CN102120771A - Preparation method of rabbit monoclonal antibody for resisting Cry1c crystal protein - Google Patents
Preparation method of rabbit monoclonal antibody for resisting Cry1c crystal protein Download PDFInfo
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- CN102120771A CN102120771A CN 201010601130 CN201010601130A CN102120771A CN 102120771 A CN102120771 A CN 102120771A CN 201010601130 CN201010601130 CN 201010601130 CN 201010601130 A CN201010601130 A CN 201010601130A CN 102120771 A CN102120771 A CN 102120771A
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- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 101710151559 Crystal protein Proteins 0.000 title abstract 3
- 210000004027 cell Anatomy 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000012216 screening Methods 0.000 claims abstract description 9
- 238000002965 ELISA Methods 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 6
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 6
- 239000006152 selective media Substances 0.000 claims abstract description 6
- 239000013598 vector Substances 0.000 claims abstract description 6
- 230000036039 immunity Effects 0.000 claims description 21
- 230000003053 immunization Effects 0.000 claims description 16
- 239000000427 antigen Substances 0.000 claims description 12
- 108091007433 antigens Proteins 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 12
- 238000010521 absorption reaction Methods 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 10
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims description 10
- 239000002671 adjuvant Substances 0.000 claims description 10
- 230000024835 cytogamy Effects 0.000 claims description 10
- 210000004408 hybridoma Anatomy 0.000 claims description 10
- 239000003547 immunosorbent Substances 0.000 claims description 10
- 230000008859 change Effects 0.000 claims description 6
- 210000000952 spleen Anatomy 0.000 claims description 6
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 claims description 5
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 claims description 5
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 5
- 229920002684 Sepharose Polymers 0.000 claims description 5
- 229960003896 aminopterin Drugs 0.000 claims description 5
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims description 5
- 239000012930 cell culture fluid Substances 0.000 claims description 5
- 239000006285 cell suspension Substances 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 5
- 238000004945 emulsification Methods 0.000 claims description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 5
- 238000002649 immunization Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000011587 new zealand white rabbit Methods 0.000 claims description 5
- -1 polyoxyethylene Polymers 0.000 claims description 5
- 230000008521 reorganization Effects 0.000 claims description 5
- 230000003248 secreting effect Effects 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 210000004988 splenocyte Anatomy 0.000 claims description 5
- 238000007920 subcutaneous administration Methods 0.000 claims description 5
- 229940104230 thymidine Drugs 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 238000001262 western blot Methods 0.000 abstract description 2
- 210000003719 b-lymphocyte Anatomy 0.000 abstract 1
- 238000001514 detection method Methods 0.000 abstract 1
- 230000001900 immune effect Effects 0.000 abstract 1
- 230000002163 immunogen Effects 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
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- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 238000002474 experimental method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a preparation method of a rabbit monoclonal antibody for resisting Cry1c crystal protein. The method comprises the following steps: high purity Cry1c crystal protein is used as immunogen immune molecule, the titer of antiserum is measured, the rabbit B lymphocyte and the myeloma cell are fused, selective medium is used to perform selective cultivation, pre-screening, preliminary screening and secondary screening are performed, positive clone is selected to extensively proliferate, recombinant vector is constructed to perform in vitro culture, and the monoclonal antibody is obtained through separation and purification. The preparation method can be used in the immunologic methods such as enzyme-linked immuno sorbent assay (ELISA) and WesternBlotting to perform the qualitative or quantitative detection of Cry1c protein.
Description
Technical field
The present invention relates to anti-Cry1c crystallin antibody, relate in particular to a kind of preparation method of anti-Cry1c crystallin rabbit monoclonal antibodies.
