CN108822203A - A kind of preparation of dog relaxain recombinant protein and its monoclonal antibody - Google Patents
A kind of preparation of dog relaxain recombinant protein and its monoclonal antibody Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
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Abstract
The invention belongs to field of biotechnology.The present invention provides a kind of dog relaxain recombinant protein, which mainly includes two dominant antigen epitopes of dog relaxain.To improve yield of the recombinant protein in prokaryotic expression system, which is converted to by corresponding nucleotide sequence using Escherichia coli preference codon, by the chemical synthesis nucleotide sequence and constructs recombinant expression carrier.The invention further relates to the preparations of the recombinant protein monoclonal antibody, monoclonal cell strain is obtained after immune, cell fusion and multi-turns screen by antigen, monoclonal antibody purification simultaneously marks colloid gold particle respectively, determines optimum monoclonal antibody combinations of pairs by orthogonal experiment, can be used for dog cyesiognosis.
Description
Technical field
The invention belongs to field of biotechnology, a kind of be related to dog relaxain recombinant protein, encode the recombinant protein nucleosides
The bacterial strain of acid sequence, the plasmid vector containing above-mentioned nucleotide sequence, conversion containing above-mentioned plasmid vector, further relates to using above-mentioned
Recombinant protein prepare dog relaxain nucleoprotein monoclonal antibody, and be applied to dog First Trimester diagnose.
Background technique
In the modern life, many people can keep a pet, especially pet dog.In order to preferably be looked after to them, Neng Gou
Dog early pregnancy is made correct diagnosis and is particularly important.The pregnancy period of dog dog generally has 9 weeks, but always will several Zhou Caihui to the end
Aobvious bosom, so pregnancy is difficult to discover early period；The reproduction mechanism of dog is different from other animals simultaneously, and main feature includes：
Maturation of ovum occurs after ovulation, embryo's attached plants delay, in serum Concentration of Progesterone in raising, pregnant dog and non-pregnant dog serum before preovulatory
Reasons, the general Testing index such as Concentration of Progesterone is close, the pregnant characteristic protein of dog lacks are difficult the use during pregnancy in dog.
Currently, for dog cyesiognosis, there are several types of methods both at home and abroad：
1. external observation method
It will appear pregnancy reaction and body variation after bitch pregnancy, people can be by observing and touch discerns whether to cherish
It is pregnant, but height, while information inaccuracy are required to operator, it is easy erroneous judgement.
2. special instrument detection method
Hospitals at Present often uses doppler instrument, A-mode ultrasonic apparatus and B-mode ultrasonic apparatus detection pregnancy, and this method accuracy rate is high, still
Equipment is expensive, requires high and testing cost high operator.
3. immunology diagnosis
By immunological method, the specific dog relaxain antigen using in monoclonal antibody recognition detection respiratory tract specimens,
This method specificity and sensitivity are all good.If association colloid gold platform generally can obtain accurate result in 10~15 minutes, fit
It closes high-volume sample quickly to detect, no special installation and personnel requirement.
Immunochromatographic method specifically uses monoclonal antibody recognition detection dog relaxain, and this method specificity and sensitivity are all
It is good.If association colloid gold platform generally can obtain accurate result in 10~15 minutes, high-volume sample is suitble to quickly to detect,
No special personnel requirement.
Currently, immunological method detects dog gestation since easy to operate, result is easy to determine, it has also become main flow direction.It should
Method usually requires to prepare the monoclonal antibody of recognizable dog relaxain, used in conventional dog relaxain monoclonal antibody preparation
Immunogene is the intact proteins of technique for gene engineering expression, due to the reason of base codon, albumen table in Escherichia coli
Up to difficult, expression quantity is extremely low, causes subsequent purification work to be difficult to carry out, its monoclonal antibody is seriously hindered to prepare.In addition, due to
Epitope amino acid sequence homology reason, the monoclonal for using dog relaxain full length sequence to be prepared as immunogene are anti-
Body poor specificity has high homology with other species albumen, so as to cause testing result distortion.
