CN110294798A - The prokaryotic expression of chIRF3 a kind of and the preparation method of polyclonal antibody - Google Patents
The prokaryotic expression of chIRF3 a kind of and the preparation method of polyclonal antibody Download PDFInfo
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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Abstract
The invention discloses the preparation methods of the prokaryotic expression of chIRF3 a kind of and polyclonal antibody, design its cDNA of the primer amplification of chIRF3, double digestion handles amplified production and PET-32a plasmid reconnects building recombinant vector, import competence DH5 α cell, screening positive clone recombinant is simultaneously transferred to BL21 cell, 37 DEG C of inversions are incubated overnight in LB solid medium of the coating containing Amp, picking white single bacterium falls in the LB culture medium containing Amp and is incubated overnight, IPTG inducing expression is added, and optimize inductive condition, White Rabbit is immunized after purifying chIRF3 albumen, prepare polyclonal antibody, polyclonal antibody passes through Western-Blot Testing and appraisal.As a result it is 1488bp that amplification, which obtains chIRF3 fragment length, and recombinant plasmid chIRF3-PET length is 7388bp, and two bands of recombinant plasmid double digestion result are 5900bp and 1488bp respectively, it can thus be concluded that chIRF3-PET is recombinated successfully.The molecular weight of albumen of recombinant plasmid prokaryotic expression is 83kDa, and the best induction time of inducing expression is 4h, and it is 0.08mmol/L that IPTG, which induces optium concentration, has obvious specificity to chIRF3 albumen using the polyclonal antibody of chIRF3 albumen preparation.
Description
Technical field
The present invention relates to the preparation method of the prokaryotic expression of chIRF3 a kind of and polyclonal antibody, more particularly to one kind are right
ChIRF3 albumen has the preparation method of specific polyclonal antibody.
Background technique
Interferon regulatory factor (IRF) be a kind of lymphocyte by organism or monocyte body by virus or
The solvable glycoprotein of the secretory generated under the action of other inducers.IRF biological activity is hematopoietic development, and cell is grown, carefully
Born of the same parents' apoptosis and tumour generation etc..Chicken source IRF3 (chIRF3) shows have unique reaction to virus infection in IRF family,
Research shows that chicken source IRF3 (chIRF3) is also important for the effect of the expression regulation ratio IRF1 of interferon gene IFN.chIRF3
Assignment of genes gene mapping 19q encodes 427 amino acid, and the molecular weight 55kDa of albumen, structure is helix turn helix.ChIRF3 is from N
The structural domain for holding C-terminal is DNA binding structural domain DBD, transcriptional activation domains TAD, reaction structure domain RD, in cell tranquillization shape
Under state, chIRF3, which is in cytoplasm, to be not involved in his protein substance and combines and show as monomer inactivated state, the reason is that TAD two
Side prevents chIRF3 from moving to nucleus in conjunction with the DNA in core there are two itself inhibiting structural domain AID to play the role of covering TAD.
Once chIRF3 activation is by the 385-386 serines and 396- in C-terminal by Viral interference invasion or dsRNA stimulation
The effect of 405 serines, under threonine regulation in IRF-3 phosphorylated molecules itself domain is inhibited to be opened after, TAD with
DBD is exposed the chIRF3 to form phosphorylation and promotes the expression of IRF-3.The chIRF3 phosphorylation when being infected with the virus,
Induce the expression of associated kinase, IFN, viral interference genetic transcription and translation, to reach prevention or limiting virus infection effect.
ChIRF3 is defendd in virus immunity, acute liver damage, has important adjustment effect in terms of tumour.The main research neck of chIRF3
Domain is the phosphorylation of chIRF3, antiviral access etc..The chIRF3 Anti-TNF-α physical efficiency of this research preparation is the chIRF3 tune of chicken
The immune fixed basis of antiviral access research honor of section.
Summary of the invention
Technical problem to be solved by the invention is to provide the preparations of the prokaryotic expression of chIRF3 a kind of and polyclonal antibody
Method has the characteristics that there is specificity to chIRF3 albumen.
