-1.5 hour for 37 ℃, wash 5 times; The horseradish peroxidase goat anti-mouse igg is diluted to 1: 10000, and every hole adds 100 μ l, hatches 1-1.5 hour for 37 ℃, washs 5 times; Add o-phenylenediamine solution 100 μ l/ holes, room temperature dark place 10-15 minute, every hole adds stop buffer 100 μ l observationss, product after the OPD oxidation is orange red, with enzyme-linked immunosorbent assay instrument record 492nm reading, survey each hole OD value 2.1 times with zeroing back, blank hole greater than negative control OD value, promptly positive.Serum titer reaches 1: 3200, can be used for cytogamy.

10. the preparation of mouse boosting cell suspension and SP2/0 cell suspension

Get a Balb/c mouse that immunity is good, extract the eyeball of mouse sacrificed by exsanguination, the positive control that serum is made ELLSA is collected in the centrifugal back of eye blood, spleen is taken out in aseptic technique, put into the glass dish that fills the incomplete substratum of 10ml, washing, reticular tissue and fatty tissue around carefully peelling off change a glass dish, spleen is pulled out, place 200 order stainless (steel) wires, with the inner core grinding of syringe, with the full substratum flushing of often toing many or too much for use, making splenocyte pass mesh enters in the solution, splenocyte is moved in the 10ml glass centrifuge tube, and the centrifugal 10min of 800rpm level removes supernatant.With too many or too much for use full substratum 10ml washed cell 1 time of method, the cell of centrifugal collecting precipitation, with the resuspended mixing of the incomplete substratum of 10ml, cell counting is about 1 * 10 with cell ⁸Individual cell.

The SP2/0 cell is taken out from liquid nitrogen, put into 37 ℃ of water-baths rapidly, constantly rock, dissolve fully until cell solution, with cell transfer in the 10ml centrifuge tube, the centrifugal 10min of 800rpm level, abandon supernatant, the resuspended precipitation of 10ml complete culture solution is transferred to cell suspension in the 50ml culturing bottle, puts in 37 ℃, 5%CO2 incubator and cultivates.Treat to screen one week of cell with the selection substratum that contains 8-AG behind the cell well-grown; Merged preceding 2 days, 1 bottle of cell is reached 4 bottles, then merge same day cell and be in logarithmic phase, vigor just in time, cell size is evenly, and is round and bright, merge the same day, the SP2/0 cell is blown down from tube wall gently, be collected in the centrifuge tube with the elbow dropper, centrifugal, abandon supernatant, precipitation is tood many or too much for use after the washing of full substratum, and the incomplete substratum of 10ml is resuspended, cell counting is about 5 * 10 ⁷

11. the preparation of nurse cell

Get a non-immune Balb/c mouse, pluck the eyeball sacrificed by exsanguination, 75% alcohol immersion sterilization 5min, cut off mouse skin, mention peritonaeum, cut an osculum with scissors with tweezers, the elbow dropper is drawn the incomplete substratum flushing abdominal cavity of precooling, and washing lotion is drawn in the 50ml centrifuge tube.With too many or too much for use full substratum flushing abdominal cavity 3 times of method, collect washing lotion, the centrifugal 10min of 1000rpm level removes supernatant under the room temperature, incomplete substratum re-suspended cell of 10ml and counting.

12. myeloma cell and spleen bone-marrow-derived lymphocyte merge

Before merging PEG1500 is placed the pre-temperature of 37 ℃ of incubators, draw 1 * 10 ⁷Individual myeloma cell's suspension and 1 * 10 ⁸Individual spleen bone-marrow-derived lymphocyte suspension (cell count 1: 10) is added the incomplete substratum of 30ml to a 50ml centrifuge tube, fully mixing, the centrifugal 10min of 1000rpm abandons supernatant, flicks the pipe end, make the loose one-tenth pasty state of cell mass, with 37 ℃ of water-baths of centrifuge tube, draw the pre-warm 50%PEG1500 solution of 0.8ml with dropper, slowly adding in the cell from the pipe about 2cm in end place along tube wall, the limit edged rotates centrifuge tube, add about 1min, leave standstill 90s then, the incomplete substratum 30ml that dropwise adds 37 ℃ of pre-temperature stops merging, add within the 3min, the slow earlier back of speed is fast, and action is soft, and centrifuge tube is left standstill 5min in 37 ℃ of incubators, take out centrifuge tube, the centrifugal 5min of 1000rpm, supernatant discarded adds 10ml HAT substratum re-suspended cell, piping and druming gently, mixing is seeded to the 96 porocyte culture plates that are covered with nurse cell with fused cell, by 100 μ l/ holes, every culture plate stays 6 holes inoculation SP2/0 cell, as the negative control that HAT selects, put 37 ℃, 5%CO ₂Cultivate in the incubator.

