CN102584958A - Purification method for 69KD outer membrane protein of pertussis bacillus - Google Patents
Purification method for 69KD outer membrane protein of pertussis bacillus Download PDFInfo
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Abstract
The invention belongs to the field of protein separating purification and relates to an industrialized large-scale separating purification method of 69KD outer membrane protein of pertussis bacillus. The method comprises extracting pertussis bacillus CS thallus in buffer solution with potential of hydrogen (pH) 8.8 and containing sodium chloride of 1mol/L to 2mol/l for thirty minutes to three hours at 40 to 60 DEG C, performing centrifugation, collecting supernatant, and obtaining thallus lysate supernatant of the 69KD outer membrane protein; b adding ammonium sulfate into the thallus lysate supernatant of the 69KD outer membrane protein to enable the ammonium sulfate to achieve 15% weight/volume to 25% weight/volume, standing the solution for one hour to eight hours, performing centrifugation, and collecting the supernatant; c adding ammonium sulfate into the supernatant collected in step b to enable the ammonium sulfate to achieve 30% weight/volume to 38% weight/volume, standing the solution for one hour to eight hours, performing centrifugation, abandoning the supernatant, and collecting deposit; d dissolving the deposit collected in the step c through Tris-HCl buffer solution, and dialyzing the solution to remove the ammonium sulfate; and e enabling the solution to pass through a diethyl aminoethyl (DEAE) sepharose ion exchange column, utilizing buffer solution with linear gradient sodium chloride concentration from 0 mol/L to 1 mol/L for washing, and collecting objective protein. The purification method for the 69KD outer membrane protein of the pertussis bacillus can fast and conveniently purify natural conformation 69 KD outer membrane protein so that the method can be applied to large-scale industrial preparation of the natural configuration 69 KD outer membrane protein.
Description
Technical field
The invention belongs to the separation and purification of protein field, relate to a kind of large-scale industrialization separation and purification of membranin, specifically is the large-scale industrialization separation purification method of the 69KD outer membrane protein of bordetella pertussis.
Background technology
Whooping cough is a kind of children acute respiratory infectious disease that is caused by bordetella pertussis, and classical symptom is unexpected paroxysmal paroxysmal spasmodic cough, and has air-breathing property coda or with vomiting, the sustainable several months.Severe complications is secondary infection, epileptic seizures, encephalopathic and death.
Bordetella pertussis is a gram-negative coccobacillus, long 0.5～1.5 μ m, and wide 0.2～0.5 μ m, how single dispersion, cultivation is early stage or cultivation is suitable can be perhaps polymorphic shape of chain.New isolating bacterium has pod membrane, no gemma, and I phase bacterium has pili.On bag ginger media surface, grow, the bacterium colony projection, neat in edge, translucent, glossy, look greyish white, and outward appearance is like the pearl shape, and periphery of bacterial colonies has tangible zone of hemolysis (the medical biotechnology goods are learned people People's Health Publisher, second edition Zhao Kai Lee Zhang Yihao river).
Bordetella pertussis mainly infects object and is mostly children below 6 years old, accounts for 50% of total incidence with interior infant's sickness rate in 1 years old, and the grownup who does not obtain immunizing power also can be infected.Whooping cough infectivity at the initial stage of a disease is the strongest, but is difficult for diagnosis, so wayward contagium cuts off the route of transmission, therefore has only the vaccination pertussis vaccine, reduces Susceptible population, just can reach control and eliminate pertussal purpose.Pertussis vaccine mainly contains WPV and APV at present; WPV forms with the deactivation of Whooping cough Bao Te Shi whole cell; Effectively controlled advancing of disease at the use initial stage, China came into effect the WPV planned immunization from 1978, reached 96% to DTP (whooping cough combined vaccine) fraction of coverage in 1997; The Whooping cough sickness rate is by carrying out 250.96/10 ten thousand before the planned immunization in 1978; Drop to below 1,/10 ten thousand in 1999,99.6% (achievement of Chinese immunoprophylaxis cause and the Chinese epidemiology magazine 1990 of prospect Wang Kean, 20:325～327) descended.The thirties in 20th century, Medson etc. processed since the WPV, and through constantly improving, used nearly 70 years countries in the world so far, and preventive effect is sure.But can produce certain side reaction after the vaccine inoculation, one or two people also possibly produce more serious cranial nerve symptom even death, receive people's resistance, and some countries even stop using cause the Whooping cough sickness rate that the trend of rising is arranged.Therefore need the new pertussis vaccine that less side reaction is arranged of development.
