CN1563406A - Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori - Google Patents
Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori Download PDFInfo
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- CN1563406A CN1563406A CN 200410022360 CN200410022360A CN1563406A CN 1563406 A CN1563406 A CN 1563406A CN 200410022360 CN200410022360 CN 200410022360 CN 200410022360 A CN200410022360 A CN 200410022360A CN 1563406 A CN1563406 A CN 1563406A
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Abstract
This invention disclose a purification technology in the preparation of reconstitution pylorus spirillum heat shock protein A gene engineering vaccines applying pressure, filtration, Ni ions affinity chromatography and purification, concentration lysis, glue filtration chromatography and renaturation to get pure reconstitution pylorus spirillus heat shock protein A.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to the purifying process in the preparation of Heat Shock Protein A Gene of Helicobactor pylori engineered vaccine.
Technical background
Helicobacter pylori (Helicobacter pylori, Hp) be a kind of Gram-negative, screw shaped, microaerobe that is colonizated in the stomach, infection rate surpasses 50% in whole world crowd, be the main paathogenic factor of disease of upper digestive tract such as chronic gastritis, peptide ulceration and non-ucler dyspepsia, and closely related with the generation of stomach malignant tumour.
Known Hp bacterial strain can both produce two kinds of heat shock protein(HSP)s, i.e. HspA and HspB.HspA is made up of 118 amino acid, and 91 amino acid of N end and intestinal bacteria GroES homology are very high, can be used as molecular chaperones, plays an important role in synthesizing, assemble at Hp albumen; 27 amino acid regions of C end are peculiar by Hp, are rich in Histidine and halfcystine, are nickel ion calmodulin binding domain CaMs and relevant with urease activity.The coexpression in intestinal bacteria with hspA and urease gene, the ureaclastic ability of urease strengthens more than 4 times.In a word, HspA participates in the assembling of urease and the performance of urease activity, is one of important virulence factor of Hp.HspA can excitating organism produces one of effective antigens of protective immune response, is the important candidate composition of Hp subunit vaccine.
Helicobacter pylori HspA is the homologous protein of HSP family member GroES (Hsp 10), compares with other bacterium, and the HspA of Hp has uniqueness, includes a metal ion binding site at the C-terminal (being the b zone of HspA) of HspA.Discover that HspA and nickel ion bonded ability are far longer than bonding force (the Kansau I with cobalt, iron, zinc, cupric ion, Guillain F, et al.Nickel binding andimmunological properties of the c-terminal domain of the Helicobacterpylori GroES homologue (HspA) .Mol Microbiol, 1996,22 (5): 1013-1023).The applicant has made up the colibacillus engineering (Ran Xiangyang that efficiently expresses recombinant helicobacterpylori heat shock protein(HSP) A, Zou Quanming etc. recombinant helicobacterpylori heat shock protein(HSP) A subunit efficiently expresses and preliminary purification. cell and molecular immunology magazine [J] .2002,18 (6): 539-542).
The applicant is groping that first purification of samples to rHspA is further purified, discovery is under non-sex change condition, ion exchange column, hydrophobic chromatography post and gel permeation chromatography post, all the foreign protein of 30KD size can not be removed, and this albumen is main foreign protein in the rHspA sample behind the affinitive layer purification.Best (the Amersham Pharmacia Biotech AB of the resolving power of employed Superdex 75 gel permeation chromatography posts in protein molecular is 3～70KD scope, Gel Filtration Principles and Methods, code No.18-1022-18), in theory should be able to be with the albumen sepn of 30KD and 16KD, yet in the Superdex75 gel permeation chromatography on the affine sample of rHspA behind the sample wash-out only formed simple spike (seeing accompanying drawing 1), before SDS-PAGE result shows the peak, the peak point, the foreign protein of tail of the peak 30KD accompanies with rHspA all the time, analyzes this foreign protein and may form polymer with rHspA.
Opening the polymeric common method of albumen is to use denaturing agent such as SDS, urea, Guanidinium hydrochloride etc. and adds reductive agent such as beta-mercaptoethanol, DTT (Ling Mingsheng, Xu Xiangyu, Ding Shubiao. the purifying of the recombinant protein that exists with inclusion body and external folding. Chinese biochemical drug magazine, 1995,16 (3): 135-139), attempted the first purification of samples of rHspA is concentrated back Guanidinium hydrochloride cracking, renaturation, repurity, can observation separate the 30KD foreign protein with rHspA.The result shows, yet both is not separated.Analysis may be that 30KD foreign protein and rHspA have separated under the sex change condition, but in renaturation process, both have formed polymer again.
