CN104109704B - A kind of isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body - Google Patents

Abstract

The present invention relates to a kind of isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body. The present invention produces after inclusion body in induction, by the broken thalline of cleavage method of optimizing, tentatively remove 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 denaturant to inclusion body such as urea, guanidine hydrochloride. And affinity chromatography makes the protein content that can purifying obtains larger, there is purity high, simple operation and other advantages. The present invention is the out destination protein of solubility of separation and purification from insoluble inclusion body, has avoided the danger of the albumen precipitation in loaded down with trivial details step and the renaturation process of sex change renaturation repeatedly, and the purity of protein obtaining can to reach electrophoresis pure.

Description

A kind of isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body

Technical field

The present invention relates to a kind of isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body.

Background technology

Pig gyrate virus II type (Porcinecircovirustype2, PCV2) is to cause that weanling pig multisystem declinesExhaust the main pathogen of syndrome (PMWS). Occurred since PMWS in Canada first from 1991, whole world great majority are supported nowAll there is the report of this disease in pig country, has caused great economic loss to whole world pig industry. 11 openings of PCV2 genomeIn reading frame (openreadingframes, ORFs), ORF2 is main ORFs. In PCV2, ORF2 is initial closeNumeral originates in the 1033rd or 1034 nucleotides, has 702 bases, encodes 234 amino acid (on gene order antisense strandORF2 terminator becomes AAG from TAA, and causes ORF2 reading frame to lengthen, total length 705bp, its terminator is TGA), coding moleculeAmount is about major function albumen-Cap albumen of 30kDa. Cap albumen, as viral major structural protein, forms PCV2'sNucleocapsid, and be the main immune protective antigen of PCV2, can produce specific immune response by induced animal body, be to grindThe desirable target antigen of PCV2 subunit vaccine processed. It is deactivation that the pig circular ring virus vaccine of selling in the market has full strain vaccineVaccine and attenuated live vaccine, and sub-single by the capsid protein that insect cell is cultivated and gland virus expression system is producedPosition vaccine. But the potential danger that full strain virus has virulence to recover, and cultivate to obtain with gland virus expression by insect cell isThe subunit vaccine that system obtains is expensive.

Escherichia coli are expression systems of a kind of cheapness and technology maturation, and its genetic background is clear, fertility strong, cultivationCycle is short, target gene expression is high, and the expression of some foreign gene in Escherichia coli can reach 5%～30 of total protein%, and downstream process simple, be easy to control, contamination resistance is strong, metabolic pathway and gene expression regulation mechanism are more clearly,There is the expression vector of a large amount of available utilizations. Be approved as safe genetic engineering receptor biological by U.S. FDA, use very wideGeneral. 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 for industrializationThe research of producing not is very thorough.

Superb [foundation of porcine circovirus 2 type ORF2 gene prokaryotic albumen indirect ELISA detection method and applicationJ ］. Chinese animal doctor's journal, 2008,28 (8): 888-891.] the EcoRI restriction enzyme site that utilizes PCV2ORF2 to include, and carrierOn the XhoI restriction enzyme site that contains, from the cloned plasmids pMD-ORF2 excision having built containing 5 ' end nuclear localization signal region, willThe ORF2 gene fragment clone of brachymemma, to pGEX-6p-1 expression vector, and is transformed into Escherichia coli sense by recombinant expression plasmidBe subject in state BL21, by IPTG abduction delivering recombinant bacterium, expression of recombinant proteins product is present in thalline with inclusion body form, entersRow 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, has tentatively removed a part of impurity, thenBy inclusion body is carried out to a series of processing, make inclusion body form solubility target protein, avoid use urea, guanidine hydrochlorideSex change dissolving and renaturation Deng denaturant to inclusion body. And affinity chromatography makes the protein content comparison can purifying obtainingGreatly, have purity high, simple operation and other advantages, for developing business-like pig circular ring virus genetic engineering subunit vaccine and phaseClosing research provides industrialized theoretical foundation and provides theoretical foundation for recombinant vaccine carries out commercialization.

