CN110194797A - One breeder MLP protein polyclone antibody and the preparation method and application thereof - Google Patents
One breeder MLP protein polyclone antibody and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention provides breeder MLP protein polyclone antibodies and the preparation method and application thereof, method includes the following steps: (1) constructs the recombinant expression plasmid containing nucleotide sequence shown in SEQ ID No:1, the recombinant expression plasmid is converted into expression bacterium and obtains recombinant protein antigen；(2) animal is immunized in the recombinant protein antigen, menses, which isolate and purify clearly, obtains chicken MLP protein polyclone antibody.The present invention also provides application of the chicken MLP protein polyclone antibody in detection chicken tissues or cell in MLP albumen.Chicken MLP protein polyclone antibody high specificity produced by the present invention, can MLP albumen in specific recognition chicken tissues or in cell, the functional study for being MLP on chicken provides important tool.
Description
Technical field
The invention belongs to the technical fields of poultry genetic engineering, more particularly, to a breeder MLP protein polyclone antibody
And the preparation method and application thereof.
Background technique
Chicken is due to having the characteristics that high protein, low cholesterol, low fat and low in calories, it has also become consumer all over the world
Generally accepted high-quality source of animal protein.In addition, compared with pig, ox, broiler chicken short, feed conversion rate with breeding cycle
The advantages that height, high raising and atomization degree, therefore, chicken is also most economical source of animal protein.In China, chicken
Meat product is mainly derived from white meat-type chickens and yellow-feather broiler two major classes.White meat-type chickens are introduced variety, belong to fast large-scale broiler chicken, tool
There is the features such as speed of growth is fast, feed conversion rate is high, meat yield is more, is suitble to industrial scale production, but mouthfeel is not good enough.Huang Yu
Broiler chicken is the special consumer food formed by the Traditional consumption habits in China, and relative to white meat-type chickens, yellow-feather broiler has disease-resistant
The features such as power is strong, delicious meat.With the improvement of living standards with the transformation of consumption idea, excellent in color and safety chicken
Meat is more and more popular with consumers.Therefore, it probes into Skeletal Muscle Growth developmental regulation mechanism, guarantee that chicken fast and safely grows simultaneously
The improvement that synchronizes for keeping meat is the most important theories and practical problems that current broiler chicken breeder faces.
The flesh generation of skeletal muscle is a sufficiently complex process, is regulated and controled by stringent development program, dependent on regulation
The accurate spatial and temporal expression of the factor.Myoblast determination family (Myogenic Regulation Factors, MRFs) is acknowledged as
It is the regulation myogenetic key factor of skeletal muscle, during MRFs activation flesh is generated with muscle specific gene expression, needs
Want the participation of some confactors.Rich in cysteine and (the Cysteine and glycine rich of glycine albumen 3
Protein 3, CSRP3) muscle LIM protein that is otherwise known as (muscle LIM protein, MLP) is that myocyte's differentiation must
The positive regulatory factor needed, lacks the myocyte of MLP, cannot be induced to differentiate；When being overexpressed MLP, then myocyte's differentiation capability
Enhancing.
The MLP antibody being commercialized at present is only the antibody for mouse or people, can not be in specific recognition chicken skeletal muscle
MLP albumen greatly limits research of the MLP on chicken.Therefore, developmental research one kind can be in specific recognition chicken skeletal muscle
MLP albumen, has great importance.
Summary of the invention
In order to solve problems in the prior art, the object of the present invention is to provide a breeder MLP protein polyclone antibodies
And the preparation method and application thereof.
Technical scheme is as follows:
The present invention provides the preparation methods of a breeder MLP protein polyclone antibody, method includes the following steps:
(1) recombinant expression plasmid containing nucleotide sequence shown in SEQ ID No:1 is constructed, by the recombinant expression plasmid
Conversion expression bacterium obtains recombinant protein antigen；
(2) animal is immunized in the recombinant protein antigen, menses, which isolate and purify clearly, obtains chicken MLP protein polyclone antibody.
Further, in step (1), the recombinant expression plasmid is will be containing nucleotide sequence shown in SEQ ID No:1
The prokaryotic expression carrier with His label is cloned into obtain.
