CN101717447A - Method for preparing antihuman recombinant tissue factor monoclonal antibody - Google Patents
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- CN101717447A CN101717447A CN200910227865A CN200910227865A CN101717447A CN 101717447 A CN101717447 A CN 101717447A CN 200910227865 A CN200910227865 A CN 200910227865A CN 200910227865 A CN200910227865 A CN 200910227865A CN 101717447 A CN101717447 A CN 101717447A
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Abstract
The invention discloses a method for preparing an antihuman recombinant tissue factor monoclonal antibody. The method comprises the following steps: combining a truncated human recombinant tissue factor obtained by purification after prokaryotic expression with a freund adjuvant immunized mouse; fusing mouse spleen cells with myeloma cells; selectively cultivating and screening an HAT selective medium; after the subcloning cultivation is subjected to limiting dilution, selecting target hybridoma cells by an ELISA method, performing massive proliferation, and injecting into an abdominal cavity of a syngeneic mouse to prepare ascites; and obtaining the antihuman recombinant tissue factor monoclonal antibody which has excellent potency and higher yield. The antihuman recombinant tissue factor monoclonal antibody prepared by the method of the invention has the advantages of high sensitivity and strong specificity, can be used for immunoaffinity purification of the human recombinant tissue factor, and also can be used for research and development of anticoagulant medicaments and medicaments for treating tumors.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of preparation method of antihuman recombinant tissue factor monoclonal antibody.
Background technology
(Tissue Factor is that molecular weight is the glycoprotein of 47KD TF) to human tissue factor, contains 263 amino-acid residues, is transmembrane receptor protein matter.TF is the acceptor and the cofactor of proconvertin, is the startup albumen of external source coagulation pathway.The TF protein molecular is divided into 3 parts, and 21 amino-acid residues of intracellular region are striden membranous part and are divided into 23 amino-acid residues, have hydrophobicity, and born of the same parents' outside part contains 219 amino-acid residues, is aminoterminal, has sugar chain.
Tissue factor is the most important startup factor in the physiological blood coagulation, is the present main component of prothrombin time detection reagent clinically, also is the principal element that influences prothrombin time.Research in recent years confirms that also TF not only plays an important role in coagulation pathway, also unconventionality expression usually in various malignant tumours can influence pathologic processes such as growth of tumor, invasion and attack and transfer by the multiple signal transduction of mediation.
Patent 200510012414.7 makes up and has expressed a kind of type human recombination factor TF that blocks
243(by striding membrane portions and the film outside part is formed) proves that simultaneously the type that the blocks human recombination factor of this disappearance intracellular region has whole blood coagulation activities.This type human recombination factor that blocks is compared with the natural tissues factor that biological extraction obtains, and the prothrombin assay reagent quality product of its preparation and measurement result are all very stable; And compare with the disclosed human recombination factor that only comprises extracellular region of 01132127.x patent application, its biological activity improves greatly.
In this patented technology, utilize the affine immune purification technique of anti-TF monoclonal antibody, be to obtain the proteic key of good human recombination factor.
Immune response also can take place with TF in anti-TF monoclonal antibody, suppresses the formation of thrombus.Huang (Science, 1997,275 (5229): 547-550.) and Versteeg (Mol Med simultaneously, 2004,10 (16): 6.) etc. Research of Animal Model for Study shows: TF antibody is with after TF combines, can the tumor vascular generation of target inhibition, cause neoplasm necrosis.
Therefore, utilize monoclonal antibody technique to prepare the anti-TF monoclonal antibody of susceptibility height, high specificity, can not only utilize the special purifying promptly of immune affine method TF albumen, for the production of novel prothrombin assay reagent provides technical guarantee; Also can be and utilize anti-TF monoclonal antibody to be applied to clinical treatments such as thrombus, cancer to lay the foundation.
And the present domestic commercial anti-TF monoclonal antibody of still not having.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of antihuman recombinant tissue factor monoclonal antibody, the antihuman recombinant tissue factor monoclonal antibody susceptibility height, the high specificity that utilize the inventive method to prepare.
