CN106834319A - A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody - Google Patents
A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody Download PDFInfo
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- CN106834319A CN106834319A CN201710111412.6A CN201710111412A CN106834319A CN 106834319 A CN106834319 A CN 106834319A CN 201710111412 A CN201710111412 A CN 201710111412A CN 106834319 A CN106834319 A CN 106834319A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The invention discloses the preparation method of a kind of coded sequence of recombinant protein, recombinant protein and its monoclonal antibody.The recombinant protein includes tetra- kinds of label protein epitopes of 6xHis, S Tag, Trx and GST, and the restructuring amino acid sequence is converted into corresponding nucleotide sequence with reference to Escherichia coli preference codon, by Protocols in Molecular Biology Prepare restructuring antigen, immune mouse, is obtained for four kinds of high-quality monoclonal cell strains of label protein respectively by Screening Platforms such as enzyme linked immunologicals.
Description
Technical field
The invention belongs to biological technical field.Specifically, the present invention relates to a kind of coded sequence of recombinant protein, restructuring
The preparation method of albumen and its monoclonal antibody.
Background technology
Label protein antibody (Tag-antibody) is mouse monoclonal antibody of the class by affinity purification.For detecting
Sequence label on extensive stock expression vector（Such as：MyC, His, GST, HA etc.）, nationality is with the table of analytical control destination protein
Up to content and its function.Its principle is antigen-antibody reaction, and these tag antibodies can combine corresponding label with high special
Fusion protein.Tag antibody is the common tool for carrying out expression of gene protein, signal transduction and gene functional research.
Conventional label protein monoclonal antibody prepares the complete egg that used immunogene is technique for gene engineering expression
In vain, but due to the reason of base codon, the albumen is difficult in expression in escherichia coli, and expression quantity is extremely low, causes follow-up pure
Chemical industry is difficult to carry out, and seriously hinders the preparation of its monoclonal antibody.In addition, label protein species is various, every kind of label protein
It is manufactured separately, time-consuming, high cost, is unfavorable for being widely popularized and using for market.
The content of the invention
It is contemplated that the weak point in avoiding background technology, by designing a kind of recombinant protein and preparing its monoclonal
Antibody, so as to realize while prepare various label protein monoclonal antibodies, both enhancing detection sensitivity, can prepare simultaneously again
Multiple Antibodies.
In order to achieve the above object, the technical scheme of present invention offer is：
The coded sequence of the recombinant protein is as shown in SEQ ID NO.1.
The amino acid sequence of the recombinant protein is as shown in SEQ ID NO.2.
The expression of recombinant proteins carrier is the plasmid vector containing coded sequence shown in SEQ ID NO.1.
Above-mentioned recombinant protein and expression of recombinant proteins carrier can be used to prepare label protein monoclonal antibody.
The preparation method of the label protein monoclonal antibody comprises the following steps：
（1）Recombinant protein coded sequence shown in synthesis SEQ ID NO.1, and recombinant protein coded sequence connection plasmid is carried
Body, and then build expression of recombinant proteins carrier；
（2）Expression of recombinant proteins carrier is converted into Escherichia coli, screening obtains expression of recombinant proteins bacterial strain；
（3）After by expression of recombinant proteins bacterial strain large-scale culture, purified acquisition recombinant protein, the amino acid of the recombinant protein
Sequence is as shown in SEQ ID NO.2；
（4）Recombinant protein is repeatedly immunized Balb/c mouse, Mouse spleen cells is taken and is merged with sp2/0 myeloma cell, passed through
Multi-turns screen and multi objective identification obtain the label protein monoclonal cell strain corresponding to recombinant protein respectively, then by label
Protein monoclonal cell line is obtained label protein monoclonal antibody.
The invention will be further described below：
The present invention is first with 4 kinds of label associated proteins（6xHis, S Tag, Trx and GST）It is target antigen, sequence comparative result shows
Show the selected epitope with other protein sequences without obvious homology.
