CN109988239A - A kind of c reactive protein genetic recombination monoclonal antibody and preparation method thereof - Google Patents
A kind of c reactive protein genetic recombination monoclonal antibody and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to biological immune technical fields, specially a kind of c reactive protein genetic recombination monoclonal antibody and preparation method thereof, the monoclonal antibody can reduce the statement of cardiac muscle cell's TGF-β caused by mCRP, and preparation method is to carry out animal immune operation using c reactive protein as antigen；It takes mouse boosting cell and murine myeloma cell SP2/0 to mix according to the ratio of 5:2, the screening of monoclonal hybridoma is carried out using ELISA method and limiting dilution assay；Prepare ascites antibody；Using the specificity of indirect ELISA and Western blot method detection antibody.Antibody of the present invention has myocardial cell injury effect caused by reducing mCPR, and preparation method has the advantages that quick, convenient, economical.
Description
Technical field
The present invention relates to biological immune technical field, specially a kind of c reactive protein genetic recombination monoclonal antibody and its
Preparation method.
Background technique
C reactive protein (CRP) is the protein material found in nineteen thirty by Tillet and Francis, and c reactive protein is
A kind of acute phase reactive protein that liver synthesizes under the stimulation of the cell factors such as interleukin-6, tumor necrosis factor α,
Change the important indicator for significantly becoming certain clinical diseases early diagnosis, antidiastole and curative effect evaluation in inflammatory reaction.
CPR has wide bioactivity, such as the equal energy in anti-infectious immunity defense reaction, inflammatory reaction and phagocytosis
Play important adjustment effect.CRP is a kind of cyclic annular pentamer albumen, and primary structure includes 5 identical subunits, sub- single
Interdigit is combined with non-covalent bond, and CRP coordination binding site is contained on its surface in each subunit.CRP is cardiovascular disease prognosis
Independent risk factor, a possibility that it can react the ingredient of atherosclerotic plaque and predict plaque rupture.In addition, more
Come more research shows that CRP is not only a biological indicator, it also takes part in atherogenesis and cardiac muscle stalk
Dead process.
CRP is a part of body nonspecific immunity mechanism, it combines C-polysaccharide, in combination with thin in the presence of Ca2+
Phosphocholine on after birth, can activating complement classical pathway, enhance the phagocytosis of leucocyte, adjust lymphocyte or monokaryon/
Macrophage system function promotes the generation of macrophage tissue factor, and CRP also can be detected in atherosclerotic plaque.Together
When, with some diseases mistaken diagnosis, increasing of failing to pinpoint a disease in diagnosis, the detection of acute phase inflammatory PROTEIN C RP may be patient's in blood plasma
Have great importance in early diagnosis and identification.
Monoclonal antibody is generated and starting immune response by being injected into antigen in host animal body.Thus
The most of operations for making monoclonal antibody are all in vitro by the pernicious myeloma of splenocyte and culture from these hosts
Cell is merged.Unique cell clone is separated, the cell survived in fusion steps is known as hybridoma.Hybridization
Tumor can be immortal due to myeloma characteristic, and is easy to breed in culture.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology, and it is anti-to provide a kind of c reactive protein genetic recombination monoclonal
Body and preparation method thereof.Antibody of the present invention has myocardial cell injury effect caused by reducing mCPR, and preparation method has fast
Fast, convenient, economic advantage.
The purpose of the present invention is achieved through the following technical solutions:
A kind of c reactive protein genetic recombination monoclonal antibody, it is thin which can reduce cardiac muscle caused by mCRP
The statement of born of the same parents' TGF-β.