Summary of the invention
Purpose of design：The shortcoming for solving immunological method detection dog relaxain, by designing, expressing dog relaxain weight
Histone simultaneously prepares its monoclonal antibody, to realize that dog relaxain specific detection identifies, it is simultaneous both to have enhanced detection sensitivity
Its broad spectrum activity is cared for, and not will lead to testing result distortion.
Design scheme：In order to realize above-mentioned purpose of design.The application：(1) using dog relaxain nucleoprotein as target antigen, analysis
And two dominant antigen epitopes of the antigen are selected, sequence comparison result shows that selected two epitopes are all hypotypes
Dog relaxation fibroin shares epitope and with other protein sequences without obvious homology.(2) it is enhancing immune effect and shortens Dan Ke
The grand Antibody preparation time passes through soft segment (continuous four after repeating selected two dominant antigen epitope sequences respectively
Glycine) connection, form dog relaxain recombinant protein amino acid sequence.(3) it to improve dog relaxain recombinant protein expression quantity, adopts
With Escherichia coli preference codon, dog relaxain recombinant protein amino acid sequence is converted into corresponding nucleotide sequence.(4) change
The nucleotide sequence that synthesis previous step obtains is learned, and is connected by digestion, the nucleotide fragments insertion expression that synthesis is obtained
Carrier PET-28a (+) constructs dog relaxain recombinant protein expression vector.(5) conversion of dog relaxain recombinant protein expression vector is big
Enterobacteria BL21 (DE3) competent cell, screening obtain dog relaxain recombinant protein expression bacterial strain.(6) dog relaxain recombinates egg
After white expression bacterial strain large-scale culture, through carrying out ultrasonic bacteria breaking and low-temperature centrifugation, solution supernatant is taken to pass through nickel agarose affinity chromatography column
Affinity chromatography affords purifying dog relaxain recombinant protein.(7) Balb/c is repeatedly immunized in the dog relaxain recombinant protein purified
After mouse, its spleen cell is taken to merge with sp2/0 myeloma cell, is excessively taken turns subclone screening and finally obtain secretion dog relaxation
The hybridoma cell strain of plain nucleoprotein monoclonal antibody.(8) hybridoma cell strain is prepared to Balb/c mouse ascites respectively, is used
Protein A affinity chromatography monoclonal antibody purification, and colloid gold particle is marked respectively.(9) orthogonal experiment screening display
8A11 monoclonal antibody coating is paired into optimum detection dog relaxain with 7G2 monoclonal antibody label and combines.
The invention discloses a kind of dog relaxain recombinant protein, the amino acid sequence of the recombinant protein such as sequence table SEQs
ID No:Amino acid sequence shown in 1.
The invention also discloses a kind of dog relaxain recombinant protein, the amino acid sequence of the recombinant protein includes such as sequence
Table SEQ ID No:2 and SEQ ID No:Amino acid sequence shown in 3.
The invention also discloses：A kind of nucleotide sequence, the nucleotide sequence such as sequence table SEQ ID No:Shown in 4, it can compile
Code dog relaxain recombinant protein described above.
The invention also discloses：One kind containing SEQ ID No:The plasmid vector of nucleotide sequence shown in 4.
Design scheme 5：A kind of bacterial strain, the bacterial strain include above-mentioned plasmid vector.
Compared with the background technology, the present invention, first is that optimizing dog relaxain recombinant protein pair using Escherichia coli preference codon
The nucleotide sequence answered, to substantially increase expression of the dog relaxain recombinant protein in Escherichia coli；Second is that conduct
The dog relaxain recombinant protein of immunogene only contains the distinctive dominant antigen epitope of dog relaxain, ensure that finally obtained Dan Ke
Grand antibody only specific recognition dog relaxation fibroin, and screen obtained optimal monoclonal antibody combinations of pairs, improve detection sensitivity；
Third is that the dominant antigen epitope that immunogene contains is the shared epitope so dog relaxation fibroin, detection wide spectrum ensure that
Property, avoid missing inspection.