This shows and adopt conventional purifying process can not obtain highly purified rHspA.
Summary of the invention
Purpose of the present invention is intended to can not be directly applied for the constructed recombinant helicobacterpylori heat shock protein(HSP) A recombinant vaccine albumen of the inventor at existing purifying process, and provide a kind of technology simple and direct, the purifying process of the purity of protein high-recovery that obtains recombinant helicobacterpylori heat shock protein(HSP) A recombinant vaccine preparation preferably.
The present invention has adopted following steps:
(1) crushes bacterium: the colibacillus engineering thalline 200-500g of the solubility expression that efficiently expresses reorganization heat shock protein(HSP) A (rHspA) is floating with nickel ion affinity chromatograph level pad 1: 10 (W/V) ratio suspendible that suspends, adopt the cell homogenates machine that it is mixed after 4 ℃ of precoolings, break bacterium 4～6 times with high pressure homogenizer, high speed centrifugation is collected supernatant;
(2) filter: directly use the nickel ion affinity chromatograph post to carry out elementary purifying by filtering with microporous membrane above-mentioned supernatant liquor.
(3) nickel ion affinity chromatograph column purification: select the nickel ion affinity chromatograph post to carry out preliminary purification, use 20mmol/L Tris under pH 7.0～9.0 conditions, target protein to be carried out purifying, collect the target protein peak.
(4) concentrate, cracking: add Guanidinium hydrochloride and DTT sex change cracking after step (3) concentrated first purification of samples;
(5) Superdex gel permeation chromatography purifying:,, use gel-filtration level pad balance with gel-filtration column Superdex purifying with the metaprotein sample that step (4) is obtained;
(6) renaturation: adopt dilution to wait step (5) purification of samples and hold dialysis method or chromatography column renaturation method renaturation, the sample after the renaturation adopts Superdex gel permeation chromatography separating monomer and aggressiveness.
The height that uses in described employing production of step (1) or the pilot scale purifying crushes the bacterium technology, and broken bacterium to bacterium is cracked rate greater than 98%, and differential centrifugation obtains the inclusion body throw out.
The described centrifuged supernatant of step (2) directly goes up the chromatography column purification behind 0.45 μ m filtering with microporous membrane.
The described nickel ion purifying of step (3) filler is one of Chelating Sepharose HP, ChelatingSepharose FF.
First purification of samples ultrafiltration and concentration to the protein concentration of step (4) is 20～30g/L, and the sex change cracking is to add Guanidinium hydrochloride to 6～7mol/L, adding DTT to 50mmol/L, 4 ℃, stirs more than 30 minutes;
The gel permeation chromatography post that step (5) is used is one of Superdex 75, Superdex 200, SuperdexHR 10/30.
The chromatography column renaturation method that step (6) is used, can adopt Sephadex G-25 desalination multiple except that refolding on urea renaturation and the nickel ion affinity chromatograph post, the nickel ion affinity chromatograph column packing is one of Chelating SepharoseHP, Chelating Sepharose FF.
The invention effect
The rHspA that the inventor is constructed, directly sample carries out under the unfavorable situation of purification effect under natural condition behind the use nickel ion affinity chromatograph, under the sex change condition, directly the affinitive layer purification sample to rHspA is further purified, adopt the nickel ion affinity chromatograph post, from the broken bacterium liquid of large intestine engineering bacteria of expressing HspA, extract rHspA, obtained the ideal purification effect, sex change rHspA to final purifying acquisition, adopt dilution refolding or chromatography on-column refolding, thereby can obtain purity greater than 98% rHspA.(rHspA purity＞75% that preliminary purification gets, the rHspA molecular weight that makes up acquisition through the evaluation inventor is about 15KD).
Referring to Fig. 2, show by SDS-PAGE, under 8mol/L urea condition, use Superdex 75 gel permeation chromatography posts to find, compare with non-sex change condition, occurred bimodal at 130ml～170ml place in the chromatography collection of illustrative plates, SDS-PAGE shows the 30KD foreign protein is separated with target protein rHspA, and rHspA purity＞98% behind the purifying, and the target protein yield is about 67%, referring to Fig. 3,1 is the low molecular protein standard among the figure; 2 is the protein sample before the purifying not; 3 is the sample behind the nickel ion affinity chromatograph purifying; 4 for Superdex under the sex change condition 75 foreign protein peak sample when secondarily purified, based on the 30KD foreign protein; 5 ~ 10 target protein peak samples for Superdex under the sex change condition 75 collection when secondarily purified.