A kind of preparation method preparing the c reactive protein genetic recombination monoclonal antibody, comprising the following steps:
S1 animal immune: using c reactive protein as antigen, with the Freund's complete adjuvant of equivalent it is fully emulsified after, through abdominal cavity
Injecting immune BALB/C mice, the incomplete freund adjuvant that same dose is added after 25-30d carry out peritoneal immunity again,
Blood is taken through mouse vein after 5-8d, antibody is detected using spot immune blotting；
S2 cell fusion: it takes mouse boosting cell and murine myeloma cell SP2/0 to mix according to the ratio of 5:2, is suspended from HAT
Selective culture solution is inoculated in 96 well culture plates, and being placed in temperature is 32-38 DEG C, and gas concentration lwevel is the culture of 6-8%
It is cultivated in case, incubation time 9-14d；
S3 ELISA screening and cloning: using ELISA method to carrying out Monoclonal hybridomas in the fused cell in S2
The screening of cell selects have the hole of positive reaction using limiting dilution assay cloning to c reactive protein, until antibody positive rate reaches
To 100%；
S4 cell cryopreservation: monoclonal hybridoma is centrifuged under the revolving speed of 1000rpm, the cell suspending liquid that will be obtained
It is put into cell cryopreservation tube, and frozen stock solution is added and freezes 8-12h at a temperature of -75 DEG C；
Production in S5 body: after the intraperitoneal continuous 7d of each mouse injects the atoleine of 0.5ml daily, inoculation is frozen
Monoclonal hybridoma after depositing extracts ascites after 7-12d, to be centrifuged 15-20min under the revolving speed of 3000rpm, collects supernatant
Liquid is saved in -25 DEG C；
S6 antibody test: using the specificity of indirect ELISA and Western blot method detection antibody.
Further, the octapeptide amino acid Yu bovine serum albumin of c reactive protein end are coupled in the step S1
Further, the murine myeloma cell SP2/0 that the step S2 takes is in logarithmic phase.
Further, in the step S2 HAT selectivity culture solution by the RPMI1640 culture solution of 85ml, the amino of 2ml
Petrin stores liquid, 1ml hypoxanthine and thymidine storage liquid and the fetal calf serum composition of 22ml.
Further, the step S3 frozen stock solution is according to weight ratio by dimethyl sulfoxide, polyethylene glycol and calf serum
2:3:5 is mixed.
Further, centrifugation time is 8-12min in the step S4.
Further, the step S6 can be using the specificity of immunofluorescent test method detection antibody.
The invention has the benefit that (1), c reactive protein genetic recombination monoclonal antibody prepared by the present invention can be right
The rat myocardial cell that mCRP intervenes plays a very good protection, and the statement by reducing cardiac muscle cell's TGF-β neutralizes
The effect of myocardial cell injury caused by mCPR.
(2), the present invention is emulsified in the animal immune stage using Freund's complete adjuvant and antigen, and is added again later not
Split completely carries out peritoneal immunity again, effectively enhances the antigenicity of antigen, and stimulation mouse body generates stronger
Immune response.
(3), hybridoma is inoculated in mouse peritoneal, in mouse peritoneal by antibody of the present invention using production in vivo
Hybridoma is grown, and generates ascites, a large amount of ascites monoclonal antibodies can be obtained, this method has operation letter compared to extracorporeal culture-ing
Single, economic and high antibody concentration advantage.
(4), the present invention uses ELISA method detection antibody specificity, and ELISA method is by antigen binding to some solid phase carrier
Surface, makes antibody and certain enzyme connects into enzyme labelled antibody, by antibody and enzyme labelled antibody by different step and surface of solid phase carriers
Antigen reacts, and final combine in the enzyme labelled antibody amount and test antibodies amount of solid phase carrier is in certain proportion, and enzyme reaction bottom is added
Object, substrate are developed the color by enzymatic, carry out qualitative or quantitative analysis according to the depth of color, this method has quick, convenient, sensitive
The characteristics of.
Detailed description of the invention
The comparison analysis chart that Fig. 1 present invention and international well-known producer H detect definite value serum sample.
Specific embodiment
Technical solution of the present invention is described in further detail combined with specific embodiments below, but protection scope of the present invention is not
It is confined to as described below.
