CN103757041A - Method for preparing swine-original interferon-gamma 3 and application - Google Patents
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Abstract
The invention discloses a method for preparing swine-original interferon-gamma 3 (IFN-gamma 3) protein and application, belonging to the field of preparation of a genetic engineered product. The method comprises the steps of: cloning a swine-original interferon-gamma 3 coding gene, building a prokaryotic expression vector which contains the swine-original interferon-gamma 3 gene having no signal peptide sequence, and transforming escherichia coli and carrying out induced expression by using the recombined prokaryotic expression vector containing the swine-original interferon-gamma 3 gene, wherein the optimal induction temperature is 20 degrees centigrade, the optimal induction time is 16 hours, and the optimal IPTG (Isopropyl-beta-d-Thiogalactoside) concentration is 0.4mM. A product prepared by the method disclosed by the invention provides basis for researching the biological activity and the action mechanism of the swine-original interferon-gamma 3, and also can be applied to preparation of swine porcine epidemic diarrhea virus prevention drugs.
Description
Technical field
The invention belongs to gene engineering product preparation field, be specifically related to a kind of pig source Interferon, rabbit-λ 3(IFN-λ 3 for preparing) methods and applications of albumen.
Background technology
Interferon, rabbit (interferon, IFN) is that humans and animals cell is subject to virus infection, or is subject to after the effects such as nucleic acid, bacterial endotoxin, cytokinin a kind of glycoprotein with high biological activity of being secreted by recipient cell.Interferon, rabbit has the advantages such as biologic activity and quick-acting multipotency, toxicological harmless, become prevention and treatment porcine viral diseases ideal biological goods.Interferon, rabbit, except having antiviral activity, also has immunoregulation effect, can be used as the immunocompetence that immunological adjuvant improves vaccine simultaneously.According to the biologic activity of Interferon, rabbit is different with antigenic activity, can be divided three classes: IFN-α, IFN-β, IFN-γ, and IFN-α and the IFN-β with common surface acceptor are belonged to I type Interferon, rabbit; And there is the IFN-γ of different surfaces acceptor with IFN-α, IFN-β, belong to II type Interferon, rabbit.
At present, Chinese scholars conducts in-depth research I type and IFN-GAMMA biological function and antivirus action, and relatively lags behind for the research of III type IFN.III type IFN is called again IFN-λ, comprises IFN-λ 1, and IFN-λ 2 and IFN-λ 3, because its gene structure is similar to IL-10, so be called again IL-29, IL-28A, IL-28B.Although IFN-λ has similar intron/exons structure to IL-10 superfamily member in genomic level, more close with I type IFN relation on protein level.Existing studies show that, IFN-λ s and IFN-α have the biologic activity such as similar antiviral, inhibition tumor cell growth and tool immunoloregulation function, but due to its bone marrow depression and toxic side effect is few and biological action has long-lasting, and people, cure clinical experimental study and obtained breakthrough, be expected to become in the near future a new genetically engineered drug.Therefore expression and the application prospect of IFN-λ s gene become Scientific Research Problem urgently to be resolved hurrily.
Summary of the invention
For overcoming the shortcoming and defect of prior art, primary and foremost purpose of the present invention is to provide a kind of pig source Interferon, rabbit-λ 3(IFN-λ 3 for preparing) method of albumen, method of the present invention is specifically utilized RT-PCR technology clone pig source IFN-λ 3, and built prokaryotic expression carrier, at prokaryotic expression system-BL21 (DE3) plysS, express IFN-λ 3.
Another object of the present invention is to provide the application of described IFN-λ 3.