13. the selectivity of fused cell is cultivated

Merging back the 5th day can be at the growing state of observation of cell under the inverted microscope, and adds HAT substratum 100 μ l, and the 10th day available indirect elisa method surveyed hybridoma and tired, and changes the HT culture medium culturing on the 14th day.

14. hybridoma screening

Merge the back 10～14 days, and treated that cell was long at the bottom of the 1/4-1/2 hole of full culture hole the time, adopt indirect elisa method detection culture supernatant, screening positive clone; With the antigen coated enzyme plate of hMIF (0.5ug/ hole), 4 ℃ are spent the night, lavation buffer solution washing 5 times, each 5min, pat dry liquid, every hole adds confining liquid 300 μ l, hatches 2h for 37 ℃, adds 100 μ l cells and supernatant, positive control selects the immune serum of mouse, negative control selects SP2/0 training supernatant, and the blank washings is hatched 2h for 37 ℃; The detersive enzyme target: every hole adds the goat-anti mouse IG antibody of the HRP mark of 100 μ l dilution in 1: 10000, hatches 2h for 37 ℃; Washing pats dry liquid, adds freshly prepared o-phenylenediamine solution 100 μ l/ holes, and room temperature dark place reaction 10--15 minute adds the every hole of stop buffer 100 μ l termination reactions, and microplate reader detects the 492nm absorbance.

The result is for being antigen with the recombinant human macrophage migration inhibition factor, and successively immune balb/c mice is 15, merges altogether 5 times, and success 3 times after 3 time cloningizations and ELISA screening, obtains the hybridoma cell strain 2D that anti-hMIF antibody is secreted in 1 strain ₁₀, its cells and supernatant is tired and is reached 1: 6400, and this strain of hybridoma is through for several times frozen, and subculture in vitro separately is cultivated all can stably excreting antibody more than 3 months.

15. the cloning of positive hybridoma cell

After filtering out positive colony, promptly should adopt limiting dilution assay that positive hybridoma cell is carried out cloning immediately cultivates, the preparation feeder cell, resuspended with the incomplete substratum of 10ml, collect positive colony cell and counting, too many or too much for use full substratum with positive colony cell dilution to 100/20ml, get 96 porocyte culture plates that have been added with nurse cell in advance, add 200 μ l cell suspensions, with remaining positive colony cell transfer enlarged culturing in 24 orifice plates, the collecting cell liquid nitrogen cryopreservation, simultaneously with culture plate at 37 ℃, 5%CO ₂Incubator is cultivated, microscopically observation of cell growing state after the 3rd day, and available ELISA method detects and tires after 10 days, and with the strongest positive colony cloning once more, reaches 100% until the cell positive rate, can decide strain; Measure the hybridoma enlarged culturing that to decide strain after the tiring of the hybridoma cell strain culture supernatant of deciding strain again and frozen in liquid nitrogen.

16. the karyotyping of hybridoma:

To decide the Hybridoma Cell Culture of strain to the logarithmic growth after date, get 1 bottle of cell and make chromosome specimen, fresh Giemsa staining fluid dyeing, 400 times of oily mirrors are observed chromosome morphology and number down, the selective staining body disperses, the non-overlapping visual field, and the digital camera shooting is filed.

With picked-up karyomit(e) result counting, 2D ₁₀Chromosome number is being 84, near mouse boosting cell and myeloma cell's chromosome number purpose summation, illustrates that this strain of hybridoma is the syzygy of mouse boosting cell and Sp2/0 cell.Most of karyomit(e)s are telocentric chromosome, and middle part and submetacentric chromosome are arranged, and this cell is to form (Fig. 8) by mouse boosting cell and SP2/0 cytogamy as can be known.