Further, prokaryotic expression carrier pCzn1.When the prokaryotic expression carrier of the band His label is pCzn1,
Recombinant protein antigen expression effect is good, and is solubility expression, convenient for the purifying of recombinant protein.Contain in recombinant protein antigen
Amino acid sequence shown in SEQ ID No:2.
Further, in step (1), the expression bacterium is Arctic-ExpressTMBacillus coli expression bacterium.The present invention couple
It is not particularly limited in used Bacillus coli expression strain class, as long as the big of recombinant expression plasmid effective expression can be suitble to
Enterobacteria, it is preferred that in order to reach better expression effect, Bacillus coli expression bacterium is Arctic-ExpressTMExpression
Bacterium.
Further, in step (2), the animal is rabbit, mouse or rat.
Further, in step (2), be immunized animal the step of are as follows: first by recombinant protein antigen and the complete Freund of equivalent
It is immunized for the first time after adjuvant mixing, recombinant protein antigen is mixed with equivalent incomplete Freund's adjuvant then and be immunized 2-3 times,
Each immunization interval time was 2 to 3 weeks.After 3-4 times immune, blood sampling detection determines that antiserum is directed to by indirect elisa method
The potency of MLP albumen is greater than the final blood sampling of 1:50000 progress to potency and prepares antiserum.
Further, it in step (2), is purified using affinity purification chromatographic column.It is affine pure the present invention provides utilizing
Change chromatographic column and obtains the chicken MLP protein polyclone antibody that purity is not less than 85%.
The present invention also provides chicken MLP protein polyclone antibodies made from the preparation method.
Invention further provides application of the chicken MLP protein polyclone antibody in detection chicken tissues or cell in MLP albumen.
Beneficial effects of the present invention: chicken MLP protein polyclone antibody high specificity produced by the present invention, it being capable of specificity knowledge
The functional study that MLP albumen in other chicken tissues or in cell is MLP on chicken provides important tool.
Detailed description of the invention
Fig. 1 is the digestion qualification result of recombinant expression plasmid pCzn1-MLP of the present invention, wherein 1 is the recombination table before digestion
Up to plasmid, 2 be the recombinant expression plasmid after digestion, M DNAmarker；
Fig. 2 is chicken MLP recombinant protein expression identification of the present invention as a result, wherein M is albumen marker, and 1 is the weight not induced
Histone expresses bacterium Arctic-ExpressTM(pCzn1-MLP), 2 be the recombinant protein expression bacterium Arctic- induced
ExpressTM(pCzn1-MLP), 3 be the recombinant protein expression bacterium Arctic-Express inducedTM(pCzn1-MLP) after being crushed
Supernatant, 4 be the recombinant protein expression bacterium Arctic-Express of inductionTM(pCzn1-MLP) it is precipitated after being crushed；
Fig. 3 is the chicken MLP recombinant protein that the present invention purifies as a result, wherein M is albumen marker, and 1 is the recombination egg of induction
White expression bacterium Arctic-ExpressTM(pCzn1-MLP) supernatant after being crushed, 2 be efflux, and 3 be the purifying collected after eluting
His-MLP recombinant protein；
Fig. 4 is the chicken MLP recombinant protein Western Blot qualification result that the present invention purifies, and wherein M is albumen marker,
1 is the His-MLP recombinant protein of purifying, and the band of arrow instruction is target protein band；
Fig. 5 is chicken MLP protein polyclone antibody Purity of the present invention as a result, wherein M is albumen marker, and 1 is purifying
Chicken MLP protein polyclone antibody；
Fig. 6 is that chicken MLP protein polyclone antibody of the invention detects MLP albumen result in chicken muscle；
Fig. 7 is that the MLP protein polyclone antibody 1 of commercialization detects MLP albumen result in chicken muscle；1B1 is 1 week old
No. 1 individual of musculus soleus, 1Z1 are No. 1 individual of 1 week old musculus extensor digitorum longus pedis；
Fig. 8 is that the MLP protein polyclone antibody 2 of commercialization detects MLP albumen result in chicken muscle；3B2 is 3 week old
No. 2 individuals of musculus soleus；3B6 is No. 6 individuals of 3 week old musculus soleus；5B3 is No. 3 individuals of 5 week old musculus soleus；7B4 is 7 weeks
No. 4 individuals of age musculus soleus.