Specific embodiment
Although following embodiment contrasts detailed verbal description to mentality of designing of the invention, these texts are retouched
State, only the simple text of mentality of designing of the present invention described, rather than the limitation to mentality of designing of the present invention, it is any without departing from
The combination, increase or modification of mentality of designing of the present invention, each falls in protection scope of the present invention.
Embodiment 1：Dog relaxation fibroin dominant antigen epitope selection
Using dog relaxation fibroin as target antigen, the parent of its epitope sequence is analyzed using biosoftware DNAssist2.0
It is aqueous and antigenic, select A dominant antigen epitope (SEQ ID No:And B dominant antigen epitope (SEQ ID No 2):3).Meanwhile
Sequence comparison result shows that two dominant antigen epitope sequences of selected A, B have broad spectrum activity, is all dog relaxation fibroins
Shared epitope；And A, B epitope and other protein sequences exist only in dog relaxation avidin sequence without obvious homology.
Embodiment 2：The series connection of dog relaxain dominant antigen epitope
Shorten monoclonal antibody preparation time to the activation effect of mouse immune system to enhance selected epitope,
After dog relaxain nucleoprotein two dominant antigen epitope sequences of A, B are passed through soft segment (continuous four glycine) connection respectively
It repeats, obtains recombinant protein amino acid sequence, particular sequence such as sequence table SEQ ID No:Shown in 1.
Embodiment 3：The nucleotide sequence of Optimized Coding Based dog relaxain recombinant protein
Under the premise of dog relaxain recombinant protein amino acid sequence is constant, in order to improve dog relaxain recombinant protein big
Expression quantity in enterobacteria converts the amino acid sequence of encoding canine relaxain recombinant protein according to Escherichia coli preference codon
For corresponding nucleotide sequence, particular sequence such as sequence table SEQ ID No:Shown in 4, and digestion position is added in downstream respectively on it
After the corresponding nucleotide sequence of point BamHI and EcoRI, synthesized by Hangzhou GoodHere Bio-Technology Co., Ltd..Purpose after synthesis
Gene is connected in pMD20-T carrier (precious bioengineering Dalian Co., Ltd).
Embodiment 4：Construct dog relaxain recombinant protein expression vector
By containing target gene pMD20-T carrier and PET-28a (+) carrier (German Novagen company) by restricted
Restriction endonuclease BamHI and EcoRI (precious bioengineering Dalian Co., Ltd) respectively at 37 DEG C double digestion 12 hours, digestion products difference
1% agarose gel electrophoresis is carried out, and gel extraction target gene and PET-28a (+) carrier (glue used in the present invention respectively
QIAquick Gel Extraction Kit is all from Ningbo Zhong Ding Bioisystech Co., Ltd).Use T4 ligase (the precious limited public affairs in bioengineering Dalian
Department) by the target gene of recycling and PET-28a (+) carrier according to a certain percentage after 4 DEG C connect 12 hours, connection product conversion
DH5 α competent cell (Hangzhou GoodHere Bio-Technology Co., Ltd.), and it is coated on that penicillin resistance (50 μ g/mL) containing card
LB plate, in 37 DEG C after constant temperature incubation 12 hours, in picking monoclonal bacterial strain on plate to containing that penicillin resistance of card (50 μ
G/mL LB liquid medium), it is 37 DEG C after constant-temperature table culture 12 hours, (used herein using plasmid purification kit
Plasmid purification kit both from Ningbo Zhong Ding Bioisystech Co., Ltd) extract plasmid, through the bis- enzymes of BamHI and EcoRI
Correct recombinant expression carrier is obtained after cutting identification.
Embodiment 5：It constructs dog relaxain recombinant protein and expresses bacterial strain
Recombinant expression carrier Transformed E .coli BL21 (DE3) competent cell that will be built, and be coated on green containing that is blocked
The LB plate of chloramphenicol resistance (50 μ g/mL), is incubated overnight in 37 DEG C.Second day, on picking plate monoclonal bacterial strain to contain card that
The LB liquid medium of penicillin resistance (50 μ g/mL), 37 DEG C after constant-temperature table culture 8 hours, add inducer isopropylthio it is thio-
Protein electrophoresis sample is prepared after 4 hours of β-D- galactoside (final concentration of 1.0mmol/L) inducing expression.13.5% polypropylene
Acrylamide gel electrophoresis result shows dog relaxain recombinant protein successful expression, obtains dog relaxain recombinant protein expression bacterial strain.