Object of the present invention is achieved through the following technical solutions: a kind of method at expression in escherichia coli pig source IFN-λ 3 genes, and concrete steps are as follows:
(1) with poly(I:C) (polyinosinic-polycytidylic acid, polycytidylic acid) inducing culture ST cell, the cell of results inducing culture, extracts cell total rna with Trizol reagent;
(2) Auele Specific Primer (S1 and S2) of design amplification pig source IFN-λ 3 full length genes, and amplification does not contain the Auele Specific Primer (P1 and P2) of the pig source IFN-λ 3 of signal peptide sequence, and primer is as follows:
Upstream primer S1:5 '-ATGGCCCTGGGTGGCTCG-3 ';
Downstream primer S2:5 '-TCAGACACACAGGTCTCC-3 ';
Upstream primer P1:5 '-CGGGTACCGTGCCTGTCCCTGAAGCCC-3 ' introduces Kpn I restriction enzyme site;
Downstream primer P2:5 '-CCGCTCGAGGACACACAGGTCTCCACT-3 ' introduces Xho I restriction enzyme site;
(3) utilize primer S1 and S2 by RT-PCR technology amplification pig source IFN-λ 3 full length genes;
(4) IFN-λ 3 gene clones of pig source are entered to pMD-18T carrier, the evaluation of checking order, obtain recombinant vectors pMD-18T-IFN-λ 3;
(5) take pMD-18T-IFN-λ 3 cloning vectors is template, utilize primer P1 and P2 amplification not to contain the gene of the pig source IFN-λ 3 of signal peptide sequence, and utilize Kpn I and these two multiple clone site of Xho I, the directed subclone of pig source IFN-λ 3 genes, to prokaryotic expression carrier pET-32a, is obtained to recombinant expression plasmid pET-32a-Po-IFN-λ 3;
(6) recombinant expression plasmid pET-32a-Po-IFN-λ 3 transforms e. coli bl21 (DE3) plysS;
(7) determine best inducing temperature, time and the IPTG concentration of expressing pig source IFN-λ 3 in prokaryotic expression system;
(8) abduction delivering of IFN-λ 3 in pig source in e. coli bl21 (DE3) plysS.
Best inducing temperature described in step (7), time and IPTG concentration are that inducing temperature is 20 ℃, and the time is 16h, and IPTG concentration is 0.4mM.
The application of pig source IFN-λ 3 albumen that aforesaid method obtains in the anti-PEDV virus of preparation (Porcine epidemic diarrhea virus, porcine epidemic diarrhea virus) medicine.
The present invention has following advantage and effect with respect to prior art:
1. pET vector expression system expression efficiency is very high.
2. pET vector expression system carries His label, is convenient to purifying.
3. when building the prokaryotic expression carrier of pET-32a, by design of primers, the signal peptide sequence of IFN-λ 3 is removed, made to build the recombinant expression plasmid pET-32a-Po-IFN-λ 3 obtaining and there is higher protein expression efficiency.
4. the IFN-λ 3 preparing is applied to anti-epidemic diarrhea virus, thereby well selects for preparing the drug provision of anti-epidemic diarrhea virus.
5. to prepare the anti-PEDV activity of PoIFN-λ 3 high in the present invention, can reach 5 * 10
3u/mg.
Accompanying drawing explanation
Fig. 1 is the schema that builds the recombinant cloning vector that contains PoIFN-λ 3 genes;
Fig. 2 is the SDS-PAGE electrophorogram of IFN-λ 3; M wherein: albumen Markers; 1: without IPTG induction control group (supernatant); 2: without IPTG induction control group (precipitation); The 3:0.4mM IPTG20 ℃ induction (supernatant) of spending the night; The 4:0.4mM IPTG20 ℃ induction (precipitation) of spending the night; The 5:0.4mM IPTG25 ℃ induction (supernatant) of spending the night; The 6:0.4mM IPTG25 ℃ induction (precipitation) of spending the night; The 7:0.4mM IPTG30 ℃ induction (supernatant) of spending the night; The 8:0.4mM IPTG30 ℃ induction (precipitation) of spending the night; 9:0.4mM IPTG37 ℃ of induction 5h(supernatant); 10:0.4mM IPTG37 ℃ of induction 5h(precipitation).
Fig. 3 is the Western-blot figure of IFN-λ 3.Wherein, 1:pET-32a empty carrier; 2:pET-32a-Po-IFN-λ 3.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The preparation method of embodiment 1, pig source IFN-λ 3, carries out following steps successively:
(1) build cloning vector:
1. use poly(I:C) induction (1.5ug, 2h) cultivation ST cell (1 * 10
5/ ml), the cell of results inducing culture, extracts cell total rna with Trizol reagent;
2. according to unique pig source IFN-λ 3 gene orders (GQ996936) of delivering on GenBank, design specific primer, the Auele Specific Primer (S1 and S2) of design amplification pig source IFN-λ 3 full length genes,
Upstream primer S1:5 '-ATGGCCCTGGGTGGCTCG-3 '
Downstream primer S2:5 '-TCAGACACACAGGTCTCC-3 ';
3. utilize RT-PCR technology amplification pig source IFN-λ 3 full length genes (carrying out pcr amplification with PrimeScript RT-PCR Kit); The reaction conditions of RT-PCR is 50 ℃ of 30min; 94 ℃ of 3min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 40s, 35 circulations; 72 ℃ of 10min;
4. by transforming Top10 intestinal bacteria, by IFN-λ 3 gene clones of pig source enter pMD-18T carrier, the evaluation of checking order, obtain correct recombinant vectors pMD-18T-IFN-λ 3.