Specific embodiment
Below by specific embodiments of the present invention will be described in detail.It should be understood that described herein
Specific embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
Instrument used in embodiment and reagent can be obtained by way of buying commercial product.
The clone of 1 chicken MLP gene of embodiment
It carries out as follows:
(1) building of chicken MLP DNA recombinant expression plasmid
In Nanjing, bronze object Bioisystech Co., Ltd is used for prokaryotic expression carrier structure by full genome synthetic method, synthesis
The DNA sequence dna built includes the sequence of nucleotide sequence and pCzn1 carrier part shown in SEQ ID No:1；By PCR and again
The nucleotide sequence of synthesis is cloned into carrier pCzn1 by group enzyme connection, constructs recombinant expression plasmid pCzn1-MLP.
The primer sequence of PCR amplification are as follows:
F:5 '-AAAGTGCATCATCATCATCATCATATGCCAA-3 ',
R:5'-TTTTAAGCAGAGATTACCTATCTAGATTATTC-3'；
Pcr amplification reaction system is 10 μm of olL-1Each 1 μ L, 100ng μ L of upstream and downstream primer-1Gene chemical synthesis product (weight
Group expression plasmid pCzn1-MLP) 0.3 0.5 μ L, 10 × Taq Pfu buffer of μ L, 5U/ μ L Pfu archaeal dna polymerase, 5 μ L, add
ddH2O to 50 μ L, response procedures are 95 DEG C of 3min；95 DEG C of 25s, 62 DEG C of 20s, 72 DEG C of 40s, 25 circulations；72℃1min.PCR
For product through gel electrophoresis analysis, product recycling is stand-by.
Recombinase coupled reaction system are as follows: 4 μ L, pCzn1 linearized vector of PCR product, the 3.5 μ L of glue purification recycling, recombination
DNA ligase (being purchased from Nanjing Vazyme Biotechnology Co., Ltd.) 2.5 μ L.Recombinase DNA ligase connects response procedures are as follows:
50 DEG C of water-bath 25min, be placed at room temperature for 2-3min make temperature reduce after, converted.
(2) building of chicken MLP recombinant protein expression bacterium
PCzn1-MLP recombinant expression plasmid in step (1) is converted into Arctic-ExpressTMIt expresses in bacterium, building weight
Group expression bacterium Arctic-ExpressTM(pCzn1-MLP).It comprises the concrete steps that: the pCzn1-MLP recombination table in aspiration step (1)
100 μ L Arctic-Express are added to up to 1 μ L of plasmidTMCompetence is expressed in bacterium, after setting 20min on ice, 42 DEG C of heat shock 90s,
5min in ice is set rapidly, 600 μ L LB culture mediums are added, and 37 DEG C of 220r/min shake 1h, and LB of the coating containing 50 μ g/mL ammonia benzyls is flat
Plate selects positive clone molecule, and digestion identification, the result is shown in Figure 1, by building, correctly recombinant expression bacterium is named as Arctic-
ExpressTM(pCzn1-MLP)。
The expression and purifying of 2 chicken MLP recombinant protein of embodiment
It carries out as follows:
(1) IPTG induces chicken MLP recombinant protein expression bacterium to express His-MLP fusion protein
The recombinant expression bacterium constructed in picking embodiment 1 is inoculated in the test tube of the 3ml LB culture solution containing 50 μ g/ml ammonia benzyls
In, 37 DEG C, revolving speed 220r/min shaking overnight；Next day is inoculated in the 30ml LB culture solution containing 50 μ g/ml ammonia benzyls by 1:100,
37 DEG C, revolving speed 220r/min shake to thallus OD600 be 0.6-0.8；Take out 1ml culture, the centrifugation of 10000r/mim room temperature
2min abandons supernatant, and bacterial sediment is resuspended with 100 μ l 1 × SDS sample-loading buffers；IPTG is added into remaining culture to end
Concentration is 0.5mM, 37 DEG C, 220r/min shaking 4h, induced fusion protein expression；Take out 1ml culture, 10000r/mim room temperature
It is centrifuged 2min, abandons supernatant, bacterial sediment, remaining culture 4000r/mim, centrifugation is resuspended with 100 μ l 1 × SDS sample-loading buffers
10min abandons supernatant, and bacterial sediment is resuspended after re-suspension liquid carries out ultrasonic disruption with PBS and takes supernatant to add with precipitated liquid respectively
Enter sample-loading buffer resuspension；12%SDS-PAGE detection and analysis are carried out, coomassie brilliant blue staining shows band, as a result as shown in Fig. 2, mesh
Mark albumen is primarily present in supernatant.
