CN105218671A - The preparation method of the full humanized antibody of a kind of infectious disease pathogens - Google Patents
The preparation method of the full humanized antibody of a kind of infectious disease pathogens Download PDFInfo
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- CN105218671A CN105218671A CN201510649970.9A CN201510649970A CN105218671A CN 105218671 A CN105218671 A CN 105218671A CN 201510649970 A CN201510649970 A CN 201510649970A CN 105218671 A CN105218671 A CN 105218671A
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Abstract
The invention discloses a kind of preparation method preparing the full humanized antibody of infectious disease pathogens, it is characterized in that, by isotopic labeling, the cytokine of methods analyst specifically expressing in infectious disease cell of multidimensional liquid chromatograph mass spectrography, carries out separation and purification to these albumen; Immunity is carried out with the cytokine of specifically expressing in the separation and purification infectious disease cell out female BAl BIc/c small white mouse to 6-8 age in week, if when antibody titers is more than 1: 15000, the immune single cell suspension of small white mouse splenocyte and the myeloma cell fusion of logarithmic phase can be screened, obtain the hybridoma of the humanized antibody of the cytokine of secreting specifically expressing in infectious disease cell; To the BALB/c mice by intraperitoneal injection hybridoma in 6-8 age in week, after injection cell, within about 15 days, collect ascites, protein? G/A pillar purifying ascites, obtains the humanized antibody of the higher treatment infectious disease cell factor of purity.
Description
Technical field
The present invention relates to biomedicine field, particularly the preparation method of the full humanized antibody of a kind of infectious disease pathogens.
Background technology
Transmissible disease (InfectiousDiseases) be by various pathogenic agent cause can in person to person, animal and the class disease mutually propagated between animal or human and animal.In pathogenic agent, major part is microorganism, and small portion is parasite, and parasite causer is also known as parasitosis.Some transmissible disease, epidemic prevention department must grasp its incidence in time, takes some countermeasures in time, should be timely to locality epidemic prevention Section report in required time after therefore finding, is called Notifiable disease.The current Notifiable disease of China has first, second, the third 3 classes, totally 39 kinds.
Transmissible disease is that one can from a people or other species, sick to the infection of another person or species through various route infection.The object that usual this disease can infect the body fluid of individual, the infected and movement by direct contact, the infected pollutes, can pass through airborne transmission, water source propagation, food transmission, contact transmission, soil-borne, vertical transmission etc.
Summary of the invention
The object of the present invention is to provide and a kind ofly prepare the preparation method that effectively can treat the humanized antibody of transmissible disease, it is for above-mentioned situation, a kind of easy and simple to handle, with low cost, efficient preparation method of invention.
Technical scheme of the present invention: a kind ofly prepare the method effectively can treating the humanized antibody of transmissible disease, is characterized in that it comprises the following steps:
(1) collect a large amount of infectious disease cell sample and normal cell sample, and by isotopic labeling, the cytokine of methods analyst specifically expressing in infectious disease cell of multidimensional liquid chromatograph mass spectrography, carries out separation and purification to these albumen;
(2) expression and purification of the cytokine humanized antibody of specifically expressing in infectious disease cell is completed;
1, animal immune is carried out by separation and purification cytokine out.Select the female BAl BIc/c small white mouse in 6-8 age in week.
According to the amount of every mouse 150ug, with isopyknic complete Freund's adjuvant mixing and emulsifying, the subcutaneous multi-point injection of emulsion.Booster immunization after 2-3 week, antigen dose is the same, with Freund's incomplete adjuvant mixing and emulsifying, the subcutaneous multiple spot of emulsion or intraperitoneal injection.
Immunity is small white mouse tail vein blood after 7-15 days, collects serum.Indirect ELISA measure in serum for antibody titers.If antibody titers is more than 1: 15000, can at fusion first three day booster immunization, the PBS of 150uL dissolves cytokine intraperitoneal injection or the intravenous injection of specifically expressing in 50ug infectious disease cell.Otherwise, proceed immunity once every two weeks, until reach higher antibody titers.