Embodiment 6：Purify dog relaxain recombinant protein
It is inoculated with dog relaxain recombinant protein and expresses bacterial strain to LB liquid medium, Jia Kana penicillin to final concentration of 50 μ
G/mL, 37 DEG C after constant-temperature table culture 8 hours, with the LB liquid medium containing 50 μ g/mL card that penicillin by the bacterium by 1：100
After dilution proportion, dispenses into bacteria culture bottle, set 37 DEG C of constant-temperature table cultures to OD600=0.8, add inducer isopropylthio sulphur
Generation-β-D- galactoside continues culture induction 4 hours to final concentration of 1.0mmol/L.After thalline were collected by centrifugation, 4 degree of low temperature are super
Sound breaks bacterium, takes supernatant by nickel agarose affinity chromatography column after low-temperature centrifugation, and washed, elution finally obtains purifying dog relaxain
Recombinant protein.
Embodiment 7：Hybridoma cell strain building
Take 4-6 week old female Balb/c mouse, the subcutaneous multi-point injection Freund's complete adjuvant emulsification of every mouse of fundamental immunity
100 μ g dog relaxain recombinant proteins, total 400ul/ only.Second of booster immunization is carried out after 20 days, method is to take 80ug dog loose
The plain recombinant protein that relaxes is emulsified with freund 's incomplete adjuvant, and total 400ul/, subcutaneous multi-point injection.Third time booster immunization was at 15 days
After, method is identical with second of booster immunization.After 20 days, 120 μ g dog relaxain recombinant protein abdominal cavity booster shots are taken, in 72
After hour, eye socket takes blood, and puts to death mouse, its spleen is taken to prepare cell suspension, cell count, by 1/5 in the quantity of splenocyte
The good sp2/0 of growth conditions (murine myeloma cell) is taken, after mixing centrifugation, polyethylene glycol (Sigma company), which is added, makes two
Person's fusion.In addition, add isometric feeder cells, 96 porocyte plates that are placed in after mixing (200 hole μ L/), in 37 DEG C,
5% carbon dioxide incubator culture.After 5 days, liquid is changed in half reservation, thin using 96 holes of indirect enzyme-linked immunosorbent assay detection after 10 days
The supernatant of hybridoma is cultivated in born of the same parents' culture plate.The specific method is as follows：
Dog relaxain recombinant protein is coated after liquid dilution (final concentration of 1 μ g/mL), and ELISA Plate is added with 100 holes μ L/
(Wuxi Guo Sheng bioengineering Co., Ltd), by DEM-3 type board-washing machine, (Zhongshan University reaches peace gene to 4 DEG C of coatings after 12 hours
Limited liability company) it is washed 1 time with cleaning solution；Confining liquid is added, 200 holes μ L/, 37 DEG C are closed 1 hour, and board-washing is machine-washed plate 1 time；
Add cells and supernatant, positive control serum and negative control sample to be checked, 100 holes μ L/, after 37 DEG C of incubation 35min, cleaning solution
Washing 3 times；The sheep anti-mouse igg for adding HRP (horseradish peroxidase) to mark, 100 holes μ L/, after 37 DEG C are incubated for 30 minutes, cleaning solution
Washing four times；Every hole adds each 50 μ L of developing solution A and developing solution B, and 37 DEG C are protected from light colour developing after ten minutes, and terminate liquid is added to terminate reaction,
OD value is read in 50 holes μ L/ behind microplate reader 450nm wavelength blank well school zero.Related solution formula is as follows：
Coating buffer：Na2CO31.59g NaHCO32.93g adds distilled water to be settled to 1000mL (pH9.6).