RHspA by above-mentioned art breading different batches makes 15%SDS-PAGE, presents single band, and molecular mass is about 15KD.The analysis of HPLC C4 post presents single peak, and purity is more than 98%.Iso-electric point is about pH6.2.Different each peak-to-peak number average of rHspA collection of illustrative plates are consistent, and the retention time at each peak all fluctuates in ± 10s, and the peptide mapping repeatability is good.RHspA behind the purifying and mucosal adjuvant CT (Toxins,exo-, cholera) or LTB (E.coli LT B subunit) be oral immunity BalB/c mouse altogether, find that IgA and IgG level that rHspA adds in mucosal adjuvant group serum, the gastrointestinal fluid are significantly higher than control group (PBS group) (P＜0.01), but prove that HspA effective stimulus body produces higher immunne response; After rHspA and mucosal adjuvant CT (Toxins,exo-, cholera) or LTB (E.coli LT B subunit) are total to the oral immunity mongolian gerbil; adopt Hp SS1 strain (Australian Lee professor A. is so kind as to give) to infect; find that it is 21% that rHspA adds mucosal adjuvants group mongolian gerbil infection rate; control group (PBS group) infection rate is 87%, and the protection ratio that calculates the anti-Hp infection of rHspA is 70%.
Description of drawings
Fig. 1 is the Superdex 75 26/60 gel permeation chromatography collection of illustrative plates of rHspA affinity purification sample under the non-sex change condition;
Fig. 2 is rHspA affinity purification sample Superdex 75 26/60 gel permeation chromatography purifying collection of illustrative plates under the sex change condition;
Fig. 3 is Superdex 75 26/60 chromatography column purifying rHspA affinity purification sample result under the sex change condition.
Embodiment
Below in conjunction with embodiment the present invention is described in detail:
Embodiment one:
With the recombinant helicobacterpylori heat shock protein(HSP) A engineering bacteria that the applicant makes up, by high density fermentation, the target protein expression rate is 23%, and centrifugal collection thalline carries out purifying according to the following steps:
(1) height crushes bacterium: the thalline 500g that efficiently expresses is floating with 1: 10 (W/V) ratio suspendible of TE nickel ion affinity chromatograph damping fluid suspension, and adopt the cell homogenates machine that it is mixed after 4 ℃ of precoolings.Adopting high pressure homogenizer is break bacterium under the condition of 75Mpa (to break bacterium 5 times altogether at pressure, crack rate greater than 98% to bacterium), after broken bacterium finishes, bacterium liquid smear staining takes a morsel, the integrity of microscope observing cell guarantees that cytoclasis is complete, with 15, the centrifugal 40min of 000g collects supernatant and abandons precipitation.
(2) filter: directly use the nickel ion affinity chromatograph post to carry out elementary purifying by 0.45 μ m filtering with microporous membrane above-mentioned supernatant liquor.
(3) nickel ion affinity chromatograph column purification: select nickel ion affinity chromatograph post Chelating SepharoseHP to carry out preliminary purification, use 20mmol/L Tris under pH 8.5 conditions, target protein to be carried out purifying, nickel ion affinity chromatograph level pad: 10mmol/L imidazoles, 20mmol/L Tir, 0.5mol/L NaCl), adopt imidazoles (0.5mol/L imidazoles, 20mmol/L Tris, 0.5mol/L NaCl) gradient elution is collected the target protein peak.
(4) concentrate, cracking: step (3) just purification of samples after ultrafiltration (film bag aperture is less than 5KD) is concentrated into protein concentration and is 30g/L, add Guanidinium hydrochloride to 6mol/L, add DTT to 50mmol/L, 4 ℃, stir more than 30 minutes.
(5) Superdex gel permeation chromatography purifying: under the sex change condition, the metaprotein sample that step (4) is obtained, adopt gel-filtration column Superdex 75 purifying (level pad: 8mol/L urea, 0.15mol/L NaCl, 20mmol Tris, pH8.5), collect target peak, when ultraviolet absorption peak is zero, stop.