Embodiment 1
A kind of c reactive protein genetic recombination monoclonal antibody, it is thin which can reduce cardiac muscle caused by mCRP
The statement of born of the same parents' TGF-β.
A kind of preparation method preparing the c reactive protein genetic recombination monoclonal antibody, comprising the following steps:
S1 animal immune: using c reactive protein as antigen, the octapeptide amino acid and bovine serum albumin of end are coupled, with
After the Freund's complete adjuvant of equivalent is fully emulsified, through immune balb/c mice is injected intraperitoneally, same dose is added after 25d
Incomplete freund adjuvant carries out peritoneal immunity again, takes blood through mouse vein after 5d, detects antibody using spot immune blotting；
S2 cell fusion: ratio of the mouse boosting cell with the murine myeloma cell SP2/0 in logarithmic phase according to 5:2 is taken
Mixing is suspended from by aminopterin-induced syndrome storage liquid, 1ml hypoxanthine and the thymidine storage of the RPMI1640 culture solution, 2ml of 85ml
The HAT selectivity culture solution of the fetal calf serum of liquid and 22ml composition, is inoculated in 96 well culture plates, and being placed in temperature is 32
DEG C, it is cultivated in the incubator that gas concentration lwevel is 6%, incubation time 9d；
S3ELISA screening and cloning: thin to Monoclonal hybridomas is carried out in the fused cell in S2 using ELISA method
The screening of born of the same parents selects have the hole of positive reaction using limiting dilution assay cloning to c reactive protein, until antibody positive rate reaches
100%；
S4 cell cryopreservation: monoclonal hybridoma is centrifuged 8min under the revolving speed of 1000rpm, obtained cell is hanged
Supernatant liquid is put into cell cryopreservation tube, and is added and is mixed by dimethyl sulfoxide, polyethylene glycol and calf serum according to weight ratio for 2:3:5
Frozen stock solution made of conjunction freezes 8h at a temperature of -75 DEG C；
Production in S5 body: after the intraperitoneal continuous 7d of each mouse injects the atoleine of 0.5ml daily, inoculation is frozen
Monoclonal hybridoma after depositing extracts ascites after 7d, to be centrifuged 15min under the revolving speed of 3000rpm, collects supernatant, in
- 25 DEG C of preservations；
S6 antibody test: using the spy of indirect ELISA and Western blot method or immunofluorescent test method detection antibody
It is anisotropic.
Embodiment 2
A kind of c reactive protein genetic recombination monoclonal antibody, it is thin which can reduce cardiac muscle caused by mCRP
The statement of born of the same parents' TGF-β.
A kind of preparation method preparing the c reactive protein genetic recombination monoclonal antibody, comprising the following steps:
S1 animal immune: using c reactive protein as antigen, the octapeptide amino acid and bovine serum albumin of end are coupled, with
After the Freund's complete adjuvant of equivalent is fully emulsified, through immune balb/c mice is injected intraperitoneally, same dose is added after 27d
Incomplete freund adjuvant carries out peritoneal immunity again, takes blood through mouse vein after 6d, detects antibody using spot immune blotting；
S2 cell fusion: ratio of the mouse boosting cell with the murine myeloma cell SP2/0 in logarithmic phase according to 5:2 is taken
Mixing is suspended from by aminopterin-induced syndrome storage liquid, 1ml hypoxanthine and the thymidine storage of the RPMI1640 culture solution, 2ml of 85ml
The HAT selectivity culture solution of the fetal calf serum of liquid and 22ml composition, is inoculated in 96 well culture plates, and being placed in temperature is 36
DEG C, it is cultivated in the incubator that gas concentration lwevel is 7%, incubation time 12d；
S3ELISA screening and cloning: thin to Monoclonal hybridomas is carried out in the fused cell in S2 using ELISA method
The screening of born of the same parents selects have the hole of positive reaction using limiting dilution assay cloning to c reactive protein, until antibody positive rate reaches
100%；
S4 cell cryopreservation: monoclonal hybridoma is centrifuged 10min under the revolving speed of 1000rpm, the cell that will be obtained
Suspension is put into cell cryopreservation tube, and addition is 2:3:5 according to weight ratio by dimethyl sulfoxide, polyethylene glycol and calf serum
The frozen stock solution mixed freezes 10h at a temperature of -75 DEG C；
Production in S5 body: after the intraperitoneal continuous 7d of each mouse injects the atoleine of 0.5ml daily, inoculation is frozen
Monoclonal hybridoma after depositing extracts ascites after 9d, to be centrifuged 17min under the revolving speed of 3000rpm, collects supernatant, in
- 25 DEG C of preservations；
S6 antibody test: using the spy of indirect ELISA and Western blot method or immunofluorescent test method detection antibody
It is anisotropic.