Antihuman recombinant tissue factor monoclonal antibody of the present invention prepares by the following method:
1) selects immunogen: the human recombination factor (rhTF that utilizes 200510012414.7 patent construction expressions
243) as immunogen, its protein specificity is to contain 243 amino-acid residues that the natural human tissue factor is striden membrane portions and born of the same parents' outside part, is the type that the blocks human recombination factor of disappearance intracellular region, has whole blood coagulation activities of natural human tissue factor.
This human recombination factor is to obtain total RNA from human placenta, and design PCR primer obtains target gene fragment by the RT-PCR method; TF gene fragment and the reorganization of phoA plasmid with collecting make up TF expression plasmid carrier pTF
243, being transformed among the intestinal bacteria MM294, screening obtains the gene recombination engineering strain, inserts the fermention medium fermentation expression, obtains rhTF by TF monoclonal antibody immunity affinity chromatography column purification at last
243, its concrete construction expression method can be consulted 200510012414.7 patents.
2) immunity: the diluent that immunogen is diluted to different concns, be mixed into the emulsification mixed solution of different immunogen concentration with Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant emulsification, to mouse carry out fundamental immunity, booster immunization, secondary booster immunization and merge before impact immunity.
Concrete immunologic process is:
Fundamental immunity: adopt 30 μ g～60 μ g rhTF
243/ ml emulsification mixed solution carries out subcutaneous multi-point injection immunity to mouse;
Booster immunization: after two weeks, adopt 15 μ g～30 μ g rhTF
243/ ml emulsification mixed solution carries out the abdominal injection immunity to mouse;
Secondary booster immunization, booster immunization adopt 15 μ g～30 μ g rhTF after two weeks
243/ ml emulsification mixed solution carries out the immunity of secondary abdominal injection to mouse;
Impact immunity before merging: the secondary booster immunization adopts 25 μ g～50 μ g rhTF after one week
243/ ml emulsification mixed solution carries out abdominal injection to mouse and impacts immunity.
Wherein, the immunogen diluent of above-mentioned different concns and Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant all are to carry out the emulsification blended according to 1: 1 volume ratio.
3) screening immune serum: adopt enzyme-linked immunosorbent assay screening immune mouse serum;
4) cytogamy and cloning: choose the highest mice spleen cell of serum titer, utilize PEG1500 reagent to carry out external fusion, obtain the hybridoma cell strain of stably excreting antihuman recombinant tissue factor through subclone repeatedly with the myeloma cell;
5) preparation ascites: after the hybridoma cell strain enlarged culturing, inject production ascites in the pretreated mouse peritoneal of whiteruss;
The preparation ascites condition be: with whiteruss as sensitiser, choose 14～22 the week age BALB/c mouse be laboratory animal, with 0.5m1/ injecting fluid paraffin, with 1 * 10
5～5 * 10
5The inoculum density inoculation hybridoma cell strain 0.5ml/ of individual/ml only.
6) collect ascites: behind inoculation hybridoma cell strain 10～12d, ascites is drawn to rapidly in the cell centrifugation pipe, the centrifugal 15min of 3000rpm collects supernatant liquor, and it is good to obtain tiring, the antihuman recombinant tissue factor monoclonal antibody that output is higher.
Compared with prior art, the advantage that has of the present invention is:
1) human recombination factor that adopts the whole blood coagulation activities with natural human tissue factor screens the anti-tissue factor monoclonal antibody specificity height that obtains as antigen;
2) optimized the preparation method of ascites, the cell strain after the enlarged culturing injects through the pretreated mouse peritoneal of whiteruss with production ascites, has improved the productive rate of ascites greatly.
3) monoclonal antibody that makes according to the inventive method can be used for researching and developing anticoagulant and anti-tumor medicine, steady quality, and the output height can be used for industrialization and commercialization.
Description of drawings
Fig. 1 is the SDS-PAGE analytical results of antihuman recombinant tissue factor monoclonal antibody;
Among the figure, 1,2 is the antihuman recombinant tissue factor monoclonal antibody of purifying, and 3 is the standard protein molecular weight.