APV has removed most objectionable impurities; Only contain some effective antigens compositions, can reach similar immune effect, but reduced the generation of side reaction greatly; Make some countries that stop using DTP after a large amount of DTaP of use, effectively reduced pertussal M & M.
Japan is the country that develops APV at first; Also all there have been the APV product in the U.S., China and other countries later on; Because different, add that the preparation method is different, thereby ingredient kind among the APV, content reaches that its specification of quality is also had very big difference to the antigenic understanding of effective protection.The mode of production of APV has two kinds: the copurification mode of production and purifying and blended mode respectively by a certain percentage.The copurification mode of production is that various effective antigen of bordetella pertussis purifying is together come out, can not be to each component accurate quantification.American-European countries has generally used respectively each component of purifying, and blended mode by a certain percentage.The present stage production of China's acellular pertussis vaccine adopts ammonium sulfate precipitation to add the copurification mode of SDGC.With culture supernatant or full culture, divide two sections to saltout or two section 33 weight % ammonium sulfate precipitation with 33 weight % ammonium sulfate and 50 weight % ammonium sulfate.With the salt precipitation thing with 0.5～1.0mol/L NaCl and damping fluid (pH8.0) extracting bacillus pertussis main protection antigen PT and FHA part (possibly contain a small amount of PRN and Agg).This purifying mode is simple and efficient, but can not be to each fraction antigen accurate quantification of acellular pertussis vaccine.In order further to improve the quality of vaccine; Exploitation is the various antigenic components of purifying respectively; The Whooping cough component vaccine of proportional mixing will become certainty then, and the preparation method who therefore gropes the suitable various effective antigenic components of large-scale production produces component pertussis vaccine necessary procedure.
The bordetella pertussis antigenic substance mainly contains Toxins, pertussis (PT), filamentous hemagglutinin (FHA), 69KDa outer membrane protein (PRN); Agglutinogen (Agg), adenylate cyclase (AC), LPS intracellular toxin (LPS); Tracheal cell toxin (TCT) and heat-labile toxin (HLT) etc., PT wherein, FHA; Agg, PRN can be used as the antigenic component of component pertussis vaccine.PRN is all a kind of non-cilium property agglutinogens that have the bordetella pertussis of virulence to produce, and is present on the thalline adventitia, belongs to autonomous translocator.It can produce the antibody of protection level, has become the important antigenic component of Whooping cough component vaccine.Present method is the method for the large-scale production that is fit to PRN.
The publication number of Connaught Laboratories Ltd. is that the patented claim of CN1193324 discloses with following method purifying 69KD outer membrane protein: 1. add Thiomersalate deactivation bacterium in the bacterium liquid of fermentation culture; 2. with the bacterial suspension of deactivation in water-bearing media such as PBS, heat 45 ℃～60 ℃, then be cooled to room temperature or 4 ℃ and so repeat to extract; 3. through precipitating action with cross Affi-gel Blue post and collect the solution that contains PRN, with the salt of high density, 0.5mol/L magnesium chloride wash-out for example; 4. after the dialysis, it is passed through the chromatofocusing carrier.This method will be passed through the twice chromatographic post, complicated operation, and increased cost, not too be useful for scale operation.
Japan's Takede Chemical Industries Ltd publication number is that the patented claim of CN1147259 discloses with following method purifying 69KD outer membrane protein: the 1. centrifugal acquisition thalline of the bacillus pertussis liquid of fermentation culture; 2. thalline is dispersed in the 0.05mol/L phosphate buffered saline buffer that contains 1mol/L sodium-chlor of 1/10th volumes (with respect to cultivating liquid measure); 3. in 60 ℃ warm water, heated 90 minutes centrifugal acquisition supernatant; 4. in supernatant, add the 1mol/L phosphate buffered saline buffer, to ultimate density be 0.1mol/L, adding calcium acetate solution to ultimate density afterwards is 1.6w/v%, stirring at room 1 hour; 5. filter this calcium phosphate gel solution, collect filtrating and gel coat.Concentrating the filtrating of gained and using the ultra-filtration membrane desalination to lead to electricity is 200 ohm; 6. through a sulfopropyl positively charged ion chromatography post, collect the solution that elutriant obtains containing PRN.This method need make electricity consumption lead desalination, and output is little, is not suitable for large-scale industrial production.