Embodiment 3
A kind of c reactive protein genetic recombination monoclonal antibody, it is thin which can reduce cardiac muscle caused by mCRP
The statement of born of the same parents' TGF-β.
A kind of preparation method preparing the c reactive protein genetic recombination monoclonal antibody, comprising the following steps:
S1 animal immune: using c reactive protein as antigen, the octapeptide amino acid and bovine serum albumin of end are coupled, with
After the Freund's complete adjuvant of equivalent is fully emulsified, through immune balb/c mice is injected intraperitoneally, same dose is added after 30d
Incomplete freund adjuvant carries out peritoneal immunity again, takes blood through mouse vein after 8d, detects antibody using spot immune blotting；
S2 cell fusion: ratio of the mouse boosting cell with the murine myeloma cell SP2/0 in logarithmic phase according to 5:2 is taken
Mixing is suspended from by aminopterin-induced syndrome storage liquid, 1ml hypoxanthine and the thymidine storage of the RPMI1640 culture solution, 2ml of 85ml
The HAT selectivity culture solution of the fetal calf serum of liquid and 22ml composition, is inoculated in 96 well culture plates, and being placed in temperature is 38
DEG C, it is cultivated in the incubator that gas concentration lwevel is 8%, incubation time 14d；
S3ELISA screening and cloning: thin to Monoclonal hybridomas is carried out in the fused cell in S2 using ELISA method
The screening of born of the same parents selects have the hole of positive reaction using limiting dilution assay cloning to c reactive protein, until antibody positive rate reaches
100%；
S4 cell cryopreservation: monoclonal hybridoma is centrifuged 12min under the revolving speed of 1000rpm, the cell that will be obtained
Suspension is put into cell cryopreservation tube, and addition is 2:3:5 according to weight ratio by dimethyl sulfoxide, polyethylene glycol and calf serum
The frozen stock solution mixed freezes 12h at a temperature of -75 DEG C；
Production in S5 body: after the intraperitoneal continuous 7d of each mouse injects the atoleine of 0.5ml daily, inoculation is frozen
Monoclonal hybridoma after depositing extracts ascites after 12d, to be centrifuged 20min under the revolving speed of 3000rpm, collects supernatant,
It is saved in -25 DEG C；
S6 antibody test: using the spy of indirect ELISA and Western blot method or immunofluorescent test method detection antibody
It is anisotropic.
Comparative example 1
Using the pairing antibody of international well-known producer H production as comparative example 1, CRP antibody of the invention is flat in colloidal gold detection
5 clinical negative whole bloods and 5 positive clinical whole bloods are detected on platform respectively, result is that 5 feminine genders are feminine gender, and 5 positives are equal
For the positive, and without Anomalies of Backgrounds.
Such as Fig. 1, hospital is detected with serum sample 25 of immunofluorescence definite value, international well-known producer H is control.Linear
(0.1 μ g/ml-200 μ g/ml) present invention is with producer H with good correlation, related coefficient 94.4% in range.