(2) purifying of His-MLP fusion protein
It is carried out according to Ni-IDA affinity chromatography glue (being purchased from An Nuolun (Beijing) Biotechnology Co., Ltd) specification, specifically
Step is: utilizing low pressure chromatography system, supernatant solution is pre- to Ni-IDA Binding-Buffer with 0.5ml/min flow velocity loading
The Ni-IDA-Sepharose CL-6B affinity column of balance；With Ni-IDA Binding-Buffer with 0.5ml/min flow velocity
It rinses, until efflux OD280 value reaches baseline；With Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazoles,
0.15M NaCl, pH8.0) with the flushing of 1ml/min flow velocity, until efflux OD280 value reaches baseline；With Ni-IDA Elution-
Buffer (20mM Tris-HCl, 250mM imidazoles, 0.15M NaCl, pH8.0) elutes destination protein with 1ml/min flow velocity, receives
Collect efflux；The efflux of above-mentioned collection is added in bag filter, uses 20mM Tris-HCl, 0.15M NaCl, pH8.0 progress
Dialysed overnight；12%SDS-PAGE analysis is carried out, as a result as shown in Figure 3, it is seen that the His-MLP fusion protein purity of purifying is high；
It is primary antibody with His antibody, the goat anti-rabbit igg polyclonal antibody (being purchased from Nanjing bronze object Bioisystech Co., Ltd) of HRP label is
Secondary antibody carries out Westren Blot analysis to the His-MLP fusion protein of purifying, as a result sees Fig. 4, occurs at expected size
Purpose band.
The preparation of 3 chicken MLP protein polyclone antibody of embodiment
It carries out as follows:
(1) animal immune
After the purifying His-MLP fusion protein BCA concentration measuring kit prepared in embodiment 2 is measured concentration, exempt from
Two New Zealand White Rabbit (2-2.5kg) of epidemic disease, 400 μ of subcutaneous inoculation g/ times are exempted from after mixing with equivalent complete Freund's adjuvant for the first time
Epidemic disease, is followed by mix with equivalent incomplete Freund's adjuvant and carries out immune 2-3 time, and each immunization interval time is 2 to 3 all.Blood sampling
Detection determines that antiserum is directed to the potency of MLP albumen by indirect ELISA method, is greater than 1:50000 to potency and is finally adopted
Blood prepares antiserum.
(2) antibody purification
By His-MLP albumen and agarose medium coupling be prepared into antigen affinity purification chromatographic column, by gained antiserum with
Slow loading after PBS mixed in equal amounts is eluted after antibody combination with glycine elution buffer to get required antibody purification is arrived,
Carry out 4 DEG C of dialysed overnights in PBS immediately, the next day carry out purity, concentration and titration.
BCA concentration measuring kit carries out concentration mensuration, result 0.68mg/mL to gained MLP antibody；Pass through SDS-
PAGE electrophoresis, coomassie brilliant blue staining observe the purity of antibody purification, as a result see Fig. 5, as seen from the figure purity after MLP antibody purification
85% or more.