(6) renaturation: use nickel ion affinity chromatograph post ChelatingSepharose FF to carry out on-column refolding method renaturation the HspA of purifying under the sex change condition, method is that chromatography column is through balance liquid (8mol/L urea, 20mmolTris, pH 8.5) after the balance, last sample sex change target protein sample, use renaturation solution (20mmol/L PBS, 2mmolGSH, 0.2mmol/L GSSG, 0.15mol/L NaCl) slowly gradient elution post to urea is removed fully, uses imidazole buffer (20mmol/LPBS, 0.5mol/L imidazoles, pH8.5) wash-out target protein is collected protein peak.
Embodiment two:
Sample preparation is used nickel ion affinity purification post Chelating Sepharose FF preliminary purification with example one, and purification condition is with example one; Adopt 8mol/L urea and 50mmol/L DTT sex change crack protein; Under the sex change condition, (level pad: the 6mol/L Guanidinium hydrochloride, 20mmolTris pH8.5), collects the target protein peak to adopt gel-filtration column Superdex 75 purifying.Metaprotein adopts gel-filtration column G-25 to carry out the post desalination and removes the denaturing agent renaturation, uses renaturation solution (20mmol NaHCO on the albumen behind the sample
3/ Na
2CO
3, 20mmol EDTA pH10), collects the target protein peak.
Embodiment three:
Sample preparation, purification condition are with example one; The sex change crack protein adopts 8mol/L urea and 50mmol/LDTT; Under the sex change condition, and employing gel-filtration column Superdex 200 purifying (level pad: the 6mol/L Guanidinium hydrochloride, 20mmol Tris pH8.5), collects the target protein peak; The HspA metaprotein, use nickel ion affinity chromatograph post Chelating Sepharose HP to carry out on-column refolding, method is that chromatography column is through balance liquid (8mol/L urea, 20mmol Tris, pH 8.5) after the balance, last sample sex change target protein sample, use renaturation solution (20mmol/L PBS, 2mmol GSH, 0.2mmol/L GSSG, 0.15mol/L NaCl) slowly gradient elution post to urea remove fully, use imidazole buffer (20mmol/L PBS, 0.5mol/L imidazoles, pH8.5) wash-out target protein is collected protein peak.
Embodiment four:
Sample preparation is used nickel ion affinity purification post Chelating Sepharose FF preliminary purification with example one, and purification condition is with example one; The sex change crack protein adopts 6mol/L Guanidinium hydrochloride and 50mmol/L DTT; Under the sex change condition, (level pad: the 6mol/L Guanidinium hydrochloride, 20mmol Tris pH8.5), collects the target protein peak to adopt gel-filtration column Superdex HR 10/30 purifying.Metaprotein adopts gel-filtration column G-25 to carry out the post desalination and removes the denaturing agent renaturation, uses renaturation solution (20mmol NaHCO on the albumen behind the sample
3/ Na
2CO
3, 20mmolEDTA pH10), collects the target protein peak.
Result: 15%SDS-PAGE, target protein present single band, and the branch quality is about 15KD.The analysis of HPLC C4 post presents single peak, and purity is 98.2%.Iso-electric point is about pH6.2.The oral immunity BALB/c mouse can stimulate mouse to produce specificity sIgA secretion, and mongolian gerbil is attacked malicious protection test and confirmed that it can effectively induce gerbil jird to prevent and removing Hp infects, and protection ratio is 70%.
Claims (8)
1. the purifying process during a recombinant helicobacterpylori heat shock protein(HSP) A recombinant vaccine prepares, it is characterized in that: after expressing rHspA with the fermentation mode high-density, adopt height to crush bacterium, filtration, the sequential combination of purification technique such as nickel ion affinity chromatograph, gel permeation chromatography obtains high purity rHspA in a large number.
