CN104109704A - Method for separating and purifying recombinant porcine circovirus capsid protein inclusion body - Google Patents
Method for separating and purifying recombinant porcine circovirus capsid protein inclusion body Download PDFInfo
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- CN104109704A CN104109704A CN201410324737.9A CN201410324737A CN104109704A CN 104109704 A CN104109704 A CN 104109704A CN 201410324737 A CN201410324737 A CN 201410324737A CN 104109704 A CN104109704 A CN 104109704A
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Abstract
The invention relates to a method for separating and purifying a recombinant porcine circovirus capsid protein inclusion body. The method for separating and purifying the recombinant porcine circovirus capsid protein inclusion body comprises that after an inclusion body is produced through induction, an optimized splitting decomposition method is adopted for crushing thalli to primarily remove a part of impurities, and then a series treatment is carried out on the inclusion body, so that the inclusion body forms the soluble target protein. By adopting the method, denatured dissolution and renaturation of the used urea, guanidine hydrochloride and the like on the inclusion body are avoided; meanwhile, nickel column affinity chromatography is carried out, so that yield of protein obtained through purification is high, and the advantages of high purity and simple operation are realized. The method for separating and purifying the recombinant porcine circovirus capsid protein inclusion body has the advantages that the soluble target protein is separated from the insoluble inclusion body and is purified, a tedious step of repeatedly carrying out denaturation and renaturation and danger of protein precipitation in a renaturation process are avoided, and the obtained protein can be electrophoretically pure.
Description
Technical field
The present invention relates to a kind of separation purification method of Recombinant Swine PCV-II capsid protein inclusion body.
Background technology
(Porcine circovirus type 2 PCV2) is the main pathogen that causes pmws (PMWS) to pig gyrate virus II type.From 1991, first since PMWS occurs in Canada, all there was the report of this disease in the great majority country of raising pigs in the whole world now, has caused great financial loss to whole world pig industry.In 11 open reading frame of PCV 2 genome (open reading frames, ORFs), ORF 2 is main open reading frame.In PCV2, ORF2 start codon initiation is in the 1033rd or 1034 Nucleotide, have 702 bases, encoding, (on gene order antisense strand, ORF2 terminator becomes AAG from TAA to 234 amino acid, and cause ORF2 reading frame to lengthen, total length 705bp, its terminator is TGA), coding molecule amount is about major function albumen-Cap albumen of 30kDa.Cap albumen, as viral major structural protein, forms the nucleocapsid of PCV2, and is the main immune protective antigen of PCV2, can produce specific immune response by induced animal body, is the desirable target antigen of development PCV2 subunit vaccine.It is inactivated vaccine and attenuated live vaccine that the pig circular ring virus vaccine of selling in the market has full strain vaccine, and by the capsid protein subunit vaccine that insect cell is cultivated and gland virus expression system is produced.But the potentially dangerous that full strain virus has virulence to recover, and it is expensive by insect cell, to cultivate the subunit vaccine obtaining with the acquisition of gland virus expression system.
Intestinal bacteria are expression systems of a kind of cheapness and technology maturation, its genetic background is clear, fecundity is strong, culture cycle is short, target gene expression level is high, the expression amount of some foreign gene in intestinal bacteria can reach 5 %～30 % of total protein, and downstream process is simple, be easy to control, contamination resistance is strong, pathways metabolism and gene expression regulation mechanism are more clearly, the expression vector of existing a large amount of available utilizations.By U.S. FDA, be approved as safe genetically engineered receptor biological, use very extensive.In addition, the pig circular ring virus 2 of report research at present virus capsid protein is a lot of as the research of subunit vaccine, but is not very thorough for the research of suitability for industrialized production.