The above is only a preferred embodiment of the present invention, it should be understood that the present invention is not limited to described herein
Form should not be regarded as an exclusion of other examples, and can be used for other combinations, modifications, and environments, and can be at this
In the text contemplated scope, modifications can be made through the above teachings or related fields of technology or knowledge.And those skilled in the art institute into
Capable modifications and changes do not depart from the spirit and scope of the present invention, then all should be in the protection scope of appended claims of the present invention
It is interior.
Claims (8)
1. a kind of c reactive protein genetic recombination monoclonal antibody, which is characterized in that the monoclonal antibody, which can reduce mCRP, to be caused
Cardiac muscle cell's TGF-β statement.
2. a kind of preparation method for preparing c reactive protein genetic recombination monoclonal antibody described in claim 1, which is characterized in that
The following steps are included:
S1 animal immune: using c reactive protein as antigen, with the Freund's complete adjuvant of equivalent it is fully emulsified after, through being injected intraperitoneally
Immune balb/c mice, the incomplete freund adjuvant that same dose is added after 25-30d carry out peritoneal immunity, 5-8d again
Blood is taken by mouse vein, antibody is detected using spot immune blotting；
S2 cell fusion: taking mouse boosting cell and murine myeloma cell SP2/0 to mix according to the ratio of 5:2, is suspended from HAT choosing
Selecting property culture solution is inoculated in 96 well culture plates, and being placed in temperature is 32-38 DEG C, and gas concentration lwevel is the incubator of 6-8%
Middle culture, incubation time 9-14d；
S3 ELISA screening and cloning: using ELISA method to carrying out monoclonal hybridoma in the fused cell in S2
Screening, select have the hole of positive reaction using limiting dilution assay cloning to c reactive protein, until antibody positive rate reaches
100%；
S4 cell cryopreservation: monoclonal hybridoma is centrifuged under the revolving speed of 1000rpm, obtained cell suspending liquid is put
Enter in cell cryopreservation tube, and frozen stock solution is added and freezes 8-12h at a temperature of -75 DEG C；
Production in S5 body: after the intraperitoneal continuous 7d of each mouse injects the atoleine of 0.5ml daily, inoculation is frozen
Monoclonal hybridoma afterwards extracts ascites after 7-12d, to be centrifuged 15-20min under the revolving speed of 3000rpm, collects supernatant
Liquid is saved in -25 DEG C；
S6 antibody test: using the specificity of indirect ELISA and Western blot method detection antibody.
3. a kind of preparation method of c reactive protein genetic recombination monoclonal antibody according to claim 2, feature exist
In octapeptide amino acid Yu the bovine serum albumin coupling of c reactive protein end in the step S1.
4. a kind of preparation method of c reactive protein genetic recombination monoclonal antibody according to claim 2, feature exist
In the murine myeloma cell SP2/0 that the step S2 takes is in logarithmic phase.
5. a kind of preparation method of c reactive protein genetic recombination monoclonal antibody according to claim 2, feature exist
In HAT selectivity culture solution stores liquid, 1ml by the RPMI1640 culture solution of 85ml, the aminopterin-induced syndrome of 2ml in the step S2
The fetal calf serum composition of hypoxanthine and thymidine storage liquid and 22ml.
6. a kind of preparation method of c reactive protein genetic recombination monoclonal antibody according to claim 2, feature exist
In the step S3 frozen stock solution is mixed by dimethyl sulfoxide, polyethylene glycol and calf serum according to weight ratio for 2:3:5.
7. a kind of preparation method of c reactive protein genetic recombination monoclonal antibody according to claim 2, feature exist
In centrifugation time is 8-12min in the step S4.
8. a kind of preparation method of c reactive protein genetic recombination monoclonal antibody according to claim 2, feature exist
In the step S6 can be using the specificity of immunofluorescent test method detection antibody.
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| CN111321121A (en) * | 2020-03-16 | 2020-06-23 | 成都大熊猫繁育研究基地 | Two-strain novel panda C-reactive protein monoclonal antibody hybridoma cell strain and application thereof |
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