2, cytogamy and screening.In order to produce the hybridoma of the humanized antibody of the secretion treatment infectious disease cell factor, the myeloma cell of logarithmic phase is mixed, then at polyoxyethylene glycol with the single cell suspension of immune small white mouse splenocyte.
(PEG) merge under the condition mediated: splenocyte and myeloma cell mix in the ratio of 15: 1.By the serum-free IMDM substratum washing at least 3 times of the mixture of cell.In 37 DEG C of water-baths, in cell mass, dropwise add 50%PEG, and shake cell precipitation gently, then wash 1 time through the IMDM substratum of serum-free.With the cell mass after the resuspended fusion of the perfect medium containing HAT, blow and beat gently to cell mass and scatter, and be inoculated in 96 porocyte culture plates by 200ul/well.Afterwards, partly measured replaced medium every 2-3 days, until hybridoma cell clone can screen enough greatly.Indirect ELISA screening secretion is for the antibody positive cell hole of the cytokine of specifically expressing in infectious disease cell.Be defined as positive cell hole first, through limiting dilution assay, at least subclone 3 times, ensures that positive rate is 150%, and obtaining can the hybridoma cell strain of the cytokine antibodies of specifically expressing in stably excreting infectious disease cell.
3, ascites preparation: the BALB/c small white mouse selecting 6-8 age in week, every mice by intraperitoneal injection 0.5ml paraffin oil, injected hybridoma after 7-15 days.Within about 15 days, collect ascites, proteinG/A pillar purifying ascites after injection cell, obtain the humanized antibody of the higher treatment infectious disease cell factor of purity.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below the form by embodiment is described in further detail foregoing of the present invention again, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only confined to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment 1: in infectious disease cell, the acquisition of the cytokine of specifically expressing is by methods analyst more than 200 infectious disease cell sample of isotopic labeling, multidimensional liquid chromatograph mass spectrography and more than 200 normal cell sample, find out the cytokine of 5-8 specifically expressing in infectious disease cell, separation and purification goes out these cytokines;
Embodiment 2: the expression of the cytokine humanized antibody of specifically expressing in infectious disease cell
1, animal immune is carried out by separation and purification cytokine out.
Select the female BAl BIc/c small white mouse in 6-8 age in week.According to the amount of every mouse 150ug, with isopyknic complete Freund's adjuvant mixing and emulsifying, the subcutaneous multi-point injection of emulsion.Booster immunization after 2-3 week, antigen dose is the same, with Freund's incomplete adjuvant mixing and emulsifying, the subcutaneous multiple spot of emulsion or intraperitoneal injection.Immunity is small white mouse tail vein blood after 7-15 days, collects serum.Indirect ELISA measure in serum for antibody titers.If antibody titers is 1: more than 1w, can at fusion first three day booster immunization, the PBS of 150 μ L dissolves cytokine intraperitoneal injection or the intravenous injection of specifically expressing in 50ug infectious disease cell.Otherwise, proceed immunity once every two weeks, until reach higher antibody titers.
2, cytogamy and screening.
In order to produce the hybridoma of the humanized antibody of the cytokine of specifically expressing in secretion infectious disease cell, the myeloma cell of logarithmic phase is mixed with the single cell suspension of immune small white mouse splenocyte, merges under the condition then mediated at polyoxyethylene glycol (PEG): splenocyte and myeloma cell mix in the ratio of 15: 1.By the serum-free IMDM substratum washing at least 3 times of the mixture of cell.In 37 DEG C of water-baths, in cell mass, dropwise add 50%PEG, and shake cell precipitation gently, then wash 1 time through the IMDM substratum of serum-free.With the cell mass after the resuspended fusion of the perfect medium containing HAT, blow and beat gently to cell mass and scatter, and be inoculated in 96 porocyte culture plates by 200ul/well.Afterwards, partly measured replaced medium every 2-3 days, until hybridoma cell clone can screen enough greatly.Indirect ELISA screening secretion is for the antibody positive cell hole of the cytokine of specifically expressing in infectious disease cell.Be defined as positive cell hole first, through limiting dilution assay, at least subclone 3 times, ensures that positive rate is 150%, and obtaining can the hybridoma cell strain of the cytokine antibodies of specifically expressing in stably excreting infectious disease cell.