17. the evaluation of anti-MIF monoclonal antibody subclass

Adopt the Mouse Monoclonal Antibody Isotyping Kit of U.S. Roche company operation by specification to carry out.Get fresh Hybridoma Cell Culture supernatant, PBS with 0.01M pH 7.4 is diluted to 1: 10, getting the culture supernatant that 150 μ l have diluted adds in the reaction tubes, room temperature leaves standstill 30s, the whirlpool 10s that shakes dissolves the latex at the bottom of the reaction tubes fully then, and test strip is inserted into reaction tubes, makes the black end of test strip bottom be immersed in the liquid fully, room temperature leaves standstill 5～10min, and the area relative subclass that blue band occurs is the subclass of IgG.Measure hybridoma monoclonal cell strain 2D through monoclonal antibody subgroup identification test strip ₁₀Excretory IgG monoclonal antibody subclass belongs to IgG ₁, light chain is the κ type.

18. the trace of exempting from service of anti-MIF monoclonal antibody is identified

Get the capable 8%Tricine SDS-PAGE of the hMIF albumen electrophoresis of final purification, electrophoresis takes off gel after finishing, one with Coomassie brilliant blue R-250 dye liquor rapid dyeing, another piece places transfer printing damping fluid balance 20min-60min, 22V constant voltage electrotransfer 1 hour, pvdf membrane after the transfer 5min that dyes in Ponceau S sloughs redness with distilled water again, and transfer film is placed confining liquid, the room temperature sealing is spent the night, discard confining liquid, with 1 * TBST rinsing film 3 times, each 10～15min, adding is with the hybridoma culture supernatant of dilution in 1: 10, hatch 4h for 37 ℃, 1 * TBST rinsing film 3 times, each 10～15min, adding is to be diluted to 1: 5000 HRP-goat anti-mouse igg, hatch 2h for 37 ℃, 1 * TBST washing 3 times, each 10～15min, add DAB colour developing liquid, jog is to developing the color distilled water rinsing termination reaction, observation analysis result and shooting.Immunoblotting is the result show, the culture supernatant of this strain monoclonal antibody can form single protein staining band with the hMIF albumen that is transferred on the pvdf membrane, proves that this strain monoclonal antibody is the monoclonal antibody specific (Fig. 9) at hMIF.

19.ELISA identify the specificity of anti-MIF monoclonal antibody

HMIF, IL-11, Insulin, IBP, GST-hMaM are wrapped the enzyme plate by ELISA respectively by every hole 0.5 μ g, one anti-culture supernatant with anti-hMIF hybridoma cell strain, the monoclonal antibody that detects this strain emiocytosis by indirect elisa method is to above-mentioned antigenic cross reaction situation.Following table is to identify the specific result of the anti-hMIF monoclonal antibody of this strain with ELISA, and this strain antibody has stronger specificity as can be known, only with the hMIF reaction, surveys the albumen no cross reaction with other.

The specificity analyses of monoclonal antibody

McAb	hMIF	GST-hMam	IL-11	Insulin	IBP
						MIF-A 2 MIF-D 5	+++ +++	- -	- -	- -	- -

Wherein antibody-antigen association reaction with-, ± ,+, ++, +++increase progressively.

20. anti-hMIF monoclonal antibody amino acid sequential analysis:

Through amino acid sequence analysis, 15 aminoacid sequences of anti-hMIF monoclonal antibody variable region of light chain aminoterminal are: MDMGGAWDVGDRVTT, 15 aminoacid sequences of variable region of heavy chain aminoterminal are: VWSGGSTVKGGRSCG, 3 aminoacid sequences of variable region of light chain carboxyl terminal are: VKR, 3 aminoacid sequences of variable region of heavy chain carboxyl terminal are: SAT.Through liquid matter logotype amino acid sequence analysis, the aminoacid sequence that obtains anti-hMIF monoclonal antibody variable region of light chain is: CRASGRNNAWYKGADGWYKGKAKRYAASSSGVSRSGSGSGTTTSSDATYYCHNDAV YSYGTKVKR, the aminoacid sequence of variable region of heavy chain is: VWSGGSTVKGGRSCGYYASGTSTYSMGMNWVRAGKGWVSSSTSVDTYYYADSVKGR TSRDNAKNSYDYDVSRADTAVYYCARDGASSGWGVDATGSRVSCYWGGTVSSWMSA T.