Confining liquid：Na2HPO4.12H2O 2.68g,NaH2PO4.2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumin
It is white, add distilled water to be settled to 1000mL (pH7.4).
Cleaning solution：Na2HPO4.12H2O 2.68g,NaH2PO4.2H2O 0.39g, NaCl 8.5g, Tween-20 0.5mL,
Distilled water is added to be settled to 1000mL (pH7.4).
Developing solution A:200mg TMB is dissolved in 100mL dehydrated alcohol, and distilled water is added to be settled to 1000mL.
Developing solution B:Citric acid 2.1g, Na2HPO4.12H2O 71g, adds distilled water to be settled to 1000mL.When use：1mL is aobvious
Color liquid A+1mL developing solution B+0.4 μ L 30%H2O2
Terminate liquid：2M H2SO4, the dense H of 21.7mL2SO4Distilled water is added to be settled to 1000mL.
For the positive hybridoma cell clone of detection, reuses limiting dilution assay and be subcloned, select individual cells
It cultivates and passes through indirect enzyme-linked immunosorbent assay (ELISA) detection.By being subcloned three times, it is thin to obtain 8 plants of monoclonals for screening altogether
Born of the same parents' strain (2D2,2A9,3F6,4B9,6C10,7G2,7B3,8A11,8F3).
Embodiment 8：Monoclonal antibody preparation and purifying
The healthy Balb/c hero mouse of 6-8 week old is taken, atoleine is injected intraperitoneally, every 500 μ L are injected intraperitoneally single after 3 days
Clone cell (about 1.2 × 106A/only), after 7-9 days, mouse web portion is heaved, and collects ascites.With 50ml equilibration buffer PBS
(pH=7.4) agarose compatible medium Protein A chromatographic column (Nanjing Genscript Biotechnology Co., Ltd.) is balanced, until computer
Nucleic acid-protein detector (Shanghai Hu Xi analysis instrument Co., Ltd., Factory) shows that absorbance is adjusted to 0.Ascites 12000rpm is centrifuged 5 points
Clock, collect supernatant cross after 0.45um filter and loading plus PBS to wash to absorbance be 0, then use 0.1M glycine (pH=3.0)
Elution collects efflux and 500Mm Tris-HCl (pH=8.5) buffer is added is neutralized to pH=7.0 or so, obtain i.e.
For monoclonal antibody.
Embodiment 9：Colloid gold particle marks monoclonal antibody
It takes 0.01% colloidal gold solution of 10ml that 0.2mol/L solution of potassium carbonate 20uL is added, 100ug is added after mixing well
Antibody, room temperature reaction added 10% bovine serum albumin(BSA) (BSA) 1ml after 2 hours, after Seal treatment 2 hours, centrifugation
(7500rpm/min, 20 minutes), precipitating is redissolved liquid with 1ml and is sufficiently dissolved after discarding supernatant, and draws film instrument (Shanghai gold using metal spraying
Mark Biotechnology Co., Ltd) liquid will be redissolved according to 10ul/cm even application in glass fibre (width 6mm), then it is placed in electric heating
37 DEG C of constant incubator (Shanghai Yiheng Scientific Instruments Co., Ltd) stand 30 minutes.
Colloidal gold mark in the above way is carried out to 2D2,2A9,3F6,4B9,6C10,7G2,7B3,8A11,8F3 monoclonal antibody respectively
Note.Related solution formula is as follows:
0.01% colloidal gold solution：1% chlorauric acid solution 1ml, 1% citric acid solution 1.4ml adds ultrapure water to dissolve by heating
It reacts and is settled to 100ml.
1% chlorauric acid solution：1gAuCL3.HCl.4H2O powder adds ultrapure water to dissolve and is settled to 100ml.
1% citric acid solution：1g Citric acid crystal adds ultrapure water to dissolve and is settled to 100ml.
Redissolve liquid：Tris alkali 6.057g is dissolved in 800ml distilled water, adjusts pH to 8.0 with appropriate HCL, adds distilled water fixed
Hold 1000ml.