Embodiment 3: in infectious disease cell, the purifying of the humanized antibody of the cytokine of specifically expressing selects the BALB/c small white mouse in 6-8 age in week, and every mice by intraperitoneal injection 0.5ml paraffin oil, injected hybridoma after 7-15 days.Within about 15 days, collect ascites, proteinG/A pillar purifying ascites after injection cell, obtain the humanized antibody of the cytokine of specifically expressing in the higher infectious disease cell of purity.
Technique means disclosed in the present invention program is not limited only to the technique means disclosed in above-mentioned embodiment, also comprises the technical scheme be made up of above technical characteristic arbitrary combination.
Claims (3)
1. a preparation method for the full humanized antibody of infectious disease pathogens, is characterized in that it comprises the following steps: the separation and purification of specifically expressing cytokine in (1) transmissible disease; (2) expression of the humanized antibody of specifically expressing cytokine in transmissible disease; (3) purifying of specifically expressing cytokine humanized antibody in transmissible disease, finds out the cytokine of specifically expressing in infectious disease cell by the method for isotopic labeling, multidimensional liquid chromatograph mass spectrography.
2. a kind of preparation method preparing the full humanized antibody of infectious disease pathogens according to claim 1, it is characterized in that, the cytokine being used in specifically expressing in infectious disease cell carries out immunity to small white mouse, if when antibody titers is more than 1: 8000-1: 12000, the myeloma cell fusion of immune small white mouse splenocyte and logarithmic phase can be screened.
3. a kind of preparation method preparing the full humanized antibody of infectious disease pathogens according to claim 1, it is characterized in that to mice by intraperitoneal injection hybridoma, within after injection cell 8-12 days, collect ascites, proteinG/A pillar purifying ascites, obtain the humanized antibody for the treatment of transmissible disease.
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Citations (4)
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| CN102216768A (en) * | 2008-09-09 | 2011-10-12 | 由卫生福利和体育大臣代表的荷兰王国 | Lcms technology and its uses |
| CN102687020A (en) * | 2009-10-09 | 2012-09-19 | 西福根有限公司 | Multiplex quantitation of individual recombinant proteins in a mixture by signature peptides and mass spectrometry |
| CN103045607A (en) * | 2012-11-27 | 2013-04-17 | 李福胜 | Preparation method of completely humanized antibody of infectious disease pathogen |
| CN103065065A (en) * | 2012-11-18 | 2013-04-24 | 浙江大学 | Active tuberculosis differential expression protein profile model and building method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102216768A (en) * | 2008-09-09 | 2011-10-12 | 由卫生福利和体育大臣代表的荷兰王国 | Lcms technology and its uses |
| CN102687020A (en) * | 2009-10-09 | 2012-09-19 | 西福根有限公司 | Multiplex quantitation of individual recombinant proteins in a mixture by signature peptides and mass spectrometry |
| CN103065065A (en) * | 2012-11-18 | 2013-04-24 | 浙江大学 | Active tuberculosis differential expression protein profile model and building method thereof |
| CN103045607A (en) * | 2012-11-27 | 2013-04-17 | 李福胜 | Preparation method of completely humanized antibody of infectious disease pathogen |
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| 张威: "运用蛋白质组学的方法研究乙肝表面抗原持续表达对宿主细胞的影响", 《中国博士学位论文全文数据库》 * |
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Application publication date: 20160106 |