It purifies MLP antibody indirect ELISA bioactivity step: using PBS coating buffer (pH7.4) by the His-MLP egg of purifying
White antigen diluent is at 5 μ g/mL, every 100 μ L of hole, 4 DEG C of refrigerator overnights；Next day discards coating buffer, and PBST board-washing 3 times, each 5min,
Then 200 μ L confining liquids, 37 DEG C of insulating box 1h are added in every hole；ELISA Plate is taken out, discards interior liquid, PBST board-washing 1 time；Antibody purification
After 1:500 dilution, 2 times of gradient dilutions, every 100 μ L of hole, if other serum antibody negative control holes, 37 DEG C of insulating box 1h；It takes out
ELISA Plate discards interior liquid, and board-washing 3 times, the goat antirabbit that the HRP that 100 μ L have diluted is marked is added into every hole, and (1:5000 is dilute
Release) ELIAS secondary antibody, 37 DEG C of insulating box 1h；ELISA Plate is taken out, discards interior liquid, PBST board-washing 4 times, each 5min；Every hole is added 100
μ L TMB developing solution, according to the depth of color determine developing time, general 37 DEG C, 15min；It is molten that 100 μ L 1M HCL are added in every hole
Liquid terminates reaction, that is, is engraved in 450nm in microplate reader and reads, OD value is greater than to 2.1 times of hole institute of the negative control OD value of setting
Corresponding dilution, it is determined as the potency of the sample；Testing result is shown in Table 1, and the MLP antibody titer purified seen from table 1 is greater than
512000。
The MLP antibody indirect ELISA testing result that table 1 purifies
MLP protein expression in the chicken MLP protein polyclone antibody detection chicken muscle that embodiment 4 purifies
With MLP protein expression situation in Western Blot technology detection chicken muscle, concrete operations are according to such as lower section
Method carries out:
Musculus soleus, biceps muscle of thigh, musculus extensor digitorum longus pedis, sartorius, the caput laterale musculi gastrocnemii, calf of acquisition adult Guangxi Sanhuang chicken
The muscle samples such as myenteron medial head, pectoralis major, musculus pectoralis minor are transferred to -80 DEG C of refrigerators preservations after being placed in liquid nitrogen flash freezer；By the flesh of preservation
The each sample of meat tissue takes 300mg, be added 0.8ml RIPA lysate (being purchased from Beijing Puli lema gene Technology Co., Ltd.) and
The protease of final concentration of 1mmol/L inhibits mixture (being purchased from Beijing Puli lema gene Technology Co., Ltd.), is homogenized with glass
Device is homogenized 20 times manually up and down, and homogenate is transferred to 1.5ml centrifuge tube, and 12000r/min, 4 DEG C of centrifugation 5min take supernatant, is used
BCA Concentration Reagent box measures protein concentration；After 4% concentration glue of preparation and 10% separation gel, every 30 μ g albumen of hole loading；Glue is concentrated
Constant pressure 90V, separation gel constant pressure 200V, when bromjophenol blue goes to glue bottom, electricity is transferred on NC film (300mA constant current, 1.5h)；Electricity
After turning, NC film is placed in 5% defatted milk confining liquid, 37 DEG C of closing 1h；The chicken that will be purified in embodiment 3 with confining liquid
MLP protein polyclone antibody is diluted to 1:2000, and rabbit-anti GAPDH internal reference primary antibody polyclonal antibody is (purchased from Nanjing Jin Sirui biology
Science and Technology Ltd.) it is diluted to 1:3000,37 DEG C are incubated for 1.5h or 4 DEG C of overnight incubation；It is cleaned 3 times after taking the film out with TBST,
Each 5min；The goat anti-rabbit igg secondary antibody that HRP is marked is diluted to 1:5000,37 DEG C of incubation 1.5h with confining liquid；After taking the film out
It is cleaned 4 times with TBST, each 5min；ECL chemiluminescence colour developing, fixing；Film is scanned, UVP gel images are then used
The gray value of processing system Labworks4.6 software analysis purpose band.As a result as shown in Fig. 6, table 2.
As shown in fig. 6, rabbit-anti chicken MLP protein antibodies prepared by the present invention, it can the specificity inspection at expected size 21KD
Measure MLP albumen.As can be seen from Table 2, MLP albumen is not expressed in pectoralis major and musculus pectoralis minor, in biceps muscle of thigh, musculus extensor digitorum longus pedis, calf
Low expression in myenteron lateral head, expresses higher in sartorius and medial head of gastrocnemius muscle, and highest is expressed in musculus soleus.