In order to solve the above technical problems, the technical solution of the present invention is as follows: the prokaryotic expression and Anti-TNF-α of a kind of chIRF3
The preparation method of body, innovative point are that the prokaryotic expression of the chIRF3 and the preparation method of polyclonal antibody include as follows
Step:
Step is 1.: design of primers, primer sequence are as follows:
ChIRF3-F:5 '-GGGGTACC(I restriction enzyme site of Kpn) ATGGCAGCACTGGACAGCGAG-3 '；
ChIRF3-R:5 '-GGGAAGCTT(III restriction enzyme site of Kind) TCAGTCTGTCTGCATGTG-3 '；
Step is 2.: the amplification of cDNA, by fresh chicken liver tissue grinder at cell, is purified after extracting total serum IgE using RNA
Kits, using HiFiScript cNDA the first chain synthetic agent box reverse transcription at cDNA；
Step is 3.: using reverse transcription product cDNA as template, the PCR amplification system amplification of 25 μ l of configuration；
Step is 4.: passing through I enzyme of Kpn respectively using the PCR product and Escherichia coli PET-32a of DNA Purification Kit
Double digestion is carried out with III enzyme of Kind, gel extraction after the detection of 1% agarose gel electrophoresis configures the linked system of 10 μ l；
Step is 5.: the collection of chIRF3-PET recombinant plasmid, takes the connection product of 10 μ l that the Escherichia coli of 100 μ l are added
DH5 α, mixing are placed on 20min on ice, and 45 DEG C of thermal shock 90s put back to stand 5min on ice rapidly, are inoculated in the solid training containing Amp
It supports and is inverted culture in base, picking single bacterium, which is fallen in the culture medium containing Amp, to be incubated overnight, and thalline were collected by centrifugation extracts chIRF3-PET
Recombinant plasmid identifies that plasmid is enough constructs successfully by digestion and PCR；
Step is 6.: the preparation of chIRF3 albumen, and the competence that 50 μ l are added in the chIRF3-PET recombinant plasmid of 3 μ l is expressed bacterium
30min, 45 DEG C of thermal shock 90s put back to rapidly 5min on ice to strain BL21 on ice, are inverted for 37 DEG C in the LB solid medium containing Amp
Night culture, picking white single bacterium fall in the LB culture medium containing Amp and are incubated overnight, and IPTG inducing expression is added, induction parameters
Temperature, the setting of time and revolution；
Step is 7.: the preparation of anti-chIRF3 protein polyclone antibody, and His label protein purification kit is utilized to carry out
The protein purification of chIRF3, chIRF3 albumen after purification prepare chIRF3 polyclonal antibody as antigen, in 13 week old male
New zealand white rabbit dorsal sc multiple spot carries out 4 times after being immunized, and arteria carotis takes blood system from serum and purifying obtains anti-chIRF3 egg
White polyclonal antibody, freeze-drying are placed on -80 DEG C of preservations.
Preferably, 3. middle PCR amplification system is 12.5 μ l of 2xTaq Mix to the step；10 μM of 1 μ l of chIRF3-R；
10 μM of 1 μ l of chIRF3-F；Without ultrapure 9.5 μ l of bacterium water；cDNA 1μl.
Preferably, the step 3. in the amplification program applied in PCR amplification system: 94 DEG C, 2min；94 DEG C, 30s；58
DEG C, 30s；72 DEG C, 30s；35 circulations；72 DEG C, 10min.
Preferably, the step 4. in linked system are as follows: the pcr amplified fragment of the chIRF3 of 7 μ l, the PET- of 1 μ l
32a, T4 ligase 1 μ l, the 10xBuffer of 1 μ l mix 4 DEG C of connections overnight.
Preferably, the step 6. in 50 μ l competence expression bacterial strain BL21 be added in the recombinant plasmid of 3 μ l mix postposition
In 30min on ice, 45 DEG C of thermal shock 90s, 5min is stood on ice rapidly, the LB culture medium of 450 μ l is added, 37 DEG C, 220r/min shakes
Shake culture 45 minutes, be inoculated in 37 DEG C of inversions in the LB solid medium containing Amp and be incubated overnight, picking single bacterium fall within containing
Be incubated overnight in the LB culture medium of Amp, in the TM culture medium containing Amp being inoculated in the ratio of 1:100 culture be to OD600
0.6, IPTG, which is added, makes final concentration of 0.4mmol/L, and 37 DEG C, 220r/min is induced 4 hours.