Background technology
From 1996, the first routine genetically modified food---fresh-keeping tomato was since the U.S. occurs, and the research and extension of genetically modified food has obtained global extensive concern.People make the diversity of genetically modified crops get over abundant to genetically modified continuous research, absorption, popularization, and simultaneously the cultivated area of genetically modified crops also significantly increases.Use International Service Organization (ISAAA) according to Agricultural biotechnologies, the whole world has 144 transgenosis projects, represent 24 kinds of crops to obtain worldwide approval, by the end of 2008, there have been 25 country plantation genetically modified crops in the whole world, has 30 countries to allow the import genetically modified crops as food and seed simultaneously in addition.In the genetically modified crops, especially with maximum to the research of Bt-Cry transgene protein, Cry1c albumen is a kind of in the Bt-Cry series albumen.Change at present
Bt-CryThe farm crop of gene mainly contain corn, potato, paddy rice, cotton etc.
The development of transgenic technology has promoted biological development with progressive, has also promoted advanced science development of productivity.Though genetically modified food can satisfy the requirement of people to aspects such as food nutrition, output, insect-resistance even pharmaceutical uses, but the while has also brought some potential to threaten to the mankind's life, import the host as some gene and can make the food toxigenicity afterwards, transgenosis food produces anaphylactogen, the people is developed immunity to drugs, and Nutritive value of food changes etc.Carrying out genetically modified food research and development and business-like while; for the security to genetically modified food gives comprehensive assessment; make the human consumer can distinguish genetically modified food and whole food fast; setting up suitable method identifies transgene component in the genetically modified food and detects; can promote agriculture genetically modified organism security control; ensure the safety of humans and animals and microorganism, can preserve the ecological environment simultaneously, promote the further research of agriculture genetically modified organism technology.For Cry1c albumen in genetically modified crops or its derivative is carried out rapid quantitative or qualitative analysis, study and obtain the proteic monoclonal antibody of the anti-Cry1c of a strain and have very large meaning.
The conventional mouse monoclonal antibody of rabbit monoclonal antibodies has more advantages.At first, rabbit can discern than mouse and more many epitope antigen determinant of immunizing antigen.Secondly,, can carry out more fusion experiment, make the positive fused cell of high flux screening become possibility because the rabbit spleen is bigger.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of preparation method of rabbit monoclonal antibodies of anti-Cry1c crystallin is provided.
The preparation method of the rabbit monoclonal antibodies of anti-Cry1c crystallin, step as follows:
1) rabbit immunity
The immunizing antigen Cry1c albumen of 400 ~ 600 μ g is mixed with the equivalent Freund's incomplete adjuvant, fully after the emulsification, adopt 2 ~ 3 new zealand white rabbits of subcutaneous 3 ~ 6 injections in back, in three, five, seven, nine weeks of beginning after the immunity, use the immunizing antigen albumen of 150 ~ 250mg to mix respectively and then carry out immunity with same method with Freund's incomplete adjuvant;
2) cytogamy
The high rabbit of serum titer carries out booster immunization earlier one time after selecting the 5th immunity, get aseptic spleen after 8 ~ 10 days, preparation single-cell suspension liquid, merge and selected the same day growth splenocyte vigorous, that be in logarithmic phase to merge mutually with the myeloma cell who is in logarithmic phase, mass percent is that 48% ~ 52% polyoxyethylene glycol is a fusogen, volume percent be 18% ~ 22% HAT as selective medium, be laid in 40 96 orifice plates, wherein H is that concentration is 0.8 * 10
-6~ 1.2 * 10
-6The xanthoglobulin of mol/L, A are that concentration is 3.5 * 10
-9~ 4.5 * 10
-9The aminopterin of mol/L, T is that concentration is 1.4 * 10
-7~ 1.8 * 10
-7The thymidine of mol/L;
3) hybridoma screening
Selected 2 blocks of plates in 40 plates to sieve in advance by indirect enzyme-linked immunosorbent absorption at random on the 8th ~ 12 day after the cytogamy, determine whether to need to change cell culture fluid cloning son; Carried out primary dcreening operation by indirect enzyme-linked immunosorbent absorption on the 15th ~ 19 day, and tentatively determined it is male clone, and the hybridoma cell transfer of correspondence gone down to posterity in 24 well culture plates cultivate a week; Carried out multiple sieve by indirect enzyme-linked immunosorbent assay on the 22nd ~ 26 day, filter out positive colony, and determine 3 ~ 5 strain growth conditions preferably and the strongest cell strain of expression specificity antibody ability enter the structure of recombinant vectors, other-20 ℃ of clone's are frozen, the cell strain that obtains through reorganization selects a strain avidity and secretory antibody ability strong, high clone's of specificity is through vitro culture, and Sepharose CL-4B Protein A column separating purification also obtains the target monoclonal antibody.