(2) construction of expression vector
1. design amplification does not contain the Auele Specific Primer (P1 and P2) of the pig source IFN-λ 3 of signal peptide sequence, and primer is as follows:
P1:5 '-CGGGTACCGTGCCTGTCCCTGAAGCCC-3 ' (containing Kpn I restriction enzyme site);
P2:5 '-CCGCTCGAGGACACACAGGTCTCCACT-3 ' (containing Xho I restriction enzyme site);
Pig source IFN-λ 3 nucleotide sequences are as follows:
ATGGCCCTGGGTGGCTCGCTGGTGCTGGTGCTGGTGCTGATGACGGTGGCTCCACCCAGGACAGGAGCGGTGCCTGTCCCTGAAGCCCTCAGGGCCCTCCCAGGAGCAAGGGGCTGCCACTTGGCCCAGTTCAAGTCTCTGTCCCCACAAGCGCTGCAGGCCTTCAAGAGGGCCAAGGATGCCTTTGAAGAGTCCCTCTTGGAGGACTGGAACTGCAGCTCCCGCATCTTCCCCAGGAGCAGGGACCTGAAGCAGCTGCAGGTGTGGGAGCGCCCCGTGGCCTTGGAGGCCGAGGTGGCCCTGACCCTCAGCGTCCTGGGCTCCTTGGCGAACTCATCCCTGCACAGCAGCCTGGACCAGCCCCTTCACACGCTGCGCCACATCCACGCCCAGCTCCAGGCCTGTGTCCCAGCTCAGCCCATGGCAGGCCCCCGGCCCCGGGGCCGCCTCCACCACTGGCTGCACCGGCTCCAGGAGGCCCAGAAGAAGGAGCCCCAGAGCTGCCTGGAAGCCTCTGTCATGTTCAACCTCTTCCGCCTCCTCACCCGGGACCTGAAATGTGTCGCCAGTGGAGACCTGTGTGTCTGA。
Pig source IFN-λ 3 aminoacid sequences following (underscore is partly signal peptide sequence):
MALGGSLVLVLVLMTVAPPRTGAVPVPEALRALPGARGCHLAQFKSLSPQALQAFKRAKDAFEESLLEDWNCSSRIFPRSRDLKQLQVWERPVALEAEVALTLSVLGSLANSSLHSSLDQPLHTLRHIHAQLQACVPAQPMAGPRPRGRLHHWLHRLQEAQKKEPQSCLEASVMFNLFRLLTRDLKCVASGDLCV。
2. pMD-18T-IFN-λ 3 cloning vectors that check order correct of take are template, utilize P1 and P2 amplification not to contain the gene of the pig source IFN-λ 3 of signal peptide sequence, the object fragment that amplification is obtained reclaims and is connected after Xho I double digestion through Kpn I respectively with prokaryotic expression carrier pET-32a, and be converted into e. coli bl21 (DE3) plysS, coat the dull and stereotyped overnight incubation of LB containing 100 μ g/ml penbritins; Get the bacterium colony of the dull and stereotyped growth of penbritin LB and identify goal gene through PCR, positive colony bacteria plasmid carries out double digestion evaluation, is accredited as positive and represents that expression vector pET-32a-PoIFN-λ 3 successfully constructs;
Wherein, Fig. 1 is shown in by the building process schematic diagram of pET-32a-PoIFN-λ 3 recombinant expression vectors.
(3) expression of recombinant protein:
Picking vector construction is successfully containing the recombinant expressed bacterium BL21 of pET-32a-Po-IFN-λ 3 (DE3) plysS, in adding isopropylthio half IPTG(isopropyl-β-D-thiogalactoside(IPTG) containing in the LB substratum of penbritin) abduction delivering, determine the best inducing temperature of expressing pig source IFN-λ 3 in prokaryotic expression system, time (20 ℃, 16h) and IPTG concentration; After a large amount of amplification cultivation 3h of recombinant expressed bacterium, to bacterium arrival logarithmic phase, bacterium is 0.6 left and right in OD600 value, and it is 0.4mM that side adds inductor IPTG, IPTG final concentration.
(4) identify recombinant protein:
After IPTG induction in step (3), tropina shows through SDS-PAGE protein electrophoresis and coomassie brilliant blue staining, wherein the SDS-PAGE electrophorogram of IFN-λ 3 is shown in Fig. 2, compares with the bacterium that transforms empty plasmid, in 32KDa left and right, has a dense newly-increased protein band dying.Through Western-blot, identify (result is as shown in Figure 3), can with His monoclonal antibody generation strong positive reaction.