2. purifying process according to claim 1 is characterized in that: purifying carries out in the following order:
(1) height crushes bacterium: the colibacillus engineering thalline of the solubility recombinant helicobacterpylori heat shock protein(HSP) A that efficiently expresses is floating with nickel ion affinity chromatograph level pad suspendible, adopt the cell homogenates machine that it is mixed after the precooling, with the broken bacterium of high pressure homogenizer, high speed centrifugation, collect supernatant:
(2) filter: above-mentioned supernatant liquor is stand-by by filtering with microporous membrane;
(3) nickel ion affinity chromatograph column purification: select the nickel ion affinity chromatograph post to carry out preliminary purification, use Tris under pH7.0～9.0 conditions, target protein to be carried out purifying, adopt the imidazoles gradient elution;
(4) concentrate, cracking: with step (3) just purification of samples add Guanidinium hydrochloride and DTT sex change cracking after concentrating;
(5) Superdex gel permeation chromatography purifying:,, use gel-filtration level pad balance with gel-filtration column Superdex purifying with the metaprotein sample that step (4) is obtained;
(6) renaturation: adopt dilution to wait step (5) purification of samples and hold dialysis method or chromatography column renaturation method renaturation, the sample after the renaturation adopts Superdex gel permeation chromatography separating monomer and aggressiveness.
3. preparation method described in claim 2 is characterized in that the 60～80Mpa height that uses in described employing production of step (1) or the pilot scale purifying crushes the bacterium technology, and broken bacterium to bacterium is cracked rate greater than 98%, and differential centrifugation obtains the inclusion body throw out.
4. the purifying process described in claim 2 is characterized in that: the described centrifuged supernatant of step (2), directly go up the chromatography column purification behind 0.45 μ m filtering with microporous membrane.
5. preparation method described in claim 2, it is characterized in that: the described nickel ion purifying of step (3) filler is one of Chelating Sepharose HP, Chelating Sepharose FF.
6. preparation method described in claim 2, it is characterized in that: first purification of samples ultrafiltration and concentration to the protein concentration of step (4) is 20～30g/L, the sex change cracking is to add Guanidinium hydrochloride to 6～7mol/L (or urea 6～8mol/L), to add DTT (or beta-mercaptoethanol) to 10～50mmol/L, 4 ℃, stir more than 30 minutes.
7. preparation method described in claim 2, it is characterized in that: the gel permeation chromatography post that step (5) is used is one of Superdex 75, Superdex 200, Superdex HR 10/30.
8. preparation method described in claim 2, it is characterized in that: the chromatography column renaturation method that step (6) is used, can adopt Sephadex G-25 desalination multiple except that refolding on urea renaturation and the nickel ion affinity chromatograph post, the nickel ion affinity chromatograph column packing is one of Chelating Sepharose HP, Chelating Sepharose FF.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962541A (en) * | 2015-03-31 | 2015-10-07 | 芜湖康卫生物科技有限公司 | Purifying process for oral recombinant helicobacter pylori vaccine |
| CN107417765A (en) * | 2017-09-26 | 2017-12-01 | 珠海联邦制药股份有限公司 | The method that recombinant protein is isolated and purified in Escherichia coli self-dissolving expression system |
| CN107459575A (en) * | 2016-06-03 | 2017-12-12 | 硕英生医股份有限公司 | Monoclonal antibody with the immune suppression function for suppressing pathogen, its antigen-binding fragment, and produce the hybridoma of the antibody |
| CN112812969A (en) * | 2021-01-12 | 2021-05-18 | 福建基诺厚普生物科技有限公司 | System purification method for recombinant expression polypeptide in genetic engineering |
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2004
- 2004-04-20 CN CN 200410022360 patent/CN1241938C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962541A (en) * | 2015-03-31 | 2015-10-07 | 芜湖康卫生物科技有限公司 | Purifying process for oral recombinant helicobacter pylori vaccine |
| CN107459575A (en) * | 2016-06-03 | 2017-12-12 | 硕英生医股份有限公司 | Monoclonal antibody with the immune suppression function for suppressing pathogen, its antigen-binding fragment, and produce the hybridoma of the antibody |
| CN107459575B (en) * | 2016-06-03 | 2021-08-31 | 硕英生医股份有限公司 | Monoclonal antibody having immunosuppressive function against pathogen, antigen-binding fragment thereof, and hybridoma producing same |
| CN107417765A (en) * | 2017-09-26 | 2017-12-01 | 珠海联邦制药股份有限公司 | The method that recombinant protein is isolated and purified in Escherichia coli self-dissolving expression system |
| CN107417765B (en) * | 2017-09-26 | 2020-12-04 | 珠海联邦制药股份有限公司 | Method for separating and purifying recombinant protein in escherichia coli autolysis expression system |
| CN112812969A (en) * | 2021-01-12 | 2021-05-18 | 福建基诺厚普生物科技有限公司 | System purification method for recombinant expression polypeptide in genetic engineering |
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