Preferably, progress SDS-PAGE detection after IPTG induction, addition 2X sample-loading buffer suspension thalline, 100 DEG C
Water-bath 10min carries out SDS-PAGE detection.
Preferably, 7. the step is detected after the preparation of anti-chIRF3 protein polyclone antibody, by SDS-
The destination protein band of PAGE detection, is transferred in NC film using half-dried transferring film system, and 5% skimmed milk power closes 3h, with more grams
Grand antibody 1:10000 is incubated overnight at 4 DEG C, and goat antirabbit lgG is incubated for half an hour using 1:10000, and cECL develops the color 3-5 points
Clock is placed in the observation of Full-automatic chemiluminescence system.
The invention has the benefit that it is 1488bp, weight that amplification, which obtains chIRF3 fragment length, by using above-mentioned steps
Group plasmid chIRF3-PET length is 7388bp, and two bands of recombinant plasmid double digestion result are 5900bp and 1488bp respectively,
It can thus be concluded that chIRF3-PET is recombinated successfully.The molecular weight of albumen of recombinant plasmid prokaryotic expression is 83kDa, and inducing expression most preferably lures
Leading the time is 4h, optium concentration 0.08mmol/L.Polyclonal antibody using the preparation of chIRF3 albumen has chIRF3 albumen
There is obvious specificity.
Interferon is one of the major defence mechanism for the resistance poisoning intrusion that host is formed during evolution, it takes part in
Antiviral natural immune response.IRF-3 as IRF family important member, be promote the key transcription of I type interferon expression because
Son.When virus infection body, chIRF3, which is phosphorylated, to be formed dimer and enters nucleus, is stimulated IFN-α and IFN-β expression, is risen
To antiviral effect.The research of chicken chIRF3 is such as avian infectious for poultry disease, especially diplornavirus class disease
The research of the pathogenic mechanism of bursal disease etc. provides important thinking and tool.
Detailed description of the invention
The present invention is described in further detail with reference to the accompanying drawings and detailed description.
Fig. 1 is chicken liver tissue in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
The RT-PCR of chIRF3 is expanded.
Fig. 2 is the extraction of recombinant plasmid in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
As a result.
Fig. 3 is chIRF-PET recombination in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
The result of plasmid double digestion.
Fig. 4 is recombinant plasmid in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
The PCR qualification result of chIRF3-PET.
Fig. 5 is chIRF-PET recombination in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
The expression of results of albumen.
Fig. 6 is that chIRF3-PET is lured in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
Lead time-optimized result.
Fig. 7 is chIRF3-PET in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
IPTG induced concentration optimum results.
Fig. 8 is polyclonal antibody in the prokaryotic expression of chIRF3 of the present invention a kind of and the preparation method of polyclonal antibody
Western-Blot qualification result.
