CN107177613A - A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity - Google Patents
A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity Download PDFInfo
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- CN107177613A CN107177613A CN201710584160.9A CN201710584160A CN107177613A CN 107177613 A CN107177613 A CN 107177613A CN 201710584160 A CN201710584160 A CN 201710584160A CN 107177613 A CN107177613 A CN 107177613A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Abstract
The invention discloses a kind of preparation method for recombinating Porcine interferon-gamma fusion protein.Its nucleotides sequence is classified as shown in SEQ ID NO.1, and coded protein amino acid sequence is shown in SEQ ID NO.2.The present invention expresses Porcine interferon-gamma gene and pig antibody CH3 segment compositions, not only it had been beneficial to the stability of increase albumen, and had extended activity time but also can efficiently be prepared by prokaryotic expression system, it the experiment proved that, the restructuring Porcine interferon-gamma expressed by the present invention is significantly increased compared with the half-life period of natural Porcine interferon-gamma, bioactivity is also superior to natural products.
Description
Technical field
The present invention relates to a kind of gene of restructuring, more particularly to restructuring Porcine Interferon-gamma Gene, the invention further relates to containing
The expression vector and engineered strain of the gene, the invention further relates to their purposes in Porcine Interferon-gamma is prepared, category
In interferon gene engineering field.
Background technology
Nearly 2 years, because the harm of livestock immunosuppressive disease is on the rise, immunopotentiator quilt in husbandry sector
Extensive use.Animal by epidemic disease threaten or in stress situation, particularly livestock and poultry be in immunosupress when use Immune-enhancing effect
Agent has remarkable effect.The need for progress and Modern Animal Husbandry with science and technology, the preventive and therapeutic effect of immunopotentiator is got over
To be more taken seriously, the master that characteristic is determined, efficient, preferable immunopotentiator stably, nontoxic will be following preventing and treating livestock and poultry
Want material.Wherein, topmost pig is exactly interferon with immunopotentiator.Interferon（IFN）It is a kind of broad-spectrum antiviral medicament,
Direct killing or suppression be not viral, and mainly cell is produced antiviral protein by cell surface receptor effect, so that
Suppress the duplication of virus；It can also strengthen NK simultaneously（NK cells）, macrophage and T lymphocytes vigor, from
And immunoregulation effect is played, and strengthen anti-virus ability.Interferon, which is one group, has the reactive protein of a variety of functions（Mainly
It is glycoprotein）, it is a kind of cell factor produced by monocyte and lymphocyte.They have wide spectrum on allogenic cell
Antiviral, influence cell growth, and break up, adjust the multiple biological activities such as immunologic function.Because interferon has nontoxic, pair
Effect and antiviral advantage in extensive range, just attract attention, by application development for many years, interferon is at the beginning of discovery
It is used for the prevention and control field of the diseases such as virosis and tumour as a kind of extensive immunopotentiator.
China is pork producing country and country of consumption maximum in the world, and pork yield accounts for the 46% of Gross World Product.Virus
Sexually transmitted disease such as swine fever, the disease such as blue otopathy is always the major issue that China's pig industry faces.And interferon has proved to be
Prevent the important preparation of viral infectious.Conventional interferon is produced in the form of wild type, although close with natural structure, but
Half-life period is shorter, it is impossible to ensure the effective active of medicine.And conventional method increases half-life period by connecting the Fc fragments of antibody, but
Because molecular weight is larger after connection Fc, it is impossible to expressed by prokaryotic system, and can only be by eukaryotic expression system, virtually
Production cost is added, causes raiser's use cost higher.Therefore, operated with genetic engineering, original series are changed
Make, be only connected into antibody constant region CH3 fragments, can both extend half-life period or albumen application prokaryotic expression system is carried out
Expression, obtains effect and more useful recombinates the Research Thinking that Porcine Interferon-gamma is this patent.