Secondly, in order to promote stimulation of the selected epitope to BALB/c mouse immune system, immune effect is strengthened, therefore
Pass through soft segment after selected four dominant antigen epitope sequences are repeated respectively（Continuous four glycine）Connection, forms
Recombinant protein amino acid sequence.
3rd step, using Escherichia coli preference codon, corresponding nucleotides is converted to by recombinant protein amino acid sequence
Sequence, is beneficial to expression of the recombinant protein in Escherichia coli to improve expression quantity.
4th step, the nucleotide sequence that chemical synthesis previous step is obtained, and connected by digestion, the core for obtaining will be synthesized
Acid fragments insert expression vector PET-28a (+), build expression of recombinant proteins carrier.
5th step, expression of recombinant proteins carrier conversion Escherichia coli ER2566 competent cells, screening obtains recombinant protein
Expression bacterial strain.
6th step, after expression of recombinant proteins bacterial strain large-scale culture, after carrying out ultrasonic bacteria breaking and low-temperature centrifugation, takes clear and coherent on solution
Nickel agarose affinity chromatography post affinity chromatography is crossed, purification of recombinant proteins is afforded.
7th step, after recombinant protein after purification is repeatedly immunized BALB/c mouse, takes its spleen cell and sp2/0 myeloma
Cell fusion, respectively obtains by multi-turns screen and finally hybridoma cell strain.
8th step, hybridoma cell strain is prepared BALB/c mouse ascites respectively, using caprylic acid-ammonium and
Protein G monoclonal antibody purifications in two steps.
Compared with prior art, beneficial effects of the present invention are：
One is repetition and the expressing in series that four kinds of label associated proteins epitopes are realized by Protocols in Molecular Biology, is increased
Strong stimulation of the purpose antigen epitope to mouse immune system, eliminates the interference that unrelated sequences may bring；
Two is only to contain four kinds of label protein epitopes as the recombinant protein of immunogene, it is ensured that the monoclonal for finally giving
Antibody only specific recognition four kinds of label proteins, and screen and obtain four kinds of label proteins simultaneously, the sensitivity of detection is improve,
Reduce experimental cost.
Three is using the corresponding nucleotide sequence of Escherichia coli preference codon optimum combination albumen, so as to substantially increase
Expression of the recombinant protein in Escherichia coli.
Specific embodiment
First, 4 kinds of label protein epitope selections
Be target antigen with 4 kinds of label proteins, using biosoftware DNAssist2.0 analyze its epitope sequence hydrophily and
Antigenicity, selects 6xHis, S Tag, Trx and GST epitope A, B, C and D respectively.Meanwhile, sequence comparative result shows selected
A, B, C and D epitope sequence specificity selected are high, with other protein sequences without obvious homology.
2nd, 4 kinds of series connection of label protein epitope
Be beneficial to the carrying out of subsequent experimental for selected stimulation of the epitope to mouse immune system of enhancing, by label protein A,
Tetra- kinds of epitope sequences of B, C and D pass through soft segment respectively（Continuous four glycine）Connection, obtains recombinant protein amino acid
Sequence, its particular sequence is as shown in sequence table SEQ ID N0.2.
3rd, the nucleotide sequence of Optimized Coding Based recombinant protein
In order to improve expression quantity of the recombinant protein in Escherichia coli, on the premise of recombinant protein amino acid sequence is constant, root
The amino acid sequence for encoding recombinant protein is converted into corresponding nucleotide sequence, particular sequence according to Escherichia coli preference codon
As shown in sequence table SEQ ID NO.1, and the corresponding nucleotides of restriction enzyme site BamHI and EcoRI is added in downstream respectively thereon
After sequence, synthesized by Hangzhou GoodHere Bio-Technology Co., Ltd..Genes of interest after synthesis is cloned in pMD19-T carriers, and (treasured is raw
Thing engineering Dalian Co., Ltd) in.