Specific embodiment
The prokaryotic expression of chIRF3 of the invention and the preparation method of polyclonal antibody, include the following steps:
Step is 1.: design of primers, primer sequence are as follows:
ChIRF3-F:5 '-GGGGTACC(I restriction enzyme site of Kpn) ATGGCAGCACTGGACAGCGAG-3 '；
ChIRF3-R:5 '-GGGAAGCTT(III restriction enzyme site of Kind) TCAGTCTGTCTGCATGTG-3 '；
Step is 2.: the amplification of cDNA, by fresh chicken liver tissue grinder at cell, is purified after extracting total serum IgE using RNA
Kits, using HiFiScript cNDA the first chain synthetic agent box reverse transcription at cDNA；
Step is 3.: using reverse transcription product cDNA as template, the PCR amplification system amplification of 25 μ l of configuration；
Step is 4.: passing through I enzyme of Kpn respectively using the PCR product and Escherichia coli PET-32a of DNA Purification Kit
Double digestion is carried out with III enzyme of Kind, gel extraction after the detection of 1% agarose gel electrophoresis configures the linked system of 10 μ l；
Step is 5.: the collection of chIRF3-PET recombinant plasmid, takes the connection product of 10 μ l that the Escherichia coli of 100 μ l are added
DH5 α, mixing are placed on 20min on ice, and 45 DEG C of thermal shock 90s put back to stand 5min on ice rapidly, are inoculated in the solid training containing Amp
It supports and is inverted culture in base, picking single bacterium, which is fallen in the culture medium containing Amp, to be incubated overnight, and thalline were collected by centrifugation extracts chIRF3-PET
Recombinant plasmid identifies that plasmid is enough constructs successfully by digestion and PCR；
Step is 6.: the preparation of chIRF3 albumen, and the competence that 50 μ l are added in the chIRF3-PET recombinant plasmid of 3 μ l is expressed bacterium
30min, 45 DEG C of thermal shock 90s put back to rapidly 5min on ice to strain BL21 on ice, are inverted for 37 DEG C in the LB solid medium containing Amp
Night culture, picking white single bacterium fall in the LB culture medium containing Amp and are incubated overnight, and IPTG inducing expression is added, induction parameters
Temperature, the setting of time and revolution；
Step is 7.: the preparation of anti-chIRF3 protein polyclone antibody, and His label protein purification kit is utilized to carry out
The protein purification of chIRF3, chIRF3 albumen after purification prepare chIRF3 polyclonal antibody as antigen, in 13 week old male
New zealand white rabbit dorsal sc multiple spot carries out 4 times after being immunized, and arteria carotis takes blood system from serum and purifying obtains anti-chIRF3 egg
White polyclonal antibody, freeze-drying are placed on -80 DEG C of preservations.
Experimental material of the invention are as follows: Escherichia coli PET-32a, bacillus coli DH 5 alpha, competence expression bacterial strain BL21 are equal
It is saved as the laboratory where applicant, 13 week old Male New Zealand White Rabbits, RNA extracts kit, RNA QIAquick Gel Extraction Kit,
HiFiScript cNDA the first chain synthetic agent box, quick Ago-Gel DNA QIAquick Gel Extraction Kit, the goat antirabbit of HRP label
LgG antibody, His label protein purification kit, it is ShiJi Co., Ltd, restriction enzyme that the reagents such as ECL kit, which are purchased from health,
It is purchased from Fermentas company, DNA Marker is purchased from TaKaRa company, and albumen Maeker is purchased from Sheng Gong company, NC film and IPTG
It is purchased from Solarbio company.
With cDNA amplification, PCR amplification system is 12.5 μ l of 2xTaq Mix for chIRF3 preparation；10 μM of chIRF3-R 1
μl；10 μM of 1 μ l of chIRF3-F；Without ultrapure 9.5 μ l of bacterium water；cDNA 1μl.Amplification program: 94 DEG C, 2min；94 DEG C, 30s；58
DEG C, 30s；72 DEG C, 30s；35 circulations；72 DEG C, 10min.
It constructs in chIRF3-PET recombinant plasmid and identification, target gene chIRF3 and large intestine bar after recovery purifying PCR
Bacterium PET-32a passes through I enzyme of Kpn and III enzyme of Kind respectively and carries out double digestion, gel extraction after the detection of 1% agarose gel electrophoresis,
Configure the linked system of 10 μ l, the pcr amplified fragment of the chIRF3 of 7 μ l, the PET-32a of 1 μ l, 1 μ l of T4 ligase, 1 μ l's
10xBuffer mixes 4 DEG C of connections overnight；Take the connection product of 10 μ l that the bacillus coli DH 5 alpha of 100 μ l is added, mixing is placed on
20min on ice, 45 DEG C of thermal shock 90s, puts back to stand 5min on ice rapidly, is inoculated in the solid medium containing Amp and is inverted culture,
Picking single bacterium, which is fallen in the culture medium containing Amp, to be incubated overnight, and thalline were collected by centrifugation extracts chIRF3-PET recombinant plasmid.