Fig. 2 is protein immunoblot (Wester Blot) analytical results of antihuman recombinant tissue factor monoclonal antibody;
Among the figure, M is the standard protein molecular weight, and 1,2,3 for blocking type human recombination factor rhTF
243, 4 is anti-people's re-organized tissue factor monoclonal antibody of purifying.
Embodiment
The preparation of antihuman recombinant tissue factor monoclonal antibody
1, immunogen
Select the human recombination factor (rhTF of 200510012414.7 patent construction expressions
243) as immunogen.
2, immune mouse
Fundamental immunity: get the rhTF that is stored in the Tris-HCl damping fluid
243(1mg/ml) be diluted to 120 μ g/ml, according to 1: 1 volume ratio with the Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant is fully emulsified mixes, with cotton ball soaked in alcohol sterilization BALB/c mouse belly, choose 5 somes mouse is carried out the subcutaneous injection immunity, injection volume 0.5ml/ is only.Immunity amount 0.5ml, 30 μ g rhTF
243/ only, injection finishes the back with the cotton ball soaked in alcohol injection site of sterilizing, and protects from infection.
Booster immunization: after two weeks, get the rhTF that is stored in the damping fluid
243Be diluted to 60 μ g/ml, with the Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant is fully emulsified mixes, with the right belly of cotton ball soaked in alcohol sterilization BALB/c mouse, draw emulsion and carry out the abdominal injection immunity, immunity amount 0.5ml, 15 μ g rhTF according to 1: 1 volume ratio
243/ only, injection finishes the back with the cotton ball soaked in alcohol injection site of sterilizing, and protects from infection.
Secondary booster immunization: after two weeks, get the rhTF that is stored in the damping fluid
243Be diluted to 60 μ g/ml, with the Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant is fully emulsified mixes, with the right belly of cotton ball soaked in alcohol sterilization BALB/c mouse, draw emulsion and carry out the abdominal injection immunity, immunity amount 0.5ml, 15 μ g rhTF according to 1: 1 volume ratio
243/ only, injection finishes the back with the cotton ball soaked in alcohol injection site of sterilizing, and protects from infection.
Impact immunity before merging: the secondary booster immunization is got the rhTF that is stored in the damping fluid after one week
243, be diluted to 100 μ g/ml,, with the right belly of cotton ball soaked in alcohol sterilization BALB/c mouse, draw emulsion and carry out the abdominal injection immunity, immunity amount 0.5ml, 25 μ g rhTF with the Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant is fully emulsified mixes according to 1: 1 volume ratio
243/ only, injection finishes the back with the cotton ball soaked in alcohol injection site of sterilizing, and protects from infection.
3, cytogamy and cloning
Immunity was impacted the back the 3rd day, got the splenocyte of immune mouse, carried out cytogamy with the myeloma cell, and concrete operations are as follows:
1) get the BALB/c mouse of immunity, extract the eyeball blood sampling, and the positive control serum of separation of serum during as antibody test.
2) immune mouse is drawn neck put to death, soaking disinfection 5min in 75% alcohol gets thymocyte (feeder cell) and spleen respectively, and spleen places the plate that fills the incomplete substratum of 10ml, gets the irrigation with syringe spleen and obtains splenocyte suspension;
3) respectively with splenocyte suspension and the centrifugal 5min of thymus cell suspension 1000rpm, supernatant discarded, splenocyte is resuspended with the DMEM high glucose medium, and thymocyte is selected 100 μ l/ holes, the resuspended back of substratum with HAT, spreads 96 well culture plates;
4) the rat bone marrow tumour cell SP2/0 that takes the logarithm vegetative period with the mixed of splenocyte with 1: 5 or 1: 10, toos many or too much for use full nutrient solution washing once, behind two kinds of enchylema mixings, with the centrifugal 8min of 1200rpm in the 50ml centrifuge tube;
5) supernatant discarded makes two kinds of cell thorough mixing;
6) add the PEG1500 of 1ml in 1min, add the DMEM of 1ml again in the 1min, the limit edged rotates centrifuge tube, continues to add in 1min the DMEM of 4ml, and the limit edged rotates centrifuge tube, the last DMEM that in 1min, adds 20ml, and the limit edged rotates centrifuge tube;
7) with liquid with the centrifugal 8min of 1200rpm, add the HAT suspendible gently contain 20% foetal calf serum in right amount;
8) add nutrient solution according to used 96 orifice plate quantity, divide to install in feeder cell 96 orifice plates (100 μ l/ hole) of preparation, or by per 2 * 10
7Individual splenic lymphocyte adds the ratio of 10mlHAT nutrient solution and adds nutrient solution, puts 37 ℃ of CO
2Cultivate in the case;
9) cultivate after 5 days, change liquid with HAT substratum half amount, change liquid with the HT substratum after 7～10 days, cultivate with common perfect medium after 14 days, when treating that hybridoma grows to hole floorage 1/3, adopt the ELISA method to detect, keep the best hybridoma cell strain of a strain after three limited dilution cloningizations of positive aperture, the strain of promptly anti-people's recombinant tissue monoclonal antibody hybridoma.