WO91/15505 is through chromatography and ultrafiltration, or through urea extract, centrifugal, or carry out the purification of 69KD outer membrane protein through IX and gel-filtration resin and ultrafiltration.The ultrafiltration step cost that this method adopts is high, and introduces urea, is unfavorable for the proteic acquisition of follow-up native conformation.
US005276142 provide the repeated multiple times of suitable commercialization purifying 69KD outer membrane protein extract, then through dyestuff part chromatography, carry out the method for chromatofocusing at last.It is higher that this method obtains purity of protein, but complex steps, length consuming time is not suitable for suitability for industrialized production.
The method of US005101014 purifying 69KD outer membrane protein comprise with on the hot deactivation of thalline, dialysis, the DEAE-Spharose post with salt gradient wash-out, dialysis, Affi-Gel Blue column chromatography and with 4mol/L urea wash-out.This method dialysis step length consuming time need to use special chromatography media, and the introducing of urea is unfavorable for obtaining native conformation albumen.
The aforesaid method step is more, needs the higher material of use cost, and length consuming time is unfavorable for obtaining the albumen of native conformation, is not suitable for heavy industrialization and prepares purifying 69KD outer membrane protein.
Summary of the invention
In order to solve the aforementioned problems in the prior, the contriver is through big quantity research, on the basis of conventional protein purification technology, invented a kind of simple, fast, the method for purifying 69KD outer membrane protein that cost is low, be suitable for large-scale industrial production.
The invention provides a kind of method of 69KD outer membrane protein of the bacillus pertussis of purifying, aforesaid method may further comprise the steps:
B. in containing the cellular lysate thing supernatant of said 69KD outer membrane protein, add ammonium sulfate and make ammonium sulfate concentrations reach 15% weight/volume to 25% weight/volume, leave standstill 1 hour to 8 hours, centrifugal, collect supernatant;
C. in the supernatant that step b collects, add ammonium sulfate and make ammonium sulfate concentrations reach 30% weight/volume to 38% weight/volume, leave standstill 1 hour to 8 hours, centrifugal, abandon supernatant, collecting precipitation;
D. remove ammonium sulfate with the deposition and the dialysis of collecting among the Tris-HCl damping fluid dissolving step c;
E. cross DEAE sepharose ion exchange column,, collect target protein with the buffer solution elution of linear gradient sodium chloride concentration from 0mol/L to 1mol/L.
In the method for the invention, the ammonium sulfate concentrations among the step b is 20% (weight/volume).
In the method for the invention, the ammonium sulfate concentrations among the step c is 33% (weight/volume).
In the method for the invention, the DEAE sepharose ion exchange column among the step e is the DEAEsepharoseCL-6B ion exchange column.
In the method for the invention, the pH of the elution buffer among the step e is 8.5 to 9.0, and flow velocity is 0.4ml/ minute to 0.6ml/ minute, and the sodium-chlor wash-out concentration of said 69KD outer membrane protein is 0.05mol/L to 0.12mol/L.
In the method for the invention, the sodium-chlor wash-out concentration of the said 69KD outer membrane protein among the step e is 0.07mol/L.
Method of the present invention also comprises step a:
A. with bordetella pertussis CS thalline with the damping fluid of the pH8.8 that contains 1mol/L to 2mol/L sodium-chlor 40 to 60 ℃ of lixiviates 30 minutes to 3 hours, centrifugal, collect supernatant, obtain containing the cellular lysate thing supernatant of said 69KD outer membrane protein.
In the method for the invention, the sodium chloride concentration among the step a is 1mol/L.
In the method for the invention, the extraction temperature among the step a is 40 to 60 ℃.
In the method for the invention, the extraction time among the step a is 1 hour to 3 hours.
The present invention is through making up conventional protein purification process method; Invented a kind of method of quick, easy, purifying natural conformation 69KD outer membrane protein that cost is low through big quantity research; Thereby can be used for large-scale industry and prepare native conformation 69KD outer membrane protein, help inoculating against efficiently Whooping cough.
Description of drawings
Fig. 1: the selection of thick extracting method, A: lixiviate, B: ultrasonication, C: refiner is broken; D: molecular weight standard.