Embodiment 10：Match monoclonal antibody screening
Dog relaxain monoclonal antibody (2D2,2A9,3F6,4B9,6C10,7G2,7B3,8A11,8F3) is coated liquid respectively
After dilution (final concentration of 1mg/ml), by metal spraying draw film instrument (Shanghai Jinbiao Bio-Tech Co., Ltd.) according to 1ul/cm by its
It uniformly is coated in nitrocellulose filter (Sartorius), this is T line.Drawing film instrument by metal spraying, (Shanghai gold mark biotechnology is limited
Company) sheep anti mouse solution (final concentration of 1mg/ml) is uniformly coated in nitrocellulose filter according to 1ul/cm, this is C line.It draws
After film is coated with, it is quiet that nitrocellulose filter is placed in 37 DEG C of electro-heating standing-temperature cultivator (Shanghai Yiheng Scientific Instruments Co., Ltd)
It sets 30 minutes.
After sample pad, glass fibre, nitrocellulose filter, filter paper are successively assembled on PVC backing plate according to common process
It is cut into wide 4mm strip, load onto reagent strip shell and is compressed.Related solution formula is as follows：
Coating buffer：Na2HPO4.7H2O 43.42g,NaH2PO4.H2O 5.244g, adds distilled water to be settled to 1000mL
(pH7.4)。
Flu-A patient clinical serum sample and normal human serum sample, 100 hole μ L/ loadings, are placed at room temperature for 15min
Afterwards, P/N value (positive sample detected value readings and is calculated respectively by chromatography readout instrument (Shanghai Jie Hao scientific instrument Co., Ltd)
With the ratio of negative sample detected value), see Table 1 for details.
Table 1 matches monoclonal antibody P/N Data-Statistics
By upper table it is found that it is optimal combination that 8A11 monoclonal antibody coating, which detects Flu-A with 7G2 label pairing,.
SEQ ID NO1：Dog relaxain recombinant protein amino acid sequence；
SEQ ID NO2：Dog relaxain nucleoprotein A dominant antigen epitope amino acid sequence；
SEQ ID NO3：Dog relaxain nucleoprotein B dominant antigen epitope amino acid sequence；
SEQ ID NO4：The nucleotide sequence of encoding canine relaxain recombinant protein；
Sequence table
<110>Hangzhou GoodHere Bio-Technology Co., Ltd.
<120>Dog relaxain recombinant protein and its monoclonal antibody
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 98
<212> PRT
<213>Artificial sequence (dog relaxain nucleoprotein dominant antigen epitope)
<400> 1
Ile Glu Val Cys Gly Ser Ile Trp Trp Gly Arg Lys Gly Gly Gly Gly
1 5 10 15
Thr Phe Leu Asn Thr Gln Phe Glu Ala Glu Asp Lys Ser Leu Gly Gly
20 25 30
Gly Gly Ile Glu Val Cys Gly Ser Ile Trp Trp Gly Arg Lys Gly Gly
35 40 45
Gly Gly Thr Phe Leu Asn Thr Gln Phe Glu Ala Glu Asp Lys Ser Leu
50 55 60
Gly Gly Gly Gly Ile Glu Val Cys Gly Ser Ile Trp Trp Gly Arg Lys
65 70 75 80
Gly Gly Gly Gly Thr Phe Leu Asn Thr Gln Phe Glu Ala Glu Asp Lys
85 90 95
Ser Leu
<210> 2
<211> 12
<212> PRT
<213> Virus
<400> 2
Ile Glu Val Cys Gly Ser Ile Trp Trp Gly Arg Lys
1 5 10
<210> 3
<211> 14
<212> PRT
<213> Virus
<400> 3
Thr Phe Leu Asn Thr Gln Phe Glu Ala Glu Asp Lys Ser Leu
1 5 10
<210> 4
<211> 294
<212> DNA
<213>Artificial sequence (preference codons of Escherichia coli)
<400> 4
Claims (6)
1. a kind of dog relaxain recombinant protein, which is characterized in that the amino acid sequence of the recombinant protein such as SEQ ID No:1 institute
Show.