4, euzymelinked immunosorbent assay (ELISA) is carried out serum titer mensuration and hybridoma screening
Enzyme-linked immunoassay method in serum titer and the hybridoma screening is as follows: choose 96 hole enzyme plates, pH9.6 carbonate buffer solution bag is by rhTF
243(10 μ g/ml, 100 μ l/ holes) add the gelatin sealing after 4 ℃ of preservations are spent the night.Add dilute serum or hybridoma culture supernatant, 1000 ELIAS secondary antibody after the drying successively, add substrate O-Phenylene Diamine termination reaction at last.The 492nm microplate reader is measured.The negative contrast of PBS, the positive contrast of PBSTB.
The preparation of different vaccination density antihuman recombinant tissue factor monoclonal antibody ascites
1, hybridoma is prepared
Select anti-people's recombinant tissue monoclonal antibody hybridoma of acquisition among the embodiment 1, stand-by after the conventional enlarged culturing of the high sugared cell culture medium of DMEM.
2, selection of laboratory animal and pre-treatment
Select 14 ages in week, female, multiparity BALB/c mouse, as sensitiser, inoculate preceding sensitization of one week of hybridoma cell strain with whiteruss, 0.5ml/ only.
3, inoculation hybridoma cell strain
Hybridoma cell strain is cultivated through the tissue culture flasks passage, treat that cell grows to logarithmic phase counting back and collects (1000rpm, 6min, the centrifugal supernatant that goes), utilize the resuspended adjustment cell of serum-free basic medium (it is anti-to avoid introducing ox) to different inoculum densities, to inoculate.Different hybridoma cell strain inoculum density ascites are produced and be the results are shown in Table 1.
Table 1 different vaccination density mouse ascites output and antibody titer
4, the Collecting and dealing of ascites
Hybridoma cell strain inoculation back 10～12d keeps a close eye on, about about 10d, mouse web portion has tangible protuberance, by the time mouse when being at death's door, draws neck to put to death, lie on the back and be fixed on the mouse frame in the super clean bench, 75% alcohol carefully exposes peritonaeum with aseptic eye scissors after handling, and with disposable syringe ascites is drawn to rapidly in the cell centrifugation pipe, the centrifugal 15min of 3000rpm ,-20 ℃ of preservations.
5, the extraction of monoclonal antibody and purifying
After the ascites centrifugal treating, supernatant liquor behind ammonium sulfate precipitation and Protein G column chromatography method purifying in-20 ℃ of preservations.
6, the preliminary evaluation of monoclonal antibody
6.1SDS-PAGE analyze
3% concentrates glue, 80V voltage stabilizing; 12% separation gel, 120V voltage stabilizing (see figure 1).
6.2 protein immunoblot (Wester Blot) is analyzed
Get rhTF
243After 30 μ g made 12%SDS-PAGE, electrotransfer was to the NC film, one anti-be the purifying mAb of 1: 1500 times of dilution, two anti-be the sheep anti-mouse igg/HRP of 1: 2000 times of dilution, ECL detects (see figure 2).
The preparation of different treatment group antihuman recombinant tissue factor monoclonal antibody ascites
Select anti-people's recombinant tissue monoclonal antibody hybridoma of acquisition among the embodiment 1, stand-by after the conventional enlarged culturing.