The content of the invention
An object of the present invention is to provide a kind of restructuring Porcine Interferon-gamma Gene, and the gene can be in prokaryotic expression system
Stable in system, efficient expression restructuring pig interferon；
The two of the object of the invention are to provide the expression vector containing above-mentioned restructuring Porcine Interferon-gamma Gene；
The three of the object of the invention are to provide the engineered strain converted by above-mentioned restructuring Porcine Interferon-gamma Gene；
The four of the object of the invention are to provide a kind of method of Prepare restructuring Porcine Interferon-gamma；
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
One kind restructuring Porcine Interferon-gamma Gene, its nucleotides sequence is classified as shown in SEQ ID NO.1, its amino acid sequence encoded
Shown in SEQ ID NO.2.
The restructuring Porcine Interferon-gamma Gene of the present invention is with pig interferon and pig source antibody CH3 regions plus the connection of His labels
It is formed by connecting, its more control sequences is to be best suitable for the nucleotide sequence that prokaryotic expression host produces by Optimizing Reconstruction.
The structure of the restructuring Porcine Interferon-gamma Gene of the present invention is connected by Porcine Interferon-gamma and 6, pig antibody CH3 areas His
Form, the gene constitutes 276 amino acid of coding by 831 nucleotides.
The present invention is also constructed containing the recombinant expression plasmid of nucleotide sequence shown in SEQ ID NO.1 and with the restructuring table
Obtained engineered strain is converted up to plasmid.
The recombinant expression plasmid of the present invention can be built-up by the conventional method of this area, i.e., by SEQ ID NO.1 institutes
The nucleotide sequence shown is inserted between the suitable restriction enzyme site of expression vector, makes the nucleosides shown in SEQ ID NO.1
Acid sequence is exercisable to be connected with expression regulation sequence.As the embodiment of a reference, for example, Recombinant Swine can be done
Disturb element-γ genes with escherichia coli prokaryotic expression carrier pET27b using restriction enzyme site BamHI and HindIII to be connected, be named as
pET-rIFN-γ。
Recombinant expression plasmid constructed by the present invention can convert host cell by various conventional methods.As reference,
Expression vector pET-rIFN- γ containing Recombinant Swine interferon beta gene can be converted Escherichia coli Rosetta and (be purchased from Beijing
Quan Shijin Bioisystech Co., Ltd, article No. catalogue CD801) obtained bacterial strain, it is named asEscherichia coli
Rosetta/pET-rIFN- γ, abbreviation Rosetta/pET-rIFN- γ.
Can be with using the E. coli recombinant stain Rosetta/pET-rIFN- γ specific methods for producing recombinant protein：
1st, the preparation of seed liquor：Single bacterium colony will be obtained after strain line culture, picking single bacterium falls within 10mL LB fluid nutrient mediums
In, while 100mg/L ampicillins are added, 37 DEG C of culture 12h.
2nd, ferment：The seed liquor of acquisition is pressed 1：100 are inoculated in fermentation medium, when culture to OD600For 0.4 or so
When, IPTG inductions are added, its final concentration of 5 mM cultivates 3h or so for 37 DEG C and receives bacterium.
The present invention also provides a kind of method of Prepare restructuring Porcine Interferon-gamma, comprises the following steps：
Build the recombinant expression plasmid containing the nucleotide sequence shown in SEQ ID NO.1；Cultivate with the recombinant expression plasmid institute
The host cell of conversion, obtains recombinant bacterial strain；Recombinant bacterial strain is cultivated, the expression of recombinant protein is induced, separates and purifies expressed
Recombinant protein.
In the method for above-mentioned Prepare restructuring albumen, described recombinant expression plasmid is preferably pET-rIFN- γ；Described place
Chief cell is Escherichia coli（Escherichia coli）Rosetta (DE3), described recombinant bacterial strain is preferablyEscherichia coli Rosetta/pET-rIFN-γ。
In the method for above-mentioned Prepare restructuring albumen, it is preferred that the separation and purifying of described recombinant protein include following step
Suddenly：
1st, the thalline of collection is suspended from phosphate buffer, low-temperature centrifugation collects supernatant after crushing, and utilizes Ni affinity columns and AKTA
Protein purification system carries out albumen and slightly purified；
2nd, recycle AKTA systems to carry out the purifying of molecular sieve in the albumen slightly purified, finally give purity and reach more than 90%
Albumen.