In the prokaryotic expression of recombinant plasmid, after the expression bacterial strain BL21 mixing of 50 μ l competence is added in the recombinant plasmid of 3 μ l
30min on ice, 45 DEG C of thermal shock 90s are placed in, stand 5min on ice rapidly, are added the LB culture medium of 450 μ l, 37 DEG C, 220r/min
Shaking culture 45 minutes, be inoculated in 37 DEG C of inversions in the LB solid medium containing Amp and be incubated overnight, picking single bacterium fall within containing
Be incubated overnight in the LB culture medium of Amp, in the TM culture medium containing Amp being inoculated in the ratio of 1:100 culture be to OD600
0.6, IPTG, which is added, makes final concentration of 0.4mmol/L, and 37 DEG C, 220r/min is induced 4 hours.IPTG carries out SDS- after inducing
PAGE detection, is added 2X sample-loading buffer suspension thalline, and 100 DEG C of water-bath 10min carry out SDS-PAGE detection.
In the preparation and detection of chIRF3 protein polyclone antibody, after the preparation of anti-chIRF3 protein polyclone antibody
It is detected, the destination protein band that SDS-PAGE is detected is transferred in NC film, 5% defatted milk using half-dried transferring film system
Powder closes 3h, is incubated overnight with polyclonal antibody 1:10000 at 4 DEG C, and goat antirabbit lgG is incubated for half an hour using 1:10000,
CECL develops the color 3-5 minutes, is placed in the observation of Full-automatic chemiluminescence system.
From the total serum IgE reverse transcription of chicken liver tissue extraction at cDNA, detected after PCR amplification through 1% agarose gel electrophoresis,
As shown in Figure 1, M is 15000bpDNA marker in Fig. 1；1,2,3 is the RT-PCR amplified production of chIRF3, at 1488bp
Apparent bright wisp band, clip size are in the same size with required chIRF3's.The extraction of recombinant plasmid is as a result, as shown in Fig. 2, figure
M is 15000bpDNA marker in 2；2,3 be recombinant plasmid chIRF3-PET after single endonuclease digestion, is had at 7388bp apparent bright
Band, it is consistent with the clip size of required recombinant plasmid.
Recombinant plasmid double digestion identification, as shown in figure 3, in Fig. 31 be 15000bpDNA marker；2 be sterile water；3 are
The double digestion of recombinant plasmid chIRF3-PET；4 have an apparent 1488bp band and 5900bp item for the double digestion of PET32a
Band, it was demonstrated that vector construction success, an only 5900bp band, it was demonstrated that digestion is normal.
The plasmid of extraction is subjected to PCR amplification as a result, as shown in figure 4,1 being 15000bp DNA marker in Fig. 4；2,3,
4,5 be chIRF3 fragment amplification in recombinant plasmid, as a result only using single 1488bp band, illustration purpose complete fragment is slotting
Enter plasmid；6 be the charge of no template, illustrates that PCR amplification is not contaminated.
In the identification of the prokaryotic expression and polyclonal antibody of recombinant plasmid, recombinant plasmid chIRF3-PET is transferred to expression bacterial strain
At 37 DEG C, 220r/min carries out SDS-PAGE electrophoretic analysis after inducing 4 hours by BL21, the IPTG through final concentration of 0.4mmol/L.
As a result as shown in figure 5, M is the double-colored pre-dyed albumen Marker of 15-150kDa in Fig. 5；1 is PET32a；2 is final concentration of for IPTG
The PET-32a of 0.4mmol/L；3,5,7 be chIRF3-PET；4,6,8 be the final concentration of 0.4mmol/L of IPTG chIRF3-
In 83kDa without band, chIRF3-PET has a little in 83kDa by PET, result PET-32a, the PET-32a through IPTG induction
Band, through IPTG induce chIRF3-PET have obviously protein band in 83kDa, with required destination protein molecule
It measures identical.
Optimize the experimental result of induction time, as shown in fig. 6, M is the double-colored pre-dyed albumen Marker of 15-150kDa in Fig. 6；
1 is PET32a；2 PET-32a induced for IPTG；3 be chIRF3-PET；4,5,6,7,8, No. 9 are respectively with final concentration IPTG
ChIRF3-PET 2h, 3h, 4h, 5h, 6h, the induction of 7h are induced, result is that chIRF3 recombinant protein is induced by IPTG, in 2h
When albumen start to express, induce 4h after albumen chIRF3 expression quantity it is most, the expression quantity of recombinant protein chIRF3 is opened in 5h
Begin to reduce, therefore the best induction time of recombinant protein is determined as 4h.