(5) purification of recombinant proteins, obtains raw product Interferon, rabbit.
The concrete steps of purification of recombinant proteins are:
1. vector construction is successfully inoculated into containing in the LB nutrient solution of 100 μ g/mL penbritins to 37 ℃ of 200r/min jolting overnight incubation containing the recombinant expressed bacterium of pET-32a-Po-IFN-λ 3.The fresh bacterium liquid of getting incubated overnight is by inoculation in 1: 100, when 37 ℃ of joltings are cultured to OD600nm and reach 0.6 left and right, with the IPTG induction of 0.2mmol/L, spends the night.4 ℃ of centrifugal 10min of 6000r/min collect thalline; Abandon supernatant, to the PBS damping fluid that adds 0.01mol/L pH7.4 in bacterial sediment, washing thalline; 4 ℃ of centrifugal 10min of 6000r/min; Abandon supernatant, in bacterial sediment, add appropriate buffer A lysate (about 500mL bacterium liquid 50mL bufferA), thalline carefully suspends; In the thalline suspending, add N,O-Diacetylmuramidase (final concentration is 100 μ g/mL), mix; Ice bath cracking 30min; Ultrasonic disruption cell (working hour 5s(power 400W), interval time 10s, continue 5min, the ultrasonoscope of take intermittently 5min is a working cycle, repeats 3 circulations); 4 ℃ of centrifugal collection inclusion bodys of the centrifugal 10min of 8000r/min;
The PBS(of buffer A:pH8.5 is containing 10 μ g/mL DNase, 0.5mmol/L PMSF).
2. supernatant liquor is transferred in another one centrifuge tube, in precipitation, added inclusion body washings (urea of 4mol/L), washing inclusion body 5min; 4 ℃ of centrifugal 10min centrifugal collecting precipitations of 8000r/min, proceed to supernatant liquor in another clean centrifuge tube; In precipitation, add the about 200mL bacterium of appropriate buffer B(liquid 10mLbuffer B), 4 ℃ are spent the night, and dissolve inclusion body.
Buffer B:Ni-Denature-GuHCl is containing 100mM NaH
2pO
4, 300mM NaCl, 4M GuHCl, pH7.0;
3. 4 ℃ of centrifugal 10min of 12000r/min, by supernatant with after 0.45 μ m membrane filtration, with Ni-NTA affinity chromatography column purification, concrete steps and solution preparation are shown in this Shanghai Sheng Gong biotechnology Ni-NTA of company limited purifying specification sheets BSP079-3, and the elutriant of collecting 60-250mM imidazoles is IFN-λ 3 recombinant proteins.
4. the renaturation of recombinant protein is with concentrated: the albumen eluting is packed in dialysis tubing, clamp dialysis tubing two ends, in renaturation solution 1,2,3,4, dialyse 4 hours successively, finally at damping fluid 10Mm Tris-Hcl, 0.15M NaCl(pH9.0) dialysis 12-24 hour in, dialysis tubing is put into a large plate concentrated with PEG-20000, collect concentrated good albumen and preserve in-80 degree.
Join renaturation solution 1:10mMTris-Hcl, 0.2M NaCl, 8M urea, adds 0.5mM reduced glutathion, 0.2mM Sleep-promoting factor B after tune pH to 7.0;
Join renaturation solution 2:10mMTris-Hcl, 0.2M NaCl, 6M urea, adds 0.5mM reduced glutathion, 0.2mM Sleep-promoting factor B after tune pH to 7.0;
Join renaturation solution 3:10mMTris-Hcl, 0.2M NaCl, 4M urea, adds 0.5mM reduced glutathion, 0.2mM Sleep-promoting factor B after tune pH to 7.0;
Join renaturation solution 4:10mMTris-Hcl, 0.2M NaCl, adds 2mM reduced glutathion, 0.4mM Sleep-promoting factor B after tune pH to 7.0.