Summary of the invention

Given this, the invention provides a kind of isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body.

The technical scheme that the present invention takes is as follows:

An isolation and purification method for Recombinant Swine PCV-II capsid protein inclusion body, comprise recombinant bacterium abduction delivering,Bacterial cell disruption and cracking, affinity chromatography purifying, dialysis.

The preparation of described recombinant bacterium: the 576bp of the ORFs ORF2 gene of pig gyrate virus II type virus is incorporated intoCarrier pET28a is upper, imports in host e. coli BL-21 (DE3), obtains recombinant bacterium.

The step of described bacterial cell disruption and cracking comprises: with PBS buffer solution washing thalline, lysozyme is processed 30min, centrifugalRear removal part foreign protein, through the cracking washing of the TritonX-100 of twice, obtains the precipitation containing destination protein, with containing0.5%(v/v) the PBS buffer solution dissolution precipitation of NaTDC, obtains solubility destination protein.

Concrete steps are as follows:

(1): washing thalline, by 15mlPBS buffer solution repeated washing for every gram of weight in wet base thalline, high speed centrifugation, abandons supernatant, fromHeart condition is 8000rpm, 4 DEG C, and 5min;

(2): lysozyme destroys cell membrane, the lysozyme lysis Escherichia coli that working concentration is 1～10mg/ml, 37 DEG C, shakeSwing 30min, high speed centrifugation, abandons supernatant, collecting precipitation;

(3) add detergent TritonX-100, making final concentration is 0.5%(v/v), fully mix, now solution becomes stickyThick, illustrate that the nucleic acid in bacterial cell discharges, bacteria lysis, adds DNase I, and final concentration 1ug/ml, fully mixes, and treatsSolution is thickness no longer, illustrates that nucleic acid is degraded, and high speed centrifugation, abandons supernatant, collecting precipitation; In order to make the abundant cracking of thalline, againBy the resuspended precipitation of PBS buffer solution, add TritonX-100, making final concentration is 0.5% (v/v), fully mixes, high speed centrifugation, receivesCollection precipitation;

(4): obtain containing inclusion body precipitation, use the PBS buffer solution dissolution precipitation that contains 0.5%v/v NaTDC, quietPut 30min, centrifugal, supernatant contains more destination protein, and this destination protein is solubility, is obtained supernatant.

Described PBS buffer solution is pH=7.4,50mMPBS buffer solution.

The step of described affinity chromatography purifying comprises: loading is the supernatant that step 4 obtains, the rear bufferA that usesBalance nickel post, bufferB washs foreign protein, and bufferC wash-out is also collected destination 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) 1MNaOH 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 particle larger impurity through high speed centrifugation, or with the filter membrane in 0.45um apertureFilter;

3. buffer solution balance columns material: gone up the bufferA balance nickel post again with 10 times of column volumes after sample;

4. bufferB washing foreign protein;

5. bufferC wash-out collect destination protein.

BufferA used is pH=7.4,20mMNa3PO4,0.5MNaCl;

BufferB is pH=7.4,20mMNa3PO4,0.5MNaCl, 100mM imidazoles;

BufferC is pH=7.4,20mMNa3PO4,0.5MNaCl, 250mM imidazoles.

Technology path of the present invention as shown in Figure 1.

The present invention is the out destination protein of solubility of separation and purification from insoluble inclusion body, has avoided sex change renaturation repeatedlyLoaded down with trivial details step and renaturation process in the danger of albumen precipitation, and the purity of protein obtaining can to reach electrophoresis pure.

Brief description of the drawings

Fig. 1 is Technology Roadmap of the present invention.

Fig. 2 is SDS-PAGE and the westernblot testing result of destination protein, wherein 1-3 loading, and percolation peak,100mM imidazoles eluting peak, 4-6250mM imidazoles eluting peak.

The SDS-PAGE of the destination protein of Fig. 3 purifying.