With natural Porcine Interferon-gamma Gene（The original series of Porcine Interferon-gamma Gene are shown in annex one）It is dry instead of Recombinant Swine
Disturb element-γ genes and carry out above step, obtain the natural Porcine Interferon-gamma available for subsequent experimental as control.
Porcine Interferon-gamma and pig antibody CH3 areas amalgamation and expression are added the stability and activity of albumen, together by the present invention
When the present invention Protein expression and purification method have that the production time is short, expression efficiency is high, expression quantity is big, be easy to purify it is excellent
Point, and the experiment proved that, the restructuring Porcine Interferon-gamma expressed by the present invention is significantly better than natural pig interference on half-life period
Element-γ.
Brief description of the drawings
Fig. 1 recombinates the PAGE gel electrophoresis result of Porcine Interferon-gamma.
The inhibitory action that Fig. 2 restructuring Porcine Interferon-gammas are bred to PK-15 cells.
Fig. 3 restructuring Porcine Interferon-gammas promote the result of the test of mouse spleen cell proliferation.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these are changed and replacement is each fallen within protection scope of the present invention.
PK-15 cells：Purchased from ATCC, CCL-33, by the culture of this laboratory passage, culture medium is containing 10% hyclone
DMEM。
Embodiment 1 recombinates the structure of Porcine Interferon-gamma Gene plasmid
1st, the restructuring Porcine Interferon-gamma Gene needed for being synthesized by the method for chemical synthesis.
2nd, the structure of restructuring Porcine Interferon-gamma Gene and pET27b carriers
The restructuring Porcine Interferon-gamma Gene that above-mentioned steps 1 are synthesized is cut with restriction enzyme BamHI and HindIII, simultaneously
With being connected by the pET27b carriers after identical cleavage and converting bacillus coli DH 5 ɑ competence, screening has ammonia benzyl mould
The transformant of plain resistance, proves that restructuring Porcine Interferon-gamma Gene has been cloned into after plasmid extraction, digestion identification, sequencing
On pET27b carriers, obtained recombinant plasmid is named as pET-rIFN- γ.
The high efficient expression of embodiment 2 recombinates the coli strain of Porcine Interferon-gamma GeneE.coli Rosetta/pET-
RIFN- γ structure
With chemical conversion process by pET-rIFN- γ convert toE.coliRosetta, in the LB flat boards containing ampicillin
Upper screening transformant, the recon for proving to obtain through plasmid extraction, digestion identification, sequencing analysisE.coli Rosetta/pET-
RIFN- γ are consistent with expection.
Embodiment 3 utilizes colibacillus engineeringE.coliRosetta/pET-rIFN- γ production Recombinant Swine interference
Element-γ
1st, the cultivation and fermentation of strain
Picking colibacillus engineeringE.coliRosetta/pET-rIFN- γ, LB liquid is inoculated in by 1% inoculum concentration by engineering bacteria
In body culture medium, 37 DEG C of incubated 12-14h, next day 1：100 to expand culture to OD values be 0.4, plus IPTG is to final concentration 0.5
MM, continues to cultivate 3h, 4000 r/min, 30 min of centrifugation collect thalline.
2nd, the purifying of Porcine Interferon-gamma is recombinated
By the thalline ultrasonication collected after centrifugation supernatant of above-mentioned collection, AKATA protein purification systems are utilized after supernatant is filtered
Purified, successively progress affinity chromatography and sieve chromatography obtain restructuring Porcine Interferon-gamma after purification.
Take a small amount of restructuring Porcine Interferon-gamma albumen after purification to add 5 × SDS sample-loading buffers of 1/5 volume, boil 10
PAGE gel electrophoresis is carried out after minute.Electrophoresis result is shown in Fig. 1.