The anti-Cry1c crystallin rabbit monoclonal antibodies of the present invention's preparation can be used for immunology means such as ELISA, Western Blotting to the proteic qualitative or quantitative examination of Cry1c.
Concrete embodiment
Embodiment 1
1) rabbit immunity
The immunizing antigen Cry1c albumen of 400 μ g is mixed with the equivalent Freund's incomplete adjuvant, fully after the emulsification, adopt 2 new zealand white rabbits of subcutaneous 3 injections in back, in three, five, seven, nine weeks of beginning after the immunity, use the immunizing antigen albumen of 150 ~ 250mg to mix respectively and then carry out immunity with same method with Freund's incomplete adjuvant;
2) cytogamy
The high rabbit of serum titer carries out booster immunization earlier one time after selecting the 5th immunity, get aseptic spleen after 8 days, preparation single-cell suspension liquid, merge and selected the same day growth splenocyte vigorous, that be in logarithmic phase to merge mutually with the myeloma cell who is in logarithmic phase, mass percent is that 48% polyoxyethylene glycol is a fusogen, volume percent be 18% HAT as selective medium, be laid in 40 96 orifice plates, wherein H is that concentration is 0.8 * 10
-6The xanthoglobulin of mol/L, A are that concentration is 3.5 * 10
-9The aminopterin of mol/L, T is that concentration is 1.4 * 10
-7The thymidine of mol/L;
3) hybridoma screening
Selected 2 blocks of plates in 40 plates to sieve in advance by indirect enzyme-linked immunosorbent absorption at random on the 8th day after the cytogamy, determine whether to need to change cell culture fluid cloning son; Carried out primary dcreening operation by indirect enzyme-linked immunosorbent absorption on the 15th day, and tentatively determined it is male clone, and the hybridoma cell transfer of correspondence gone down to posterity in 24 well culture plates cultivate a week; Carried out multiple sieve by indirect enzyme-linked immunosorbent assay on the 22nd day, filter out positive colony, and determine 3 strain growth conditions preferably and the strongest cell strain of expression specificity antibody ability enter the structure of recombinant vectors, other-20 ℃ of clone's are frozen, the cell strain that obtains through reorganization selects a strain avidity and secretory antibody ability strong, high clone's of specificity is through vitro culture, and Sepharose CL-4B Protein A column separating purification also obtains the target monoclonal antibody.
Measure by analysis, the immunology type of the monoclonal antibody of this method preparation is IgG, and the monoclonal antibody of this method preparation is 2.33 * 10 to the proteic affinity costant of Cry1c
9M
-1, its molecular weight is 150KDa.
Embodiment 2
1) rabbit immunity
The immunizing antigen Cry1c albumen of 600 μ g is mixed with the equivalent Freund's incomplete adjuvant, fully after the emulsification, adopt 3 new zealand white rabbits of subcutaneous 6 injections in back, in three, five, seven, nine weeks of beginning after the immunity, mix with Freund's incomplete adjuvant with the immunizing antigen albumen of 250mg respectively and then carry out immunity with same method;
2) cytogamy
The high rabbit of serum titer carries out booster immunization earlier one time after selecting the 5th immunity, get aseptic spleen after 10 days, preparation single-cell suspension liquid, merge and selected the same day growth splenocyte vigorous, that be in logarithmic phase to merge mutually with the myeloma cell who is in logarithmic phase, mass percent is that 52% polyoxyethylene glycol is a fusogen, volume percent be 22% HAT as selective medium, be laid in 40 96 orifice plates, wherein H is that concentration is 1.2 * 10
-6The xanthoglobulin of mol/L, A are that concentration is 4.5 * 10
-9The aminopterin of mol/L, T is that concentration is 1.8 * 10
-7The thymidine of mol/L;
3) hybridoma screening
Selected 2 blocks of plates in 40 plates to sieve in advance by indirect enzyme-linked immunosorbent absorption at random on the 12nd day after the cytogamy, determine whether to need to change cell culture fluid cloning son; Carried out primary dcreening operation by indirect enzyme-linked immunosorbent absorption on the 19th day, and tentatively determined it is male clone, and the hybridoma cell transfer of correspondence gone down to posterity in 24 well culture plates cultivate a week; Carried out multiple sieve by indirect enzyme-linked immunosorbent assay on the 26th day, filter out positive colony, and determine 5 strain growth conditions preferably and the strongest cell strain of expression specificity antibody ability enter the structure of recombinant vectors, other-20 ℃ of clone's are frozen, the cell strain that obtains through reorganization selects a strain avidity and secretory antibody ability strong, high clone's of specificity is through vitro culture, and Sepharose CL-4B Protein A column separating purification also obtains the target monoclonal antibody.