Superb [foundation and the application ［ J ］ of porcine circovirus 2 type ORF2 gene prokaryotic albumen indirect ELISA detection method. Chinese animal doctor's journal, 2008,28 (8): 888-891.] E that utilizes PCV2 ORF2 to include
coRi restriction enzyme site, and the X containing on carrier
hoi restriction enzyme site, from the cloned plasmids pMD-ORF2 excision having built, containing 5 ', hold nuclear localization signal region, by the ORF2 gene fragment clone of brachymemma to pGEX-6p-1 expression vector, and recombinant expression plasmid is transformed in intestinal bacteria competence BL21, by IPTG abduction delivering recombinant bacterium, expression of recombinant proteins product is present in thalline with inclusion body form, carries out ultrasonic treatment thalline, inclusion body washing, dissolving and Urea Gradient dialysis renaturation, last electroelution method purification of recombinant proteins.The present invention produces after inclusion body in induction, by the broken thalline of cleavage method of optimizing, tentatively removed a part of impurity, then by inclusion body is carried out to a series of processing, make inclusion body form solubility target protein, avoided using sex change dissolving and the renaturation of denaturing agent to inclusion body such as urea, Guanidinium hydrochloride.And affinity chromatography makes the protein content that can purifying obtains larger, there is purity high, simple operation and other advantages, provides industrialized theoretical basis and provides theoretical foundation for recombiant vaccine carries out commercialization for developing business-like pig circular ring virus genetic engineering subunit vaccine and correlative study.
Summary of the invention
Given this, the invention provides a kind of separation purification method of Recombinant Swine PCV-II capsid protein inclusion body.
The technical scheme that the present invention takes is as follows:
A separation purification method for Recombinant Swine PCV-II capsid protein inclusion body, comprises abduction delivering, bacterial cell disruption and cracking, affinity chromatography purifying, the dialysis of recombinant bacterium.
The preparation of described recombinant bacterium: it is upper that the 576bp of the open reading frame ORF2 gene of pig gyrate virus II type virus is incorporated into carrier pET28a, imports in host e. coli BL-21 (DE3), obtains recombinant bacterium.
The step of described bacterial cell disruption and cracking comprises: with PBS damping fluid washing thalline, N,O-Diacetylmuramidase is processed 30min, centrifugal rear removal part foreign protein, cracking washing through the TritonX-100 of twice, obtain the precipitation containing target protein, with containing 0.5%(v/v) the PBS damping fluid dissolution precipitation of Sodium desoxycholate, obtain solubility target protein.
Concrete steps are as follows:
(1): washing thalline, by 15ml PBS damping fluid repeated washing for every gram of weight in wet base thalline, high speed centrifugation, abandons supernatant, and centrifugal condition is 8000rpm, 4 ℃, 5 min;
(2): N,O-Diacetylmuramidase destroys cell walls, the lysozyme lysis intestinal bacteria that working concentration is 1～10mg/ml, 37 ℃, concussion 30min, high speed centrifugation, abandons supernatant, collecting precipitation;
(3) add stain remover TritonX-100, making final concentration is 0.5%(v/v), fully mix, now the solution thickness that becomes, illustrates that the nucleic acid in bacterial cell discharges, bacteria lysis, add DNase I, final concentration 1ug/ml, fully mixes, and treats no longer thickness of solution, illustrate that nucleic acid is degraded, high speed centrifugation, abandons supernatant, collecting precipitation; In order to make the abundant cracking of thalline, again use the resuspended precipitation of PBS damping fluid, add TritonX-100, making final concentration is 0.5% (v/v), fully mixes high speed centrifugation, collecting precipitation;
(4): obtain containing inclusion body precipitation, use the PBS damping fluid dissolution precipitation that contains 0.5% v/v Sodium desoxycholate, standing 30min, centrifugal, supernatant contains more target protein, and this target protein is solubility, is obtained supernatant liquor.
Described PBS damping fluid is pH=7.4,50mM PBS damping fluid.
The step of described affinity chromatography purifying comprises: loading is the supernatant liquor that step 4 obtains, and rear with bufferA balance nickel post, bufferB washs foreign protein, and bufferC wash-out is also collected target protein.