Select with buy goods wholesale into 14 age in week 21 of BALB/c mouse, respectively 7 of female, male, female multiparity mouse are divided into 3 groups.With whiteruss sensitization, 0.5ml/ only before one week for the inoculation hybridoma cell strain.One week back inoculation equal densities (5 * 10
5Individual/ml) hybridoma cell strains cell suspension, when being dying shape, mouse gathers ascites.
Ascites is produced and be the results are shown in Table 2.
Table 2 different treatment group mouse ascites output and antibody titer
The preparation of difference mouse anti human recombinant tissue factor monoclonal antibody ascites in all ages
Select anti-people's recombinant tissue monoclonal antibody hybridoma of acquisition among the embodiment 1, stand-by after the conventional enlarged culturing.
Choose with buy goods wholesale into 25 of female multiparity BALB/c mouse, be divided into 5 groups at random, since 14 ages in week, every one group of mouse inoculation equal densities (5 * 10 of 2 weekly selections
5Individual/ml) hybridoma cell strains cell suspension, when being dying shape, mouse gathers ascites.
Ascites is produced and be the results are shown in Table 3.
Table 3 difference mouse ascites output in age in week and antibody titer
Claims (4)
1. the preparation method of an antihuman recombinant tissue factor monoclonal antibody may further comprise the steps:
1) selects immunogen: the type that the blocks human recombination factor rhTF that selects the disappearance intracellular region
243As immunogen, this blocks the type human recombination factor and contains 243 amino-acid residues that the natural human tissue factor is striden membrane portions and born of the same parents' outside part;
2) immunity: the diluent that immunogen is diluted to different concns, be mixed into the emulsification mixed solution of different immunogen concentration with Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant emulsification, to mouse carry out fundamental immunity, booster immunization, secondary booster immunization and merge before impact immunity;
3) screening immune serum: adopt enzyme-linked immunosorbent assay screening immune mouse serum;
4) cytogamy and cloning: choose the highest mice spleen cell of serum titer, carry out external fusion, obtain the hybridoma cell strain of stably excreting antihuman recombinant tissue factor through subclone repeatedly with the myeloma cell;
5) preparation ascites: after the hybridoma cell strain enlarged culturing, inject production ascites in the pretreated mouse peritoneal of whiteruss;
6) collect ascites: behind inoculation hybridoma cell strain 10～12d, ascites is drawn to rapidly in the cell centrifugation pipe, the centrifugal 15min of 3000rpm obtains antihuman recombinant tissue factor monoclonal antibody.
2. the preparation method of antihuman recombinant tissue factor monoclonal antibody according to claim 1 is characterized in that step 2) immunologic process be:
Fundamental immunity: adopt 30ug～60ug rhTF
243/ ml emulsification mixed solution carries out subcutaneous multi-point injection immunity to mouse;
Booster immunization: after two weeks, adopt 15ug～30ug rhTF
243/ ml emulsification mixed solution carries out the abdominal injection immunity to mouse;
Secondary booster immunization, booster immunization adopt 15ug～30ug rhTF after two weeks
243/ ml emulsification mixed solution carries out the immunity of secondary abdominal injection to mouse;
Impact immunity before merging: the secondary booster immunization adopts 25ug～50ug rhTF after one week
243/ ml emulsification mixed solution carries out abdominal injection to mouse and impacts immunity.
3. the preparation method of antihuman recombinant tissue factor monoclonal antibody according to claim 1, the immunogen diluent that it is characterized in that described different concns and Fu Shi Freund's complete adjuvant or freund 's incomplete adjuvant all mix according to 1: 1 volume ratio emulsification.
4. according to the preparation method of the described antihuman recombinant tissue factor monoclonal antibody of claim 1, it is characterized in that described preparation ascites is that to choose the female multiparity mouse of 14～22 week BALB/c in age be laboratory animal, with 0.5ml/ injecting fluid paraffin, with 1 * 10
5～5 * 10
5The inoculum density inoculation hybridoma cell strain 0.5ml/ of individual/ml only.
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