Measure by analysis, the immunology type of the monoclonal antibody of this method preparation is IgG, and the monoclonal antibody of this method preparation is 2.45 * 10 to the proteic affinity costant of Cry1c
9M
-1, its molecular weight is 150KDa.
Embodiment 3
1) rabbit immunity
The immunizing antigen Cry1c albumen of 500 μ g is mixed with the equivalent Freund's incomplete adjuvant, fully after the emulsification, adopt 3 new zealand white rabbits of subcutaneous 5 injections in back, in three, five, seven, nine weeks of beginning after the immunity, mix with Freund's incomplete adjuvant with the immunizing antigen albumen of 200mg respectively and then carry out immunity with same method;
2) cytogamy
The high rabbit of serum titer carries out booster immunization earlier one time after selecting the 5th immunity, get aseptic spleen after 10 days, preparation single-cell suspension liquid, merge and selected the same day growth splenocyte vigorous, that be in logarithmic phase to merge mutually with the myeloma cell who is in logarithmic phase, mass percent is that 50% polyoxyethylene glycol is a fusogen, volume percent be 20% HAT as selective medium, be laid in 40 96 orifice plates, wherein H is that concentration is 1.0 * 10
-6The xanthoglobulin of mol/L, A are that concentration is 4.0 * 10
-9The aminopterin of mol/L, T is that concentration is 1.6 * 10
-7The thymidine of mol/L;
3) hybridoma screening
Selected 2 blocks of plates in 40 plates to sieve in advance by indirect enzyme-linked immunosorbent absorption at random on the 10th day after the cytogamy, determine whether to need to change cell culture fluid cloning son; Carried out primary dcreening operation by indirect enzyme-linked immunosorbent absorption on the 17th day, and tentatively determined it is male clone, and the hybridoma cell transfer of correspondence gone down to posterity in 24 well culture plates cultivate a week; Carried out multiple sieve by indirect enzyme-linked immunosorbent assay on the 24th day, filter out positive colony, and determine 3 strain growth conditions preferably and the strongest cell strain of expression specificity antibody ability enter the structure of recombinant vectors, other-20 ℃ of clone's are frozen, the cell strain that obtains through reorganization selects a strain avidity and secretory antibody ability strong, high clone's of specificity is through vitro culture, and Sepharose CL-4B Protein A column separating purification also obtains the target monoclonal antibody.
Measure by analysis, the immunology type of the monoclonal antibody of this method preparation is IgG, and the monoclonal antibody of this method preparation is 2.40 * 10 to the proteic affinity costant of Cry1c
9M
-1, its molecular weight is 150KDa.