Concrete steps are as follows:
1. the balance of nickel post
The bufferA washing nickel post of (1) 5 times of column volume;
(2) 1M NaOH washing pillar, guarantees that NaOH and nickel post be at least combined 30min;
(3) deionized water rinsing nickel post, makes nickel post pH be less than 9;
The bufferA balance nickel post of (4) 5 times of column volumes;
2. loading: sample need to be removed the larger impurity of particle through high speed centrifugation, or with the membrane filtration in 0.45um aperture;
3. damping fluid balance columns material: gone up after sample the bufferA reequilibrate nickel post with 10 times of column volumes;
4. bufferB washs foreign protein;
5. bufferC wash-out collect target protein.
BufferA used is pH=7.4,20mM Na3PO4,0.5M NaCl;
BufferB is pH=7.4,20mM Na3PO4,0.5M NaCl, 100mM imidazoles;
BufferC is pH=7.4,20mM Na3PO4,0.5M NaCl, 250mM imidazoles.
Technological line of the present invention as shown in Figure 1.
The present invention is the separation and purification target protein of solubility out from insoluble inclusion body, has avoided the loaded down with trivial details step of sex change renaturation repeatedly and the danger of the albumen precipitation in renaturation process, and the purity of protein obtaining can to reach electrophoresis pure.
Accompanying drawing explanation
Fig. 1 is Technology Roadmap of the present invention.
Fig. 2 is SDS-PAGE and the western blot detected result of target protein, 1-3 loading wherein, percolation peak, 100mM imidazoles elution peak, 4-6 250mM imidazoles elution peak.
The SDS-PAGE of the target protein of Fig. 3 purifying.
Embodiment
The abduction delivering of 1 recombinant bacteria
This laboratory virus stain used, from great Bei agriculture bio tech ltd, Foochow, obtains this by amplification
The 576bp of the open reading frame ORF2 gene of pig gyrate virus II type virus, is wherein incorporated into Nco on this gene
and Xho
two restriction enzyme sites, this gene of double digestion, and cut system digestion pET28a carrier with identical enzyme, enzyme connects ORF2 gene and pET28a carrier, be transformed in clone's t bacteria OP10, sequence verification, then this plasmid is imported in host e. coli BL-21 (DE3), IPTG abduction delivering, by SDS-PAGE and Western blot technical Analysis, the expression of target protein detected, and this target protein exists with inclusion body form.
The fermentation of 2 engineering bacterias
By the setting-out on the LB culture medium flat plate of the kana resistance containing of the engineering strain of preservation, 37 ℃ of activation are spent the night, picking smooth surface, and the bacterium colony that particle is larger, 37 ℃ of enlarged culturing 10h, culture condition is 37 ℃, 200rpm.Bacterium liquid is transferred in the LB liquid nutrient medium that contains kana resistance by 1:100.Until thalli growth, adding inductor IPTG, final concentration during to logarithmic phase is 1mg/ml, continues to cultivate 10h, and culture condition is 37 ℃, 200rpm.
Kana antibiotic concentration used is 50ug/ml.
LB culture medium prescription used is as follows:
Yeast extract （0.5% w/w）
NaCl （1% w/w）
Tryptone （1% w/w）。
The optimization breaking method of 3 bacteriums
High speed centrifugation results thalline, the about mycetome 0.5 ~ 0.7g of every 100ml nutrient solution wherein, broken thalline.Wherein broken thalline method is more, high-pressure homogeneous cracking process, excusing from death cracking process, chemical cracking liquid cracking process, enzymolysis process, multigelation method etc. the whole bag of tricks.
This experiment is obtained solubility target protein by a kind of new method from inclusion body.
Step is as follows in detail:
(1): washing thalline, by 15ml PBS gravity treatment washing for every gram of weight in wet base thalline.High speed centrifugation, abandons supernatant.Centrifugal condition is 8000rpm, 4 ℃, and 5 min.