2 Western Blot of table detects MLP protein expression result in chicken muscle
MLP protein expression in the MLP protein polyclone antibody detection chicken muscle that comparative example 1 is commercialized
With MLP protein expression situation in Western Blot technology detection chicken muscle, concrete operations are according to such as lower section
Method carries out:
Musculus soleus, biceps muscle of thigh, musculus extensor digitorum longus pedis, sartorius, the caput laterale musculi gastrocnemii, calf of acquisition adult Guangxi Sanhuang chicken
The muscle samples such as myenteron medial head, pectoralis major, musculus pectoralis minor are transferred to -80 DEG C of refrigerators preservations after being placed in liquid nitrogen flash freezer；By the flesh of preservation
The each sample of meat tissue takes 300mg, be added 0.8ml RIPA lysate (being purchased from Beijing Puli lema gene Technology Co., Ltd.) and
The protease of final concentration of 1mmol/L inhibits mixture (being purchased from Beijing Puli lema gene Technology Co., Ltd.), is homogenized with glass
Device is homogenized 20 times manually up and down, and homogenate is transferred to 1.5ml centrifuge tube, and 12000r/min, 4 DEG C of centrifugation 5min take supernatant, is used
BCA Concentration Reagent box measures protein concentration；After 4% concentration glue of preparation and 10% separation gel, every 30 μ g albumen of hole loading；Glue is concentrated
Constant pressure 90V, separation gel constant pressure 200V, when bromjophenol blue goes to glue bottom, electricity is transferred on NC film (300mA constant current, 1.5h)；Electricity
After turning, NC film is placed in 5% defatted milk confining liquid, 37 DEG C of closing 1h；With confining liquid respectively by the MLP albumen of commercialization
Polyclonal antibody 1 (the rabbit anti-human polyclonal antibody purchased from Sangon Biotech (Shanghai) Co., Ltd.) and MLP albumen are more
Clonal antibody 2 (the rabbit anti-human polyclonal antibody purchased from Beijing Bo Aosen Bioisystech Co., Ltd) is diluted to 1:500, rabbit-anti
GAPDH internal reference primary antibody polyclonal antibody (being purchased from Nanjing Genscript Biotechnology Co., Ltd.) is diluted to 1:3000,37 DEG C of incubations
1.5h or 4 DEG C of overnight incubation；It is cleaned 3 times after taking the film out with TBST, each 5min；The goat-anti rabbit for being marked HRP with confining liquid
IgG secondary antibody is diluted to 1:5000,37 DEG C of incubation 1.5h；It is cleaned 4 times after taking the film out with TBST, each 5min；ECL chemiluminescence
Colour developing, fixing；Film is scanned.Obtained test result is as shown in Figure 7,8.
As shown in fig. 7, (the rabbit-anti purchased from Sangon Biotech (Shanghai) Co., Ltd. of MLP protein polyclone antibody 1
People's polyclonal antibody), purpose band cannot be detected at expected 21KD.As shown in figure 8, (the purchase of MLP protein polyclone antibody 2
From the rabbit anti-human polyclonal antibody of Beijing Bo Aosen Bioisystech Co., Ltd), band is detected near expected 20KD, it will be
Doubtful band near 20KD serves Hai Aiao Biotechnology Co., Ltd and carries out Mass Spectrometric Identification, is free of chicken MLP egg as the result is shown
White, therefore, the doubtful band in Western Blot result is not chicken MLP protein band.
To sum up, the MLP antibody of commercialization cannot detect MLP protein expression in chicken muscle, can not specificity knowledge
MLP albumen in other chicken skeletal muscle.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement and simple modifications etc., should all be included in the protection scope of the present invention in content.