Claims (1)
1. the preparation method of the rabbit monoclonal antibodies of an anti-Cry1c crystallin is characterized in that its step is as follows:
1) rabbit immunity
The immunizing antigen Cry1c albumen of 400 ~ 600 μ g is mixed with the equivalent Freund's incomplete adjuvant, fully after the emulsification, adopt 2 ~ 3 new zealand white rabbits of subcutaneous 3 ~ 6 injections in back, in three, five, seven, nine weeks of beginning after the immunity, use the immunizing antigen albumen of 150 ~ 250mg to mix respectively and then carry out immunity with same method with Freund's incomplete adjuvant;
2) cytogamy
The high rabbit of serum titer carries out booster immunization earlier one time after selecting the 5th immunity, get aseptic spleen after 8 ~ 10 days, preparation single-cell suspension liquid, merge and selected the same day growth splenocyte vigorous, that be in logarithmic phase to merge mutually with the myeloma cell who is in logarithmic phase, mass percent is that 48% ~ 52% polyoxyethylene glycol is a fusogen, volume percent be 18% ~ 22% HAT as selective medium, be laid in 40 96 orifice plates, wherein H is that concentration is 0.8 * 10
-6~ 1.2 * 10
-6The xanthoglobulin of mol/L, A are that concentration is 3.5 * 10
-9~ 4.5 * 10
-9The aminopterin of mol/L, T is that concentration is 1.4 * 10
-7~ 1.8 * 10
-7The thymidine of mol/L;
3) hybridoma screening
Selected 2 blocks of plates in 40 plates to sieve in advance by indirect enzyme-linked immunosorbent absorption at random on the 8th ~ 12 day after the cytogamy, determine whether to need to change cell culture fluid cloning son; Carried out primary dcreening operation by indirect enzyme-linked immunosorbent absorption on the 15th ~ 19 day, and tentatively determined it is male clone, and the hybridoma cell transfer of correspondence gone down to posterity in 24 well culture plates cultivate a week; Carried out multiple sieve by indirect enzyme-linked immunosorbent assay on the 22nd ~ 26 day, filter out positive colony, and determine 3 ~ 5 strain growth conditions preferably and the strongest cell strain of expression specificity antibody ability enter the structure of recombinant vectors, other-20 ℃ of clone's are frozen, the cell strain that obtains through reorganization selects a strain avidity and secretory antibody ability strong, high clone's of specificity is through vitro culture, and Sepharose CL-4B Protein A column separating purification also obtains the target monoclonal antibody.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108314711A (en) * | 2018-02-28 | 2018-07-24 | 吉林省农业科学院 | A kind of cry1C recombinant proteins with immunogenicity, the nucleic acid molecules of separation and its application |
| CN109781992A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of insect resistance protein Cry1C |
| CN109781993A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of enzyme linked immunological kit of quantitative detection insect resistance protein Cry1C |
| CN109777785B (en) * | 2018-12-27 | 2021-05-04 | 中国农业科学院生物技术研究所 | Hybridoma cell strain and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831441A (en) * | 2010-03-26 | 2010-09-15 | 中国农业大学 | Escherichia coli engineering bacteria for expressing recombinant CrylC genes |
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2010
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101831441A (en) * | 2010-03-26 | 2010-09-15 | 中国农业大学 | Escherichia coli engineering bacteria for expressing recombinant CrylC genes |
Non-Patent Citations (1)
| Title |
|---|
| 《中国优秀博硕士学位论文全文数据库（硕士）基础科学辑》 20041215 乔艳红 生物转基因成分检测方法的研究 参见第20-47页第三章和第四章，尤其是第3.2和第4.2部分 1 , * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108314711A (en) * | 2018-02-28 | 2018-07-24 | 吉林省农业科学院 | A kind of cry1C recombinant proteins with immunogenicity, the nucleic acid molecules of separation and its application |
| CN109781992A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of insect resistance protein Cry1C |
| CN109781993A (en) * | 2018-12-27 | 2019-05-21 | 中国农业科学院生物技术研究所 | A kind of enzyme linked immunological kit of quantitative detection insect resistance protein Cry1C |
| CN109781992B (en) * | 2018-12-27 | 2021-04-06 | 中国农业科学院生物技术研究所 | Colloidal gold immunochromatographic assay rapid test card for insect-resistant protein Cry1C |
| CN109777785B (en) * | 2018-12-27 | 2021-05-04 | 中国农业科学院生物技术研究所 | Hybridoma cell strain and application thereof |
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Application publication date: 20110713 |