4th, expression of recombinant proteins carrier is built
Contain purpose in 37 DEG C of difference double digestions with restriction enzyme BamHI and EcoRI (precious bioengineering Dalian Co., Ltd)
The pMD19-T carriers and PET-28a (+) carrier of gene（German Novagen companies）12 hours, the digestion products fine jade of row 1% respectively
Sepharose electrophoresis, and gel extraction genes of interest and PET-28a (+) carrier respectively（Glue reclaim reagent used in the present invention
Box is all from Ningbo Zhong Ding Bioisystech Co., Ltd）.To be reclaimed using T4 ligases (precious bioengineering Dalian Co., Ltd)
Genes of interest and PET-28a (+) carrier according to a certain percentage in after 4 DEG C of connections 12 hours, connection product conversion DH5 α impression
State cell（Hangzhou GoodHere Bio-Technology Co., Ltd.）, and coat containing the LB flat boards for blocking that penicillin resistance (50 μ g/mL), in
37 DEG C incubated 12 hours afterwards, in picking monoclonal bacterial strain on flat board to containing block that penicillin resistance (50 μ g/mL) LB
After fluid nutrient medium, 37 DEG C of constant-temperature table cultures 12 hours, using plasmid purification kit（Plasmid purification used in the present invention
Kit is both from Ningbo Zhong Ding Bioisystech Co., Ltd）Plasmid is extracted, after being identified through BamHI and EcoRI double digestions
To correct recombinant expression carrier.
5th, recombinant protein E expression bacterial strains are built
The recombinant expression carrier conversion that will be builtE.coliER2566 competent cells, and coat anti-containing that penicillin is blocked
The LB flat boards of property (50 μ g/mL), in 37 DEG C of incubated overnights.Second day, monoclonal bacterial strain extremely that penicillin containing card on picking flat board
After the LB fluid nutrient mediums of resistance (50 μ g/mL), 37 DEG C of constant-temperature table cultures 8 hours, plus the thio-β-D- of inducer isopropylthio half
Lactoside（Final concentration of 1.0mmol/L）Induced expression prepares protein electrophoresis sample after 4 hours.13.5% polyacrylamide coagulates
Gel electrophoresis result shows recombinant protein successful expression, obtains expression of recombinant proteins bacterial strain.
6th, purification of recombinant proteins F
It is inoculated with expression of recombinant proteins bacterial strain to LB fluid nutrient mediums, Jia Kana penicillin to final concentration of 50 μ g/mL, 37 DEG C of constant temperature
After shaking table culture 8 hours, with the LB fluid nutrient mediums containing 50 μ g/mL cards that penicillin by the bacterium by 1：After 100 dilution proportions, point
It is filled in bacteria culture bottle, puts 37 DEG C of constant-temperature table cultures to OD600=0.8, plus the thio-β-D- galactolipins of inducer isopropylthio
Glycosides continues to cultivate induction 4 hours to final concentration of 1.0mmol/L.After thalline is collected by centrifugation, low temperature ultrasonic breaks bacterium, low-temperature centrifugation
After take supernatant by nickel agarose affinity chromatography post, scrubbed, wash-out finally gives purification of recombinant proteins.
7th, the coupling protein for detecting is built
Two epitope sequences of label protein A, B are connected into a cysteine in N-terminal respectively, synthesizes G, H sequences polypeptide, carried
Body protein selects BSA（Roche companies）, use SPDP（PIERCE companies）The polypeptide that connection method will synthesize is coupled with BSA respectively：
4.6mg SPDP dissolving 740ul DMSO, final concentration of 20mM.0.1008g BSA are dissolved in 2ml PBS-EDTA solution, room
Temperature stands 1h.HiTrapTM Deaslting column desalting columns elute unnecessary SPDP.4mg polypeptides add what is be coupled
Ambient temperature overnight in BSA-SPDP systems, obtains product BSA-G, BSA-H（Synthesized by Hangzhou GoodHere Bio-Technology Co., Ltd.）.Will
Protein G ST and TRX（Buy in Hangzhou Bi Kenlaibo bio tech ltd）It is named as I, L.