2. a kind of dog relaxain recombinant protein according to claim 1, which is characterized in that the amino acid of the recombinant protein
Sequence includes sequence table SEQ ID No:2 and SEQ ID No:Amino acid sequence shown in 3.
3. one kind can encode the nucleotide sequence of dog relaxain recombinant protein of any of claims 1-2, special
Sign is, the nucleotide sequence such as SEQ ID No:Shown in 4.
4. one kind contains SEQ ID No:The plasmid vector of nucleotide sequence shown in 4.
5. the bacterial strain that a kind of bacterial strain uses plasmid vector as claimed in claim 4.
6. a kind of prepare dog relaxain nucleoprotein monoclonal using the described in any item dog relaxain recombinant proteins of claim 1-2
The method of antibody, includes the following steps:
(a) synthesis such as SEQ ID No:Nucleotide sequence shown in 4, and plasmid vector is connected, construct dog relaxain recombinant protein
Expression vector；
(b) the dog relaxain recombinant protein expression vector in step (a) is transformed into Escherichia coli, screening obtains dog relaxain weight
Histone expresses bacterial strain；
(c) after large-scale culture dog relaxain recombinant protein expression bacterial strain, purified acquisition dog relaxain recombinant protein；
(d) after repeatedly Balb/c mouse is immunized in dog relaxain recombinant protein, its spleen cell is taken to melt with sp2/0 myeloma cell
It closes, obtains monoclonal cell strain by multi-turns screen；
(e) monoclonal antibody purification and colloid gold particle is marked respectively, optimum monoclonal antibody combinations of pairs is determined by orthogonal experiment.
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| CN201810857284.4A CN108822203A (en) | 2018-07-31 | 2018-07-31 | A kind of preparation of dog relaxain recombinant protein and its monoclonal antibody |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2005247332A2 (en) * | 2004-04-30 | 2005-12-08 | Corthera, Inc. | Methods and compositions for control of fetal growth via modulation of relaxin |
| CN102659937A (en) * | 2012-03-16 | 2012-09-12 | 杭州贤至生物科技有限公司 | Preparation method and application of recombinant protein and monoclonal antibody thereof |
| CN106632619A (en) * | 2016-12-02 | 2017-05-10 | 杭州贤至生物科技有限公司 | Influenza A virus recombinant protein and preparation of monoclonal antibody thereof |
| CN106811470A (en) * | 2015-11-30 | 2017-06-09 | 四川农业大学 | A kind of preparation method of dog relaxation precipitinogen recombinant protein and dog relaxation precipitinogen polyclonal antibody |
-
2018
- 2018-07-31 CN CN201810857284.4A patent/CN108822203A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2005247332A2 (en) * | 2004-04-30 | 2005-12-08 | Corthera, Inc. | Methods and compositions for control of fetal growth via modulation of relaxin |
| CN102659937A (en) * | 2012-03-16 | 2012-09-12 | 杭州贤至生物科技有限公司 | Preparation method and application of recombinant protein and monoclonal antibody thereof |
| CN106811470A (en) * | 2015-11-30 | 2017-06-09 | 四川农业大学 | A kind of preparation method of dog relaxation precipitinogen recombinant protein and dog relaxation precipitinogen polyclonal antibody |
| CN106632619A (en) * | 2016-12-02 | 2017-05-10 | 杭州贤至生物科技有限公司 | Influenza A virus recombinant protein and preparation of monoclonal antibody thereof |
Non-Patent Citations (3)
| Title |
|---|
| DEBRA A. CARLSON ET AL.: ""Relaxin as a diagnostic tool for pregnancy in the coyote (Canis latrans)"", 《ANIMAL REPRODUCTION SCIENCE》 * |
| 任志华 等: "犬松弛素原基因原核表达及其多克隆抗体的制备", 《农业生物技术学报》 * |
| 黄韧 等: "《生物信息学网络资源与应用》", 30 June 2003, 中山大学出版社 * |
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