(2): N,O-Diacetylmuramidase destroys cell walls.Working concentration is the lysozyme lysis intestinal bacteria of 1～10mg/ml, 37 ℃, and concussion 30min.High speed centrifugation, abandons supernatant, collecting precipitation.Because intestinal bacteria are Gram-negative bacterias, use separately N,O-Diacetylmuramidase effect undesirable.
(3) the resuspended precipitation of stain remover cracking intestinal bacteria PBS, adds stain remover TritonX-100, and final concentration is 0.5%, fully mix, now the solution thickness that becomes, illustrates that the nucleic acid in bacterial cell discharges, bacteria lysis, add DNase I, final concentration 1ug/ml, fully mixes, and now can accelerate mixing of solution with lower-powered ultrasonic cleaner, but must add ice, guarantee that temperature is unlikely to raise too fast.Treat no longer thickness of solution, illustrate that nucleic acid is degraded, high speed centrifugation, abandons supernatant.Collecting precipitation.In order to make the abundant cracking of thalline, again use the resuspended precipitation of PBS, add TritonX-100, fully mix high speed centrifugation, collecting precipitation.
(4): through the cracking thalline of former steps and the use of stain remover, can obtain containing inclusion body precipitation, use the PBS dissolution precipitation of the Sodium desoxycholate that contains 0.5%, standing 30min, centrifugal, supernatant contains more target protein, and this target protein is solubility.Although used N,O-Diacetylmuramidase when the advantage of the method is broken thalline, this foreign protein is removed after centrifugal, and through the cracking washing of the TritonX-100 of twice, removed more foreign protein, for later purifying has brought convenience.
The phosphate buffered saline buffer that wherein PBS damping fluid is 50mM, pH=7.4.
4 affinity chromatographies
Detailed step is as follows:
1. the balance of nickel post
The bufferA washing nickel post of (1) 5 times of column volume;
(2) 1M NaOH washing pillar, guarantees that NaOH and nickel post be at least combined 30min;
(3) deionized water rinsing nickel post, makes nickel post pH be less than 9;
The bufferA balance nickel post of (4) 5 times of column volumes.
2. loading, sample need to be removed the larger impurity of particle through high speed centrifugation, or with the membrane filtration in 0.45um aperture.For every milliliter of post material flow velocity, be no more than 0.2ml/min.
3. damping fluid balance columns material, after upper complete sample with the bufferA reequilibrate nickel post of 10 times of column volumes.
4. bufferB washs foreign protein.Through the groping of great many of experiments, find the imidazoles of 100mM can wash-out under most foreign proteins.
5. bufferC wash-out collect target protein.
SDS-PAGE and western blot detected result are as shown in Figure 2.Arrowed is the target protein of purifying, and western blot detected result.
BufferA used is pH=7.4; 20mM Na3PO4; 0.5M NaCl;
BufferB is pH=7.4; 20mM Na3PO4; 0.5M NaCl; 100mM imidazoles;
BufferC is pH=7.4; 20mM Na3PO4; 0.5M NaCl; 250mM imidazoles;
6. the dialysis of the target protein of purifying and concentrated
The target protein of purifying is put in dialysis tubing, at PBS damping fluid dialysis 4h, then again changed damping fluid dialysed overnight.Subsequently dialysis tubing is taken out, and concentrate with PEG 20000, the protein concentration of acquisition is higher, reaches 0.3mg/ml, as Fig. 3.
5 sum up prospect
To insoluble protein, especially the purifying of inclusion body is still a difficult problem at present, and this experiment is by optimizing broken thalline, and the pollution of the foreign protein while having reduced purifying, secondly, is used relatively mild surfactant dissolves inclusion body.By this step, can make 50% solubilization of inclusion bodies.And by affinity chromatography, obtain very pure recombinant protein, for industrialized purification inclusion body provides new thinking.