8th, the acquisition of hybridoma cell strain
Take 5-7 week old female BAl BIc/c mouse, 70 μ of the subcutaneous multi-point injection Freund's complete adjuvant emulsification of every mouse of fundamental immunity
G recombinant proteins；Booster immunization is carried out after 15 days, after method is to take same amount of recombinant protein freund 's incomplete adjuvant emulsification,
Subcutaneous multi-point injection；Third time booster immunization is after 15 days, and method is identical with second.After 30 days, 120 μ g recombinant proteins are taken
Abdominal cavity booster shots, and in after abdominal cavity booster shots 72 hours, eye socket takes blood, and puts to death mouse, take its spleen and prepare cell and hang
Liquid, cell count takes the good sp2/0 murine myeloma cells of growth conditions by 1/5 in the quantity of splenocyte, mixes centrifugation
Afterwards, polyethylene glycol is added（PEG-4000）Merge the two.In addition, adding isometric feeder cells, it is placed in after mixing
96 porocyte plates（200 μ L/ holes）, in 5% CO2gas incubator culture.After 5 days, liquid is changed in half reservation, using indirect after 10 days
Enzyme linked immunosorbent assay detects the Hybridoma Cell Culture supernatant in 96 porocyte culture plates.
Specific method is as follows：
Protein B SA-G, BSA-H, I, L are respectively after coating buffer dilutes（Final concentration of 1 μ g/mL）, enzyme is added with 100 μ L/ holes
Target（Shenzhen Jin Canhua Industrial Co., Ltd.s）, 4 DEG C coating 12 hours after washed once and patted dry with cleaning solution；Add closing
Liquid, 150 μ L/ holes, 37 DEG C are closed 2 hours, abandon liquid in hole, are patted dry；Respectively plus cells and supernatant to be checked and control serum,
100 μ L/ holes, after 37 DEG C are incubated 1 hour, cleaning solution is washed three times and patted dry；Plus HRP（Horseradish peroxidase）The goat-anti of mark
Mouse IgG, 100 μ L/ holes, after 37 DEG C are incubated 30 minutes, cleaning solution is washed four times and patted dry；Add nitrite ion A and nitrite ion B each per hole
50 μ L, after 37 DEG C of lucifuges develop the color 10 minutes, plus terminate liquid terminating reaction, 50 μ L/ holes, ELIASA 450nm wavelength blank wells school zero
OD values are read afterwards.Using the serum of immune mouse as positive control, related solution formula is as follows：
Coating buffer：Na2CO31.5g, NaHCO32.9g, plus distilled water is settled to 1000mL (pH9.6).
Confining liquid：Na2HPO4.12H2O 2.68g, NaH2PO4.2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumins
In vain, plus distilled water is settled to 1000mL (pH7.4).
Cleaning solution：Na2HPO4.12H2O 2.68g, NaH2PO4.2H2O 0.39g, NaCl 8.5g, Tween-20
0.5mL, plus distilled water is settled to 1000mL (pH7.4).
Nitrite ion A:200mg TMB are dissolved in 100mL absolute ethyl alcohols, plus distilled water is settled to 1000mL.
Nitrite ion B:Citric acid 2.1g, Na2HPO4.12H2O 71g, plus distilled water is settled to 1000mL.
When using：1mL nitrite ion A+1mL nitrite ion B+0.4 μ L 30%H2O2
Terminate liquid：2M H2SO4, the dense H of 21.7mL2SO4Plus distilled water is settled to 1000mL.
The hybridoma cell clone positive for detection, reuses limiting dilution assay and is subcloned.By three Asias gram
Grand, screening respectively obtains the strain of the strain of hybridoma of 6xHis 3（1F4、8B6、3D7、）, the strain of the strain of hybridoma of S Tag 2（5D6、
2C3）；The strain of the strain of hybridoma of Trx 5（4F4、6B7、3G7、6T5、7H5）；The strain of GST3 strain of hybridoma（7F4、2C7、
6D7）.
Hybridoma cell strain is finally prepared BALB/c mouse ascites respectively, caprylic acid-ammonium and Protein G is used
Monoclonal antibody purification in two steps.
Claims (6)
1. a kind of coded sequence of recombinant protein, it is characterised in that the coded sequence is as shown in SEQ ID NO.1.