Fig. 2: the selection of lixiviate salt concn, A: standard substance; B:2mol/L NaCl lixiviate; C:1mol/L NaCl lixiviate; D:0.5mol/L NaCl lixiviate.
Fig. 3: the selection of extraction time, A: lixiviate 1 hour; B: lixiviate 2 hours; C: lixiviate 3 hours; D: standard substance.
Fig. 4: the selection of extraction temperature, A:4 ℃ of lixiviate; B:37 ℃ of lixiviate; C:60 ℃ of lixiviate; D: molecular weight standard.
Fig. 5: the selection of ammonium sulfate precipitation (specifically seeing embodiment).
Fig. 6 AGB column chromatography purification, A～D:PBS wash-out is collected sample; E～J:Tris-HCl buffer solution elution is collected sample.
The color atlas of DEAE-Sepharose CL-6B before Fig. 7 optimizes.
Each peak sample electrophoresis result of DEAE-Sepharose CL-6B chromatogram before Fig. 8 optimizes.
Fig. 9: the DEAE-Sepharose CL-6B color atlas of condition optimizing.
Figure 10: DEAE-Sepharose CL-6B column chromatography result.
Figure 11: the electrophorogram of the target protein of the inventive method purifying.
Figure 12: the PRN target protein of identifying the inventive method purifying through protein immunoblotting.
Figure 13-1 is to Figure 13-9: be respectively through the qualitative response parallel method and calculate the computer screen shot (probit=probit) that the protection of the PRN target protein of the inventive method purifying is tired.
Embodiment
Abbreviation of part T.T. and Chinese full name are following among the present invention:
Acellular pertussis vaccine: APV
Whole cell pertussis vaccine: WPV
Full cell whooping cough combined vaccine: DTwP
Acellular whooping cough combined vaccine: DTaP
Toxins, pertussis: PT
Filamentous hemagglutinin: FHA
Whooping cough adhesin (69KD outer membrane protein): PRN
Agglutinogen: Agg
The purification process of Whooping cough 69KD outer membrane protein of the present invention comprises from the bordetella pertussis of fermentation through slightly mentioning that ion column one goes on foot essence and carries early stage; Promptly can obtain the Whooping cough 69KD outer membrane protein of the above purity of 95 weight %; This method is comparatively simple and feasible, is that the component pertussis vaccine is produced indispensable step.
The inventor tests through a large amount of on existing multiple protein purification process basis; The separation and purification condition is optimized; Finally set up the purification process of Whooping cough 69KD outer membrane protein of the present invention, the inventive method purity is high, step is easy, cost is low, is fit to the heavy industrialization preparation.
Said method comprises slightly puies forward step and essence is put forward step, and the said step of slightly carrying is two sections fractionation precipitations of ammonium sulfate; It is the DEAE-Sepharose ion-exchange chromatography that said essence is put forward step, for example DEAE-Sepharose CL-6B ion-exchange chromatography.
Wherein, two sections fractionation precipitations of ammonium sulfate comprise low-concentration sulfuric acid ammonium settling step and high-concentration sulfuric acid ammonium settling step.Ammonium sulfate concentrations in the low-concentration sulfuric acid ammonium settling step can for 15% (weight/volume) to 25% (weight/volume), be preferably 18% (weight/volume) to 22% (weight/volume), most preferably be 20% (weight/volume).The ammonium sulfate of said concentration range is precipitated impurities albumen to greatest extent, and when ammonium sulfate concentrations was lower than 15% (weight/volume), then foreign protein was more, and when ammonium sulfate concentrations was higher than 25% (weight/volume), then target protein can partly precipitated.The carrying out time of low-concentration sulfuric acid ammonium settling step is 1 to 8 hour, is preferably 2 to 6 hours.
The ammonium sulfate concentrations of high-concentration sulfuric acid ammonium settling step can be preferably 33 weight % for 30 weight %-38 weight %.The ammonium sulfate of said concentration range can precipitate target protein to greatest extent, and ammonium sulfate concentrations is lower than at 30% o'clock, and the target protein deposition not exclusively when ammonium sulfate concentrations is higher than 38 weight %, can precipitate more foreign protein.The carrying out time of high-concentration sulfuric acid ammonium settling step is 1 to 8 hour, is preferably 2 to 6 hours.
The DEAE-Sepharose ion-exchange chromatography can use the DEAE-Sepharose ion exchange resin of any model, preferably uses DEAE-Sepharose CL-6B ion exchange resin (U.S. GE company).