The inhibitory action that the mtt assay of embodiment 4 detection pig restructuring Porcine Interferon-gamma albumen is bred to porcine kidney cell PK-15
Pig PK-15 cells in exponential phase are collected after Trypsin Induced, cell suspension is prepared into, blown and beaten
96 orifice plates are inoculated in after uniform, in 37 DEG C, 5% CO2Overnight incubation in incubator.Meanwhile, take fresh pig blood 10ml, the body such as add
Product PBS, dilutes blood sample.Isometric lymphocyte separation medium is drawn to be placed in 50ml centrifuge tubes.Blood sample after dilution is pressed and divided
The amount of 2 times of volumes of chaotropic is slowly added into the upper strata of lymphocyte separation medium.Make separating liquid：Blood sample：The volume of balanced salt solution
Than for 1：1：1,2200r centrifugation 22min.Quick tunica albuginea of drawing is into centrifuge tube, and the PBS for adding at least 3 times volumes is extremely centrifuged
Guan Zhong, is mixed, 2200r centrifugation 8-10min, is abandoned supernatant, is hanged with 10ml serum free mediums（Big bottle）, counted.Platform
Expect blue dyeing statistics living cells>When 95%, 200 μ L cell suspensions are added per hole in 96 well culture plates, 37 DEG C, 5%CO are put2Training
Support in case and cultivate.
Complete after step 1, remove culture medium, be divided into 6 groups, every group of experimental port respectively adds 2,10,50,250,1250,6250
The ng μ L of restructuring Porcine Interferon-gamma protein 10 0, and lymphoid cell of pig bl ood 107, control wells add 100 μ L DMEM.37 DEG C, 5%
CO2Under conditions of cultivate 48 h.
Complete after step 2,20 μ L MTT solution are added per hole（5 mg/mL）, after 4 h, nutrient solution is discarded, is added per hole
After 150 μ L DMSO, 10 min of concussion, OD values are determined for 570 nm in measure wavelength with ELISA instrument, cell is calculated
The inhibiting rate of growth.
Inhibiting rate %=（Control group OD averages-treatment group's OD values）/ control group OD average × 100%
As a result Fig. 2 is seen.From figure 2 it can be seen that with albumen buffer control group and natural Porcine Interferon-gamma protein control group phase
Than restructuring Porcine Interferon-gamma after purification has obvious inhibitory action to PK-15 cell growths, right with the increase of concentration
PK-15 cell inhibitory rates gradually increase (* * p<0.01).
The spleen lymphocyte proliferation of embodiment 5 is tested
1st, fresh pig blood 10ml is taken, isometric PBS is added, blood sample is diluted.Isometric lymphocyte separation medium is drawn to be placed in
In 50ml centrifuge tubes.
The 2nd, blood sample after dilution is slowly added into the upper strata of lymphocyte separation medium by the amount of 2 times of volumes of separating liquid.Make
Separating liquid：Blood sample：The volume ratio of balanced salt solution is 1：1：1,2200r centrifugation 22min.
3., quick tunica albuginea of drawing into centrifuge tube, add the PBS of at least 3 times volumes into centrifuge tube, mix, 2200r
8-10min is centrifuged, supernatant is abandoned, is hanged with 10ml serum free mediums（Big bottle）, counted.Trypan Blue statistics is living thin
Born of the same parents>When 95%, 200 μ L cell suspensions are added per hole in 96 well culture plates, 37 DEG C, 5%CO are put2Cultivated in incubator.
4th, Recombinant Swine rIFN- γ albumen and natural porcine IFN γ albumen are separately added into cell after culture 1h, and set cell pair
According to group.
5th, cultivating after 72h, then add MTT in every hole makes its final concentration of 5mg/mL, continues to cultivate 4h, takes out and cultivate
Plate, gently upset drains liquid in hole, is added per hole after 150 μ L DMSO vibrations 20min, with ELIASA test sample sample wells OD570
Value.As a result SPSS statistical softwares are used, statistical procedures are carried out to surveyed data, shown using one-factor analysis of variance method
Work property is examined.