Claims (10)

1. human macrophage tumor metastasis suppressor gene monoclonal antibody and preparation method thereof is characterized in that:

A. the prokaryotic expression of recombinant human macrophage tumor metastasis suppressor gene: through reverse transcriptase polymerase chain reaction, use upstream primer CGCGGATCCTGCGGCTCTTAGGCGAAGG and downstream primer CGCCATATGCCGATGTTCATCGTAAACAC, amplify human macrophage tumor metastasis suppressor gene gene from human breast cancer cell strain MDA-MB453 and human breast carcinoma tissue, make up the pET11b/hMIF recombinant plasmid, transformed into escherichia coli BL21 (DE3) carries out the expression of recombinant human macrophage tumor metastasis suppressor gene;

B. recombinant human macrophage tumor metastasis suppressor gene purifying: with cation exchange medium and hydrophobic chromatoghaphy medium separation and purification;

C. anti-recombinant human macrophage tumor metastasis suppressor gene MONOCLONAL ANTIBODIES SPECIFIC FOR: with the human macrophage tumor metastasis suppressor gene immune mouse of purifying, obtain hybridoma by hybridoma technology through cytogamy repeatedly, select the hybridoma cell strain 2D of the anti-human macrophage tumor metastasis suppressor gene monoclonal antibody of secretion through the specificity testing sieve of antibody subgroup identification and antibody ₁₀, to the evaluation of hybridoma cell cloneization and secretory antibody subclass, 2D ₁₀The excretory monoclonal antibody belongs to IgG, and light chain is the κ type;

The aminoacid sequence of d. anti-human macrophage tumor metastasis suppressor gene monoclonal antibody variable region of light chain is: CRASGRNNAWYKGADGWYKGKAKRYAASSSGVSRSGSGSGTTTSSDATYYCHNDAV YSYGTKVKR, the aminoacid sequence of variable region of heavy chain is: VWSGGSTVKGGRSCGYYASGTSTYSMGMNWVRAGKGWVSSSTSVDTYYYADSVKGR TSRDNAKNSYDYDVSRADTAVYYCARDGASSGWGVDATGSRVSCYWGGTVSSWMSA T.

2. recombinant human macrophage tumor metastasis suppressor gene purifying according to claim 1 is characterized in that with cation exchange medium be SP Sepharose Fast Flow, and hydrophobic chromatoghaphy medium is Hitrap butyl Fast Flow.

3. anti-recombinant human macrophage tumor metastasis suppressor gene MONOCLONAL ANTIBODIES SPECIFIC FOR according to claim 1 is characterized in that the hybridoma cell strain 2D that obtains ₁₀Secrete IgG monoclonal antibody subclass and belong to IgG ₁

4. the aminoacid sequence of anti-human macrophage tumor metastasis suppressor gene monoclonal antibody variable region of light chain according to claim 1 is characterized in that this aminoacid sequence is used to prokaryotic cell prokaryocyte and the eukaryotic cell expression purifying obtains, and synthetic.

5. the aminoacid sequence of anti-human macrophage tumor metastasis suppressor gene monoclonal antibody variable region of heavy chain according to claim 1 is characterized in that this aminoacid sequence is used to prokaryotic cell prokaryocyte and the eukaryotic cell expression purifying obtains, and synthetic.

6. a human macrophage tumor metastasis suppressor gene monoclonal antibody is used for humanization modified.

7. according to claim 6 humanization modified, it is characterized in that utilizing human macrophage tumor metastasis suppressor gene monoclonal antibody amino acid sequence to carry out the homology transformation that computer mould is built transformation and alternate manner.

8. a human macrophage tumor metastasis suppressor gene monoclonal antibody is used for detecting animal and human's body Macrophagocyte transfer inhibition factor content.

9. Macrophagocyte transfer inhibition factor content in the detection animal and human body according to claim 7, it is characterized in that using human macrophage tumor metastasis suppressor gene monoclonal antibody, detect the serum of animal and human's body and the Macrophagocyte transfer inhibition factor content in the tissue by the specificity immunology reaction method between antibody and the antigen.

10. a human macrophage tumor metastasis suppressor gene monoclonal antibody is mainly used in the systemic inflammatory response syndrome that the clinical treatment wound causes.

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