DEAE-Sepharose ion-exchange chromatography elution requirement is: pH8.0 to 9.0, the elution buffer of preferred pH8.8; The elution buffer flow velocity is 0.5ml/ minute to 2ml/ minute, preferred 1ml/ minute.NaCl concentration is 0.07mol/L in the damping fluid of wash-out target protein.
When the pH of elution buffer is in said scope, can better target protein be separated with foreign protein.
When the elution buffer flow velocity is in said scope, target protein and foreign protein are further effectively separated.
Those skilled in the art can know that the currently known methods that from the fermentation cell, discharges target protein has a lot, and this is not a key point of the present invention.In order to discharge target protein, can adopt any ordinary method in this area to carry out the collection and the fragmentation of Whooping cough thalline.From helping the angle of subsequent extracted, the target protein that from the Whooping cough thalline, discharges of the inventive method preferably comes broken thalline through the dense saline solution lixiviate, and after fragmentation the centrifuging and taking supernatant.
Said dense saline solution is preferably 0.5ml/ minute to 2mol/L, more preferably neutral salt such as the sodium chloride solution of 1mol/L.When said dense saline solution concentration during less than 0.5mol/L, effective lixiviate target protein then; When said dense saline solution concentration during, then waste starting material and increase protein-denatured risk greater than 2mol/L.
Said extraction temperature can be 40 to 60 ℃, is preferably 50 to 60 ℃, most preferably 60 ℃.When extraction temperature during less than 40 ℃, effective lixiviate target protein; When extraction temperature during, increase the risk of target protein sex change greater than 60 ℃.
Said extraction time can be for more than 30 minutes, and preferred 1 to 3 hour, most preferably 1 hour.Extraction time can not fully make target protein discharge during less than 30 minutes; Extraction time can not further improve the release of target protein, and expend time in during greater than 3 hours.
The inventor has also attempted the thick extraction of additive method, the smart method (embodiment sees below) of extracting and discharging target protein, and all known method is for the object of the invention albumen effect and bad in these prior aries.Therefore, the inventor selects and condition optimizing from a large amount of existing separation and purification of protein methods, thereby has invented the purification process of Whooping cough 69KD outer membrane protein of the present invention.This purification process of setting up through the inventor can be simply, the Whooping cough 69KD outer membrane protein of mass preparation purity high (more than the 95 weight %) cheaply.
The selection of embodiment 1. thalline treatment processs and the optimization of condition
1.1 the acquisition of thalline:
With CS bacterial strain (CMCC58003; Available from ATCC (U.S. representative microbial preservation center)) in 10L S-S substratum, to be cultured to cell concentration be 22,000,000,000/ml; Obtained thalline in centrifugal 10 minutes at 8000rpm; Use the Tris-HCl damping fluid of the 0.025mol/L of the pH 8.8 that contains 0.035mol/L sodium-chlor, 1mmol/L YD 30 (EDTA), 1mmol/L PMSF (PMSF) to dissolve the thalline that looses, obtain the bacteria suspension of 0.05g/ml, and handle according to following method respectively.
1.2 three kinds of modes of handling thalline:
A. supernatant is got in the dense saline solution lixiviate: thalline is suspended in containing the Tris-HCl damping fluid of 0.5mol/LNaCl, and 60 ℃ of water-baths 1 hour, 4 ℃, 8500r/ minute centrifugal 10 minutes, get supernatant.
B. supernatant is got in ultrasonication: bacteria suspension is placed ice-water bath, and ultrasonication (12w), each broken 10s, intermittently 10s is an one-period, totally 10 cycles.4 ℃, 10000r/ minute centrifugal 20 minutes, get supernatant.
C. supernatant is got in the refiner fragmentation: pour bacteria suspension into refiner (Denmark APV2000, model 04010009), pressure regulation power is to 1000Bar, and broken twice (every all over 15s), 10000r/ minute centrifugal 20 minutes, gets supernatant by 4 ℃.