Sequence table
<110>Jiangsu Inst. of Fowls Science
<120>one breeder MLP protein polyclone antibodies and the preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 603
<212> DNA
<213>chicken (Gallus gallus domesticus)
<400> 1
catcatcatc atcatcatat gccaaactgg ggaggtggag ccaaatgtgg tgcctgtgaa 60
aagacagtgt accatgctga ggaaatccag tgcaatggaa ggagttttca caagacgtgc 120
ttcctctgca tggcttgcag aaaggctctg gacagcacca cagtggcagc tcacgaatct 180
gaaatctact gcaagacttg ctacgggagg aaatacggcc ccaagggcgt tggctttgga 240
caaggggctg gatgtctcag cactgacacc ggggaccatc tgggcctgaa cctgcaacaa 300
gggtcaccaa agcctgctcg cccctccaca ccaactaacc cttcaaagtt tgccaaaaag 360
atggttgacg tggataaatg tccccgctgt ggcaaatcgg tgtacgctgc agagaagata 420
atgggaggag gaaaaccttg gcacaaaaca tgcttccgct gtgctatttg tgggaagagt 480
ttagaatcta caaatgttac agacaaagac ggagagctct actgtaaagt ttgctacgca 540
aagaattttg gtcccaaagg aattggtttt ggcggcctca ctcaagtgga aaagaaagaa 600
taa 603
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<211> 200
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<213>chicken (Gallus gallus domesticus)
<400> 2
His His His His His His Met Pro Asn Trp Gly Gly Gly Ala Lys Cys
1 5 10 15
Gly Ala Cys Glu Lys Thr Val Tyr His Ala Glu Glu Ile Gln Cys Asn
20 25 30
Gly Arg Ser Phe His Lys Thr Cys Phe Leu Cys Met Ala Cys Arg Lys
35 40 45
Ala Leu Asp Ser Thr Thr Val Ala Ala His Glu Ser Glu Ile Tyr Cys
50 55 60
Lys Thr Cys Tyr Gly Arg Lys Tyr Gly Pro Lys Gly Val Gly Phe Gly
65 70 75 80
Gln Gly Ala Gly Cys Leu Ser Thr Asp Thr Gly Asp His Leu Gly Leu
85 90 95
Asn Leu Gln Gln Gly Ser Pro Lys Pro Ala Arg Pro Ser Thr Pro Thr
100 105 110
Asn Pro Ser Lys Phe Ala Lys Lys Met Val Asp Val Asp Lys Cys Pro
115 120 125
Arg Cys Gly Lys Ser Val Tyr Ala Ala Glu Lys Ile Met Gly Gly Gly
130 135 140
Lys Pro Trp His Lys Thr Cys Phe Arg Cys Ala Ile Cys Gly Lys Ser
145 150 155 160
Leu Glu Ser Thr Asn Val Thr Asp Lys Asp Gly Glu Leu Tyr Cys Lys
165 170 175
Val Cys Tyr Ala Lys Asn Phe Gly Pro Lys Gly Ile Gly Phe Gly Gly
180 185 190
Leu Thr Gln Val Glu Lys Lys Glu
195 200
Claims (9)
1. the preparation method of a breeder MLP protein polyclone antibody, which is characterized in that method includes the following steps:
(1) recombinant expression plasmid containing nucleotide sequence shown in SEQ ID No:1 is constructed, the recombinant expression plasmid is converted
It expresses bacterium and obtains recombinant protein antigen；
(2) animal is immunized in the recombinant protein antigen, menses, which isolate and purify clearly, obtains chicken MLP protein polyclone antibody.
2. the preparation method of chicken MLP protein polyclone antibody according to claim 1, which is characterized in that in step (1),
The recombinant expression plasmid is will to be cloned into the prokaryotic expression with His label containing nucleotide sequence shown in SEQ ID No:1 to carry
Body obtains.
3. the preparation method of chicken MLP protein polyclone antibody according to claim 2, which is characterized in that prokaryotic expression carries
Body is pCzn1.
4. the preparation method of chicken MLP protein polyclone antibody according to claim 1, which is characterized in that in step (1),
The expression bacterium is Arctic-ExpressTMBacillus coli expression bacterium.
5. the preparation method of chicken MLP protein polyclone antibody according to claim 1, which is characterized in that in step (2),
The animal is rabbit, mouse or rat.