As a result Fig. 3 is seen.From figure 3, it can be seen that adding restructuring porcine IFN γ albumen than adding natural porcine IFN γ albumen
There are higher proliferation function (* p to lymphocyte<0.05) not adding the cell of IFN-γ albumen, and does not have proliferation function.
Annex one
ATGCAGGCGC CCTTTTTTAA AGAAATAACG ATCCTAAAGG ACTATTTTAA 50
TGCAAGTACC TCAGATGTAC CTAATGGTGG ACCTCTTTTC TTAGAAATTT 100
TGAAGAATTG GAAAGAGGAG AGTGACAAAA AAATAATTCA GAGCCAAATT 150
GTCTCCTTCT ACTTCAAATT CTTTGAAATC TTCAAAGATA ACCAGGCCAT 200
TCAAAGGAGC ATGGATGTGA TCAAGCAAGA CATGTTTCAG AGGTTCCTAA 250
ATGGTAGCTC TGGGAAACTG AATGACTTCG AAAAGCTGAT TAAAATTCCG 300
GTAGATAATC TGCAGATCCA GCGCAAAGCC ATCAGTGAAC TCATCAAAGT 350
GATGAATGAT CTGTCACCAA GATCTAACCT AAGAAAGCGG AAGAGAAGTC 400
AGACTATGTT CCAAGGCCAG AGAGCATCAA AAGGTGGTGG TGGTAGCGGT 450
GGTGGTGGTA GCGGTGGTGG TGGTAGCGCC AAAGGGCAGA CCCGGGAGCC 500
GCAGGTGTAC ACCCTGCCCC CACACGCCGA GGAGCTGTCC AGGAGCAAAG 550
TCAGCATAAC CTGCCTGGTC ATTGGCTTCT ACCCACCTGA CATCGATGTC 600
GAGTGGCAAA GAAACGGACA GCCGGAGCCA GAGGGCAATT ACCGCACCAC 650
CCCGCCCCAG CAGGACGTGG ACGGGACCTA CTTCCTGTAC AGCAAGTTCT 700
CGGTGGACAA GGCCAGCTGG CAGGGTGGAG GCATATTCCA GTGTGCGGTG 750
ATGCACGAGG CTCTGCACAA CCACTACACC CAGAAGTCTA TCTCCAAGAC 800
TCCGGGTAAA CATCATCATC ATCATCATTA A
Sequence table
<110>Harbin purple light bio tech ltd
<120>A kind of method for improving restructuring Porcine Interferon-gamma fusion protein antiviral activity
<160> 2
<210> SEQ ID NO: 1
<211> 831
<212> DNA
<220>
<223>
<400> SEQ ID NO: 1
1 ATGCAGGCTC CGTTCTTCAA AGAAATCACC ATCCTGAAAG ACTACTTCAA
51 CGCTTCTACC TCTGACGTTC CGAACGGTGG TCCGCTGTTC CTGGAAATCC
101 TGAAAAACTG GAAAGAAGAA TCTGACAAAA AAATCATCCA GTCTCAGATC
151 GTTTCTTTCT ACTTCAAATT CTTCGAAATC TTCAAAGACA ACCAGGCTAT
201 CCAGCGTTCT ATGGACGTTA TCAAACAGGA CATGTTCCAG CGTTTCCTGA
251 ACGGTTCTTC TGGTAAACTG AACGACTTCG AAAAACTGAT CAAAATCCCG
301 GTTGACAACC TGCAGATCCA GCGTAAAGCT ATCTCTGAAC TGATCAAAGT
351 TATGAACGAC CTGTCTCCGC GTTCTAACCT GCGTAAACGT AAACGTTCTC
401 AGACCATGTT CCAGGGTCAG CGTGCTTCTA AAGGTGGTGG TGGTTCTGGT
451 GGTGGTGGTT CTGGTGGTGG TGGTTCTGCT AAAGGTCAGA CCCGTGAACC
501 GCAGGTTTAC ACCCTGCCGC CGCACGCTGA AGAACTGTCT CGTTCTAAAG
551 TTTCTATCAC CTGCCTGGTT ATCGGTTTCT ACCCGCCGGA CATCGACGTT
601 GAATGGCAGC GTAACGGTCA GCCGGAACCG GAAGGTAACT ACCGTACCAC
651 CCCGCCGCAG CAGGACGTTG ACGGTACCTA CTTCCTGTAC TCTAAATTCT
701 CTGTTGACAA AGCTTCTTGG CAGGGTGGTG GTATCTTCCA GTGCGCTGTT
751 ATGCACGAAG CTCTGCACAA CCACTACACC CAGAAATCTA TCTCTAAAAC
801 CCCGGGTAAA CACCACCACC ACCACCACTA A
<210> SEQ ID NO: 2
<211> 276
<212> PRT
<220>
<223>
<400> SEQ ID NO: 2
1 MET Gln Ala Pro Phe Phe Lys Glu Ile Thr Ile Leu Lys Asp Tyr Phe Asn Ala Ser Thr
21 Ser Asp Val Pro Asn Gly Gly Pro Leu Phe Leu Glu Ile Leu Lys Asn Trp Lys Glu Glu