The supernatant that three kinds of methods are handled carries out SDS-PAGE (polyacrylamide gel electrophoresis); Electrophoresis result is seen Fig. 1, and the A swimming lane is the supernatant that obtains after the dense salt lixiviate, the supernatant that the B swimming lane obtains for the Ultrasonic Cell Disruptor fragmentation; The supernatant that the C swimming lane obtains for the refiner fragmentation, D swimming lane are the lower molecular weight standard.Can find out among Fig. 1 that broken and lixiviate all can obtain to contain the target protein sample of a great deal of, but the foreign protein of broken gained sample is more, target protein does not have showed increased, and fragmentation is more loaded down with trivial details.Through the AlphaDigiDoc software analysis, the target protein concentration in three kinds of supernatants is respectively 12.5 weight %, 8 weight % and 9.3 weight %.
Take all factors into consideration from cost and efficient, select the method for dense saline solution lixiviate.And this method carried out following optimization.1.3 the condition optimizing of supernatant is got in the dense saline solution lixiviate:
The thalline five equilibrium that spinning is arrived; And stir with the Tris-HCl damping fluid dissolving that contains 0.5mol/L, 0.75mol/L, 1mol/L, 1.25mol/L, 1.5mol/L, 2mol/L NaCl respectively; Respectively being divided into three parts is put into respectively in 4 ℃, 37 ℃, 60 ℃; Every in continuous 3 hours at a distance from 0.5 hour sampling centrifugal detection supernatant of 8500rpm (that is dense saline solution vat liquor).
Each sample supernatant is carried out SDS-PAGE, and Fig. 2 has shown that the sample supernatant that various concentration sodium-chlor obtained 60 ℃ of lixiviates in 1 hour carries out SDS-PAGE (sample of the only exemplary 0.5mol/L of providing of Fig. 2,1mol/L and 2mol/L condition).Electrophoresis result shows: each salts solution extracting effect of 1mol/L, 1.25mol/L, 1.5mol/L and 2mol/L is more or less the same, but the total protein content of lixiviate all is higher than 0.5mol/L and 0.75mol/L salts solution, and extracting effect is better.As shown in table 1, can know that through the analysis of band scan-image the target protein ratio is more or less the same in the protein solution that each salts solution of 1mol/L to 2mol/L is leached into.The NaCl concentration of selecting 1mol/L is as lixiviate concentration.
Table 1 pair each concentration sodium-chlor band image scan is analyzed target protein purity in each swimming lane
1mol/L sodium-chlor is carried out SDS-PAGE at the sample supernatant that 60 ℃ of lixiviate different times obtain; The selection of extraction time such as Fig. 3: can know through image scan analysis (seeing the following form 2); Extraction time is to obtain 19.1 weight %, 20.2 weight %, the 21.2 weight % that target protein accounts for total protein respectively under 1 hour, 2 hours, 3 hours the situation; Difference is little, is suitable extracting condition in 1 hour in order to raise the efficiency selection.
The band image scan of table 2 pair different time is analyzed, and target protein purity is following in each swimming lane:
The sample supernatant that 1mol/L sodium-chlor was obtained in the differing temps lixiviate in 1 hour carries out SDS-PAGE; The selection of extraction temperature such as Fig. 4: along with the rising of temperature, target protein content raises, and does not almost have proteic generation in the time of 4 ℃; Image scan is analyzed the band of (seeing the following form 3) 37 ℃ and 60 ℃; It is thus clear that target protein accounts for 19 weight % in the time of 60 ℃, target protein accounts for 11.4 weight % in the time of 37 ℃, therefore selects 60 ℃ as suitable extraction temperature.
Table 3 pair different extraction temperature band image scans are analyzed, and the target protein parameter is following in each swimming lane:
The selection of embodiment 2. thick extracting methods and the optimization of condition
2.1 the thick extracting method of ethanol sedimentation:
Obtain the dense saline solution vat liquor through above optimal conditions (1mol/LNaCl, 60 ℃ incubation 1 hour), 2 times of volume of ethanol-20 ℃ of precoolings 30 minutes, are slowly added vat liquor with ethanol then, stir ,-20 ℃ left standstill 30 minutes.10000r/ minute; 4 ℃; Centrifugal 30 minutes, the centrifugal deposition that obtains was dissolved with the Tris-HCl damping fluid of the 0.025mol/L of the pH 8.8 that contains 0.035mol/L sodium-chlor, 1mmol/L YD 30 (EDTA), 1mmol/L PMSF (PMSF), and with this damping fluid dialysed overnight.
2.2 ammonium sulfate precipitation:
Get ammonium sulfate and add the dense saline solution vat liquor that aforesaid