Detailed description of the invention

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 ORFs ORF2 gene of pig gyrate virus II type virus, is wherein incorporated into Nco on this geneAnd XhoTwo restriction enzyme sites, this gene of double digestion, and cut system digestion pET28a carrier with identical enzyme, enzyme connects ORF2 baseCause and pET28a carrier, be transformed in clone's t bacteria OP10, and sequence verification, then imports to host e. coli by this plasmidIn BL-21 (DE3), IPTG abduction delivering, by SDS-PAGE and Westernblot technical Analysis, has detected object eggWhite expression, and this destination 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 DEG C of activation are spent the night, and chooseGet smooth surface, the bacterium colony that particle is larger, 37 DEG C expand cultivation 10h, and condition of culture is 37 DEG C, 200rpm. Bacterium liquid is pressed to 1:100Be transferred in the LB fluid nutrient medium that contains kana resistance. Add inducer IPTG, final concentration during to logarithmic phase until thalli growthFor 1mg/ml, continue to cultivate 10h, condition of culture is 37 DEG C, 200rpm.

Kana antibiotic concentration used is 50ug/ml.

LB culture medium prescription used is as follows:

Yeastextract（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 wherein about mycetome 0.5 ~ 0.7g of every 100ml nutrient solution, broken thalline. Wherein brokenThalline method is more, high-pressure homogeneous cracking process, excusing from death cracking process, chemical cracking liquid cracking process, enzymatic isolation method, multigelation method etc.The whole bag of tricks.

This experiment is obtained solubility destination protein by a kind of new method from inclusion body.

Step is as follows in detail:

(1): washing thalline, by 15mlPBS gravity treatment washing for every gram of weight in wet base thalline. High speed centrifugation, abandons supernatant. Centrifugal conditionFor 8000rpm, 4 DEG C, 5min.

(2): lysozyme destroys cell membrane. Working concentration is the lysozyme lysis Escherichia coli of 1～10mg/ml, 37 DEG C, and shakeSwing 30min. High speed centrifugation, abandons supernatant, collecting precipitation. Because Escherichia coli are Gram-negative bacterias, use separately lysozyme effectFruit is undesirable.

(3) the resuspended precipitation of detergent cracking Escherichia coli PBS, adds detergent TritonX-100, and final concentration is 0.5%,Fully mix, now solution becomes thickness, illustrates that the nucleic acid in bacterial cell discharges, and bacteria lysis, adds DNase I, eventuallyConcentration 1ug/ml, fully mixes, and now can accelerate mixing of solution with lower-powered ultrasonic cleaner, but necessaryAdd 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, at a high speed fromThe heart, collecting precipitation.

(4): through the cracking thalline of former steps and the use of detergent, can obtain containing inclusion body precipitation, use containsThe PBS dissolution precipitation of 0.5% NaTDC, leaves standstill 30min, and centrifugal, supernatant contains more destination protein, and this objectAlbumen is solubility. Although the advantage of the method has been used lysozyme while being broken thalline, this foreign protein is gone after centrifugalRemove, and through the cracking washing of the TritonX-100 of twice, removed more foreign protein, for later purifying has broughtConvenient.

The phosphate buffer that wherein PBS buffer solution 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) 1MNaOH 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 particle larger impurity through high speed centrifugation, or with the filter membrane in 0.45um apertureFilter. Be no more than 0.2ml/min for every milliliter of post material flow velocity.

3. buffer solution balance columns material, after upper complete sample with the bufferA balance nickel post again of 10 times of column volumes.

4. bufferB washing foreign protein. Through the groping of great many of experiments, find that the imidazoles of 100mM is large absolutely under can wash-outMost foreign proteins.

5. bufferC wash-out collect destination protein.

SDS-PAGE and westernblot testing result are as shown in Figure 2. Arrowed is the destination protein of purifying,And westernblot testing result.