6. the preparation method of chicken MLP protein polyclone antibody according to claim 1, which is characterized in that in step (2),
The step of immune animal are as follows: be immunized after first mixing recombinant protein antigen with equivalent complete Freund's adjuvant for the first time, then will
Recombinant protein antigen is mixed with equivalent incomplete Freund's adjuvant be immunized 2-3 times, and each immunization interval time was 2 to 3 weeks.
7. the preparation method of chicken MLP protein polyclone antibody according to claim 1, which is characterized in that in step (2),
It is purified using affinity purification chromatographic column.
8. chicken MLP protein polyclone antibody made from the described in any item preparation methods of claim 1~7.
9. application of the chicken MLP protein polyclone antibody according to any one of claims 8 in detection chicken tissues or cell in MLP albumen.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763256A (en) * | 2019-08-07 | 2020-10-13 | 北京市农林科学院 | Cloning of chicken CR2 gene, expression and purification of protein and preparation of polyclonal antibody thereof |
| CN112759645A (en) * | 2021-02-23 | 2021-05-07 | 江西农业大学 | Chicken RPS14 polyclonal antibody and preparation method and application thereof |
| CN112812181A (en) * | 2021-02-19 | 2021-05-18 | 江西农业大学 | Chicken JAZF1 gene polyclonal antibody and preparation method and application thereof |
| CN113151289A (en) * | 2021-03-09 | 2021-07-23 | 河南省农业科学院畜牧兽医研究所 | Preparation method of chicken PTF1a gene recombinant protein and polyclonal antibody |
| CN114751976A (en) * | 2021-08-31 | 2022-07-15 | 河南农业大学 | Polypeptide, artificial antigen, polyclonal antibody and application for preparing chicken TFPI2 polyclonal antibody |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105924525A (en) * | 2016-05-11 | 2016-09-07 | 广西大学 | Preparation method and application of porcine TLR4 polyclonal antibody |
| CN107033245A (en) * | 2017-04-21 | 2017-08-11 | 华南农业大学 | The preparation method and purposes of the polyclonal antibodies of one breeder caspase 1 |
| CN108276488A (en) * | 2018-01-26 | 2018-07-13 | 四川农业大学 | One breeder myeloid cell triggers receptor B2 polyclonal antibodies and its preparation method and application |
-
2019
- 2019-06-12 CN CN201910505799.2A patent/CN110194797A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105924525A (en) * | 2016-05-11 | 2016-09-07 | 广西大学 | Preparation method and application of porcine TLR4 polyclonal antibody |
| CN107033245A (en) * | 2017-04-21 | 2017-08-11 | 华南农业大学 | The preparation method and purposes of the polyclonal antibodies of one breeder caspase 1 |
| CN108276488A (en) * | 2018-01-26 | 2018-07-13 | 四川农业大学 | One breeder myeloid cell triggers receptor B2 polyclonal antibodies and its preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| SHUNSHUN HAN等: ""Knockdown of CSRP3 inhibits differentiation of chicken satellite cells by promoting TGF-β/Smad3 signaling"", 《GENE》 * |
| 佚名: ""NM_001199486.1"", 《GENBANK》 * |
| 胡海龙等: ""MLP在肉鸡、蛋鸡及其与鹌鹑属间杂交禽胚胎肌肉发育过程中的差异表达"", 《中国兽医学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763256A (en) * | 2019-08-07 | 2020-10-13 | 北京市农林科学院 | Cloning of chicken CR2 gene, expression and purification of protein and preparation of polyclonal antibody thereof |
| CN112812181A (en) * | 2021-02-19 | 2021-05-18 | 江西农业大学 | Chicken JAZF1 gene polyclonal antibody and preparation method and application thereof |
| CN112759645A (en) * | 2021-02-23 | 2021-05-07 | 江西农业大学 | Chicken RPS14 polyclonal antibody and preparation method and application thereof |
| CN113151289A (en) * | 2021-03-09 | 2021-07-23 | 河南省农业科学院畜牧兽医研究所 | Preparation method of chicken PTF1a gene recombinant protein and polyclonal antibody |
| CN114751976A (en) * | 2021-08-31 | 2022-07-15 | 河南农业大学 | Polypeptide, artificial antigen, polyclonal antibody and application for preparing chicken TFPI2 polyclonal antibody |
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