Optimize the experimental result of IPTG concentration, as shown in fig. 7, M is the double-colored pre-dyed albumen Marker of 15-150kDa in Fig. 7；
1 is PET32a；2 be the PET-32a of the final concentration of 0.4mmol/L of IPTG；3 be chIRF3-PET；4,5,6,7,8,9, No. 10 are
ChIRF3-PET difference IPTG final concentration 0.05mmol/L, 0.08mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L,
The induction of 0.8mmol/L, 1mmol/L.It as a result is the best induced concentration final concentration under 37 DEG C, 220r/min, 4h induction time
0.08mmol/L。
ChIRF3 by purifying is detected using the chIRF3 albumen derived as antigen using Western-Blo method
The specificity of the polyclonal antibody of immune 13 week old Male New Zealand White Rabbits preparation, as a result as shown in figure 8, M is 10- in Fig. 8
180kDa albumen Marker；1 is the detection of chIRF3 protein polyclone antibody, is detected at 83kDa in Western-Blot
There is apparent band, the polyclonal antibody of the application has specificity to chIRF3 albumen.
In summary by using above-mentioned steps, it is 1488bp, recombinant plasmid that amplification, which obtains chIRF3 fragment length,
ChIRF3-PET length is 7388bp, and two bands of recombinant plasmid double digestion result are 5900bp and 1488bp, PCR amplification respectively
Plasmid only has single 1488bp band, it can thus be concluded that chIRF3-PET is recombinated successfully.Recombinant plasmid prokaryotic expression obtains protein molecular
Amount is about 83kDa, and the best induction time of inducing expression is 4h, and best IPTG concentration is 0.08mmol/L.Utilize chIRF3 egg
The polyclonal antibody of white preparation has obvious specificity to chIRF3 albumen.
Finally, it should be noted that property technical side the above examples are only used to illustrate the technical scheme of the present invention and are not limiting
Case, those skilled in the art should understand that, modification or equivalent replacement of the technical solution of the present invention are made for those, and
The objective and range for not departing from the technical program, are intended to be within the scope of the claims of the invention.
Sequence table
<110>Guangxi National Univ.
<120>preparation method of the prokaryotic expression of chIRF3 a kind of and polyclonal antibody
<130> 2019
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<170> SIPOSequenceListing 1.0
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<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
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ggggtaccat ggcagcactg gacagcgag 29
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<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gggaagcttt cagtctgtct gcatgtg 27
Claims (7)
1. the prokaryotic expression of chIRF3 a kind of and the preparation method of polyclonal antibody, which is characterized in that the protokaryon of the chIRF3
The preparation method of expression and polyclonal antibody includes the following steps:
Step is 1.: design of primers, primer sequence are as follows:
ChIRF3-F:5 '-GGGGTACC(I restriction enzyme site of Kpn) ATGGCAGCACTGGACAGCGAG-3 '；
ChIRF3-R:5 '-GGGAAGCTT(III restriction enzyme site of Kind) TCAGTCTGTCTGCATGTG-3 '；
Step is 2.: the amplification of cDNA, by fresh chicken liver tissue grinder at cell, uses RNA purified reagent after extracting total serum IgE
Box purifying, using HiFiScript cNDA the first chain synthetic agent box reverse transcription at cDNA；
Step is 3.: using reverse transcription product cDNA as template, the PCR amplification system amplification of 25 μ l of configuration；
Step is 4.: using the PCR product and Escherichia coli PET-32a of DNA Purification Kit pass through respectively I enzyme of Kpn with
III enzyme of Kind carries out double digestion, and gel extraction after the detection of 1% agarose gel electrophoresis configures the linked system of 10 μ l；
Step is 5.: the collection of chIRF3-PET recombinant plasmid, takes the connection product of 10 μ l that the bacillus coli DH 5 alpha of 100 μ l is added,
Mixing is placed on 20min on ice, and 45 DEG C of thermal shock 90s put back to stand 5min on ice rapidly, are inoculated in the solid medium containing Amp
Middle inversion culture, picking single bacterium, which is fallen in the culture medium containing Amp, to be incubated overnight, and thalline were collected by centrifugation extracts chIRF3-PET recombination
Plasmid identifies that plasmid confirmation constructs successfully by digestion and PCR；
Step is 6.: the preparation of chIRF3 albumen, and the competence that 50 μ l are added in the chIRF3-PET recombinant plasmid of 3 μ l is expressed bacterial strain
30min, 45 DEG C of thermal shock 90s put back to rapidly 5min on ice to BL21 on ice, are inverted overnight for 37 DEG C in the LB solid medium containing Amp
Culture, picking white single bacterium fall in the LB culture medium containing Amp and are incubated overnight, and IPTG inducing expression, the temperature of induction parameters is added
Degree, the setting of time and revolution；
Step is 7.: the preparation of anti-chIRF3 protein polyclone antibody, and His label protein purification kit is utilized to carry out chIRF3's
Protein purification, chIRF3 albumen after purification prepares chIRF3 polyclonal antibody as antigen, big in 13 week old Male New Zealands
White rabbit dorsal sc multiple spot carries out 4 times after being immunized, and arteria carotis takes blood system from serum and purifying obtains anti-chIRF3 protein polyclone
Antibody, freeze-drying are placed on -80 DEG C of preservations.