2. the recombinant protein that coded sequence as claimed in claim 1 is encoded, it is characterised in that the amino acid sequence of the recombinant protein
Row are as shown in SEQ ID NO.2.
3. a kind of expression of recombinant proteins carrier, it is characterised in that the expression of recombinant proteins carrier is to contain SEQ ID NO.1 institutes
Show the plasmid vector of coded sequence.
4. application of the recombinant protein as claimed in claim 2 in label protein monoclonal antibody is prepared.
5. application of the expression of recombinant proteins carrier as claimed in claim 3 in label protein monoclonal antibody is prepared.
6. a kind of preparation method of label protein monoclonal antibody, it is characterised in that methods described comprises the following steps：
(1) the recombinant protein coded sequence shown in synthesis SEQ ID NO.1, and recombinant protein coded sequence connection plasmid is carried
Body, and then build expression of recombinant proteins carrier；
(2) expression of recombinant proteins carrier is converted into Escherichia coli, screening obtains expression of recombinant proteins bacterial strain；
(3) after by expression of recombinant proteins bacterial strain large-scale culture, purified acquisition recombinant protein, the amino acid of the recombinant protein
Sequence is as shown in SEQ ID NO.2；
(4) recombinant protein is repeatedly immunized Balb/c mouse, takes Mouse spleen cells and merged with sp2/0 myeloma cell, passed through
Multi-turns screen and multi objective identification obtain the label protein monoclonal cell strain corresponding to recombinant protein respectively, then by label
Protein monoclonal cell line is obtained label protein monoclonal antibody.
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| CN201710845833.1A CN107603996A (en) | 2017-02-28 | 2017-09-19 | A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603996A (en) * | 2017-02-28 | 2018-01-19 | 中南大学湘雅医院 | A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody |
| CN109752561A (en) * | 2019-01-23 | 2019-05-14 | 日照岚山生化制品有限公司 | The enzyme-linked immunologic detecting kit of one boar relaxain |
| CN113234161A (en) * | 2021-06-24 | 2021-08-10 | 福州迈新生物技术开发有限公司 | anti-CD 3 protein monoclonal antibody and cell strain, preparation method and application thereof |
| WO2022057696A3 (en) * | 2021-09-08 | 2022-07-28 | 中南大学湘雅医院 | Recombinant protein coding sequence, recombinant protein, and method for preparing monoclonal antibodies |
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| CN102364342A (en) * | 2011-08-05 | 2012-02-29 | 北京康为世纪生物科技有限公司 | Method for quickly detecting expression of recombinant protein |
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| CN102659937B (en) * | 2012-03-16 | 2014-01-08 | 杭州贤至生物科技有限公司 | Preparation method and application of recombinant protein and monoclonal antibody thereof |
| CN106834319A (en) * | 2017-02-28 | 2017-06-13 | 中南大学湘雅医院 | A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody |
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| CN102364342A (en) * | 2011-08-05 | 2012-02-29 | 北京康为世纪生物科技有限公司 | Method for quickly detecting expression of recombinant protein |
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| 徐竹蔚等: "抗3种标签蛋白单克隆抗体的制备及特性鉴定", 《细胞与分子免疫学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107603996A (en) * | 2017-02-28 | 2018-01-19 | 中南大学湘雅医院 | A kind of preparation method of the coded sequence of recombinant protein, recombinant protein and its monoclonal antibody |
| CN109752561A (en) * | 2019-01-23 | 2019-05-14 | 日照岚山生化制品有限公司 | The enzyme-linked immunologic detecting kit of one boar relaxain |
| CN113234161A (en) * | 2021-06-24 | 2021-08-10 | 福州迈新生物技术开发有限公司 | anti-CD 3 protein monoclonal antibody and cell strain, preparation method and application thereof |
| WO2022057696A3 (en) * | 2021-09-08 | 2022-07-28 | 中南大学湘雅医院 | Recombinant protein coding sequence, recombinant protein, and method for preparing monoclonal antibodies |
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