BufferA used is pH=7.4; 20mMNa3PO4; 0.5MNaCl;

BufferB is pH=7.4; 20mMNa3PO4; 0.5MNaCl; 100mM imidazoles;

BufferC is pH=7.4; 20mMNa3PO4; 0.5MNaCl; 250mM imidazoles;

6. the dialysis of the destination protein of purifying and concentrated

The destination protein of purifying is put in bag filter, at PBS buffer solution dialysis 4h, then again changed buffer solution dialysisSpend the night. Subsequently bag filter 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 bacteriumBody, the pollution of the foreign protein while having reduced purifying, secondly, is used relatively mild surfactant dissolves inclusion body. By this stepSuddenly can make 50% solubilization of inclusion bodies. And obtaining very pure recombinant protein by affinity chromatography, is industrialized purificationInclusion body provides new thinking.

Claims (5)

1. an isolation and purification method for Recombinant Swine PCV-II capsid protein inclusion body, is characterized in that: comprise recombinant bacteriumAbduction delivering, bacterial cell disruption and cracking, affinity chromatography purifying, dialysis; The step of described bacterial cell disruption and cracking comprises: usePBS buffer solution washing thalline, lysozyme is processed 30min, and centrifugal rear removal part foreign protein, through the TritonX-of twice100 cracking washing, obtains the precipitation containing destination protein, and it is heavy to dissolve with the PBS buffer solution that contains 0.5%v/v NaTDCForm sediment, obtain solubility destination protein;

The step of described bacterial cell disruption and cracking, concrete steps are as follows:

(1): washing thalline, PBS buffer solution repeated washing for thalline, high speed centrifugation, abandons supernatant, and centrifugal condition is8000rpm，4℃，5min；

(2): lysozyme destroys cell membrane, the lysozyme lysis Escherichia coli that working concentration is 1～10mg/ml, 37 DEG C, shakeSwing 30min, high speed centrifugation, abandons supernatant, collecting precipitation;

(3) add detergent TritonX-100, making final concentration is 0.5%v/v, fully mixes, and now solution becomes thickness, saysNucleic acid in detailed bacterial cell discharges, and bacteria lysis, adds DNase I, and final concentration 1ug/ml, fully mixes, and treats that solution is notThickness again, illustrates that nucleic acid is degraded, and high speed centrifugation, abandons supernatant, collecting precipitation; In order to make the abundant cracking of thalline, again use PBSThe resuspended precipitation of buffer solution, adds TritonX-100, and making final concentration is 0.5% (v/v), fully mix, and high speed centrifugation, it is heavy to collectForm sediment;

(4): obtain containing inclusion body precipitation, use the PBS buffer solution dissolution precipitation that contains 0.5%v/v NaTDC, quietPut 30min, centrifugal, get supernatant, obtain solubility destination protein.

2. the isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 1, its feature existsIn: described PBS buffer solution is pH=7.4,50mMPBS buffer solution.

3. the isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 1, its feature existsIn: the step of described affinity chromatography purifying comprises: after loading, with bufferA balance nickel post, bufferB washs foreign protein,BufferC wash-out is also collected destination protein.

4. the isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 3, its feature existsIn: bufferA used is pH=7.4,20mMNa3PO4,0.5MNaCl;

BufferB is pH=7.4,20mMNa3PO4,0.5MNaCl, 100mM imidazoles;

BufferC is pH=7.4,20mMNa3PO4,0.5MNaCl, 250mM imidazoles.

5. the isolation and purification method of Recombinant Swine PCV-II capsid protein inclusion body according to claim 3, its feature existsIn: described affinity chromatography purifying, concrete steps are as follows:

1. the balance of nickel post

The bufferA washing nickel post of (1) 5 times of column volume;

(2) 1MNaOH 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 particle larger impurity through high speed centrifugation, or by the filter membrane mistake in 0.45um apertureFilter;

3. buffer solution balance columns material: gone up the bufferA balance nickel post again with 10 times of column volumes after sample;

4. bufferB washing foreign protein;

5. bufferC wash-out collect destination protein.