The activity calibrating of the pig source IFN-λ 3 of embodiment 2, the embodiment of the present invention 1 preparation:
By few cells pathology, suppress method, take MDBK cell (MDBK bovine kidney cells)/VSV(bubble stomatitis virus) be basic detection system.The high dilution that suppresses the Interferon, rabbit of 50% cytopathy (CPE) is defined as to Yi Ge Interferon, rabbit unit (U).Active detected result shows, the biologic activity of pig source IFN-λ 3 is 2 * 10
3u/mg.The I of current all expression, II type Interferon, rabbit are all with MDBK cell/VSV, to detect the expression activity of Interferon, rabbit, so whether we also first have biological activity with we the IFN-λ 3 of expression of this systems analysis, and then analyze the activity of anti-other viruses
Anti-PEDV virus (Porcine epidemic diarrhea virus) effect of embodiment 3, pig source IFN-λ 3 albumen
Measure PoIFN-λ 3(wherein ST clone (ST pig testis cell) is upper, what Po was porcine writes a Chinese character in simplified form) effect of anti-PEDV, adopt cytopathic-effect inhibition assay.ST cell suspension is pressed to 1 * 10
5/ mL density is inoculated 96 porocyte culture plates, every pore volume 100 μ L, incubated overnight; The DMEM substratum that PoIFN-λ 3 use after renaturation are contained to 2%FBS is with 2
1-2
8gradient dilution, every hole adds 100 μ L, 8 repeating holes of each gradient, 37 ℃, the CO of volume fraction 5%
2after inducing culture 24h, inhale and abandon nutrient solution, add 100TCID
50pEDV, after 24h, start to observe pathology, Continuous Observation 3 days is established cell contrast and virus control simultaneously; With reference to viral titer computing method, with pathology, suppress hole and replace pathology hole in malicious valency mensuration, by Reed-Muench method, calculate the activity unit of PoIFN-λ 3 anti-PEDV, wherein PoIFN-λ 3 biological activity titrations the results are shown in Table 1.
Table 1PoIFN-λ 3 biological activity titrations
Wherein, CPE hole is for suppressing 50% cytopathy hole.
By can be calculated, distance proportion is: (75%-50%)/(75%-37.5%)=0.67, PoIFN-λ 3 tires and can reach 10 * 2 in MDBK/VSV system
4.67u/0.1mL, protein concentration is 0.5mg/mL, therefore the anti-PEDV activity of PoIFN-λ 3 is: (10 * 2
4.67u/0.1mL)/0.5mg/mL=5 * 10
3u/mg.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (3)
1. in a method for expression in escherichia coli pig source IFN-λ 3 genes, it is characterized in that concrete steps are as follows:
(1) with poly(I:C) inducing culture ST cell, the cell of results inducing culture, extracts cell total rna with Trizol reagent;
(2) specificity upstream primer S1 and the downstream primer S2 of design amplification pig source IFN-λ 3 full length genes, and amplification is containing specificity upstream primer P1 and the downstream primer P2 of the pig source IFN-λ 3 of signal peptide sequence, primer is as follows:
Upstream primer S1:5 '-ATGGCCCTGGGTGGCTCG-3 ';
Downstream primer S2:5 '-TCAGACACACAGGTCTCC-3 ';
Upstream primer P1:5 '-CGGGTACCGTGCCTGTCCCTGAAGCCC-3 ';
Downstream primer P2:5 '-CCGCTCGAGGACACACAGGTCTCCACT-3 ';
(3) utilize primer S1 and S2 by RT-PCR technology amplification pig source IFN-λ 3 full length genes;
(4) IFN-λ 3 gene clones of pig source are entered to pMD-18T carrier, the evaluation of checking order, obtain recombinant vectors pMD-18T-IFN-λ 3;
(5) take pMD-18T-IFN-λ 3 cloning vectors is template, utilize primer P1 and P2 amplification not to contain the gene of the pig source IFN-λ 3 of signal peptide sequence, and utilize KpnI and these two multiple clone site of XhoI, the directed subclone of pig source IFN-λ 3 genes, to prokaryotic expression carrier pET-32a, is obtained to recombinant expression plasmid pET-32a-Po-IFN-λ 3;
(6) recombinant expression plasmid pET-32a-Po-IFN-λ 3 transforms e. coli bl21 (DE3) plysS;
(7) determine best inducing temperature, time and the IPTG concentration of expressing pig source IFN-λ 3 in prokaryotic expression system;
(8) abduction delivering of IFN-λ 3 in pig source in e. coli bl21 (DE3) plysS.
2. the method at expression in escherichia coli pig source IFN-λ 3 genes according to claim 1, is characterized in that the best inducing temperature described in step (7), time and IPTG concentration are that inducing temperature is 20 ℃, and the time is 16h, and IPTG concentration is 0.4mM.
3. the application of pig source IFN-λ 3 albumen that described in claim 1 or 2, method obtains in preparing porcine epidemic diarrhea resisting virus drugs.
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