2. the prokaryotic expression of chIRF3 as described in claim 1 a kind of and the preparation method of polyclonal antibody, it is characterised in that:
3. middle PCR amplification system is 12.5 μ l of 2xTaq Mix to the step；10 μM of chIRF3-R1 μ l；10 μM of 1 μ of chIRF3-F
l；Without ultrapure 9.5 μ l of bacterium water；cDNA 1μl.
3. the prokaryotic expression of chIRF3 as claimed in claim 2 a kind of and the preparation method of polyclonal antibody, it is characterised in that:
The step 3. in the amplification program applied in PCR amplification system: 94 DEG C, 2min；94 DEG C, 30s；58 DEG C, 30s；72 DEG C, 30s；
35 circulations；72 DEG C, 10min.
4. the prokaryotic expression of chIRF3 as described in claim 1 a kind of and the preparation method of polyclonal antibody, it is characterised in that:
The step 4. in linked system are as follows: the pcr amplified fragment of the chIRF3 of 7 μ l, the PET-32a of 1 μ l, T4 ligase 1 μ l, 1 μ
The 10xBuffer of l mixes 4 DEG C of connections overnight.
5. the prokaryotic expression of chIRF3 as described in claim 1 a kind of and the preparation method of polyclonal antibody, it is characterised in that:
The step 6. in 50 μ l competence expression bacterial strain BL21 be added in the recombinant plasmid of 3 μ l mix and be placed on 30min on ice, 45 DEG C
Thermal shock 90s stands rapidly 5min on ice, and the LB culture medium of 450 μ l is added, 37 DEG C, 220r/min shaking culture 45 minutes, is inoculated with
37 DEG C of inversions are incubated overnight in the LB solid medium containing Amp, and picking single bacterium is fallen in the LB culture medium containing Amp overnight
It cultivates, culture is 0.6 to OD600 in the TM culture medium containing Amp being inoculated in the ratio of 1:100, and addition IPTG makes dense eventually
Degree is 0.4mmol/L, and 37 DEG C, 220r/min is induced 4 hours.
6. the prokaryotic expression of chIRF3 as claimed in claim 5 a kind of and the preparation method of polyclonal antibody, it is characterised in that:
IPTG carries out SDS-PAGE detection after inducing, and 2X sample-loading buffer suspension thalline is added, and 100 DEG C of water-bath 10min are carried out
SDS-PAGE detection.
7. the prokaryotic expression of chIRF3 as described in claim 1 a kind of and the preparation method of polyclonal antibody, it is characterised in that:
7. the step is detected after the preparation of anti-chIRF3 protein polyclone antibody, the destination protein that SDS-PAGE is detected
Band is transferred in NC film using half-dried transferring film system, and 5% skimmed milk power closes 3h, with polyclonal antibody 1:10000 at 4 DEG C
It is incubated overnight, goat antirabbit lgG is incubated for half an hour using 1:10000, and cECL develops the color 3-5 minutes, is placed in full-automatic chemical hair
Photosystem observation.
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