41 Ser Asp Lys Lys Ile Ile Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Phe Phe Glu Ile
61 Phe Lys Asp Asn Gln Ala Ile Gln Arg Ser MET Asp Val Ile Lys Gln Asp MET Phe Gln
81 Arg Phe Leu Asn Gly Ser Ser Gly Lys Leu Asn Asp Phe Glu Lys Leu Ile Lys Ile Pro
101 Val Asp Asn Leu Gln Ile Gln Arg Lys Ala Ile Ser Glu Leu Ile Lys Val MET Asn Asp
121 Leu Ser Pro Arg Ser Asn Leu Arg Lys Arg Lys Arg Ser Gln Thr MET Phe Gln Gly Gln
141 Arg Ala Ser Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
161 Lys Gly Gln Thr Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro His Ala Glu Glu Leu Ser
181 Arg Ser Lys Val Ser Ile Thr Cys Leu Val Ile Gly Phe Tyr Pro Pro Asp Ile Asp Val
201 Glu Trp Gln Arg Asn Gly Gln Pro Glu Pro Glu Gly Asn Tyr Arg Thr Thr Pro Pro Gln
221 Gln Asp Val Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Phe Ser Val Asp Lys Ala Ser Trp
241 Gln Gly Gly Gly Ile Phe Gln Cys Ala Val MET His Glu Ala Leu His Asn His Tyr Thr
261 Gln Lys Ser Ile Ser Lys Thr Pro Gly Lys His His His His His His ***
Claims (7)
1. one kind restructuring Porcine Interferon-gamma Gene, it is characterised in that：Its nucleotides sequence is classified as shown in SEQ ID NO.1.
2. the protein coded by the restructuring Porcine Interferon-gamma Gene of claim 1, it is characterised in that：Its amino acid sequence is
Shown in SEQ ID NO.2.
3. contain the recombinant expression carrier that Porcine Interferon-gamma Gene is recombinated described in claim 1.
4. according to the recombinant expression carrier described in claim 3, it is characterised in that：It is prokaryotic expression carrier.
5. obtained recombinant bacterial strain is converted by the recombinant expression carrier of claim 3 or 4.
6. a kind of method of Prepare restructuring Porcine Interferon-gamma, comprises the following steps：
Build containing the recombinant expression plasmid that Porcine Interferon-gamma Gene is recombinated described in claim 1；Cultivate with the recombination expression matter
The host cell that grain is converted, obtains recombinant bacterial strain；Recombinant bacterial strain is cultivated, the expression of recombinant protein is induced, separates and purify institute's table
The recombinant protein reached.
7. in accordance with the method for claim 6, it is characterised in that the separation and purifying of described recombinant protein include following step
Suddenly：
（1）The thalline of collection is suspended with phosphate buffer, 0-4 DEG C of low-temperature centrifugation collects supernatant after crushing, affine using Ni
Post and AKTA protein purification systems carry out albumen and slightly purified；
（2）Recycle AKTA systems to carry out the purifying of molecular sieve in the albumen slightly purified, produce.
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