Claims (6)
1. a separation purification method for Recombinant Swine PCV-II capsid protein inclusion body, is characterized in that: comprise abduction delivering, bacterial cell disruption and cracking, affinity chromatography purifying, the dialysis of recombinant bacterium; The step of described bacterial cell disruption and cracking comprises: with PBS damping fluid washing thalline, N,O-Diacetylmuramidase is processed 30min, centrifugal rear removal part foreign protein, cracking washing through the TritonX-100 of twice, obtain the precipitation containing target protein, with the PBS damping fluid dissolution precipitation that contains 0.5%v/v Sodium desoxycholate, obtain solubility target protein.
2. the separation purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 1, is characterized in that: the step of described bacterial cell disruption and cracking, and concrete steps are as follows:
(1): washing thalline, PBS damping fluid repeated washing for thalline, high speed centrifugation, abandons supernatant, and centrifugal condition is 8000rpm, 4 ℃, 5 min;
(2): N,O-Diacetylmuramidase destroys cell walls, the lysozyme lysis intestinal bacteria that working concentration is 1～10mg/ml, 37 ℃, concussion 30min, high speed centrifugation, abandons supernatant, collecting precipitation;
(3) add stain remover TritonX-100, making final concentration is 0.5%v/v, fully mixes, now the solution thickness that becomes, illustrates that the nucleic acid in bacterial cell discharges, bacteria lysis, add DNase I, final concentration 1ug/ml, fully mixes, and treats no longer thickness of solution, illustrate that nucleic acid is degraded, high speed centrifugation, abandons supernatant, collecting precipitation; In order to make the abundant cracking of thalline, again use the resuspended precipitation of PBS damping fluid, add TritonX-100, making final concentration is 0.5% (v/v), fully mixes high speed centrifugation, collecting precipitation;
(4): obtain containing inclusion body precipitation, use the PBS damping fluid dissolution precipitation that contains 0.5% v/v Sodium desoxycholate, standing 30min, centrifugal, get supernatant, obtain solubility target protein.
3. the separation purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 1 and 2, is characterized in that: described PBS damping fluid is pH=7.4,50mM PBS damping fluid.
4. the separation purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 1, it is characterized in that: the step of described affinity chromatography purifying comprises: after loading, use bufferA balance nickel post, bufferB washs foreign protein, and bufferC wash-out is also collected target protein.
5. the separation purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 4, is characterized in that: bufferA used is pH=7.4,20mM Na
3pO
4, 0.5M NaCl;
BufferB is pH=7.4,20mM Na
3pO
4, 0.5M NaCl, 100mM imidazoles;
BufferC is pH=7.4,20mM Na
3pO
4, 0.5M NaCl, 250mM imidazoles.
6. the separation purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 4, is characterized in that: described affinity chromatography purifying, and concrete steps are as follows:
1. the balance of nickel post
The bufferA washing nickel post of (1) 5 times of column volume;
(2) 1M NaOH washing pillar, guarantees that NaOH and nickel post be at least combined 30min;
(3) deionized water rinsing nickel post, makes nickel post pH be less than 9;
The bufferA balance nickel post of (4) 5 times of column volumes;
2. loading: sample need to be removed the larger impurity of particle through high speed centrifugation, or with the membrane filtration in 0.45um aperture;
3. damping fluid balance columns material: gone up after sample the bufferA reequilibrate nickel post with 10 times of column volumes;
4. bufferB washs foreign protein;
5. bufferC wash-out collect target protein.
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| CN110004132A (en) * | 2019-04-11 | 2019-07-12 | 山东理工大学 | A kind of purification process of inclusion body protein and its application |
| CN110551703A (en) * | 2019-09-10 | 2019-12-10 | 天津大学 | Preparation and application methods of novel material BEM |
| CN113088499A (en) * | 2021-03-11 | 2021-07-09 | 嘉兴玖肽生物技术有限公司 | High-purity purification method of gene recombinant protein Tat-hMsrA |
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| CN113088499A (en) * | 2021-03-11 | 2021-07-09 | 嘉兴玖肽生物技术有限公司 | High-purity purification method of gene recombinant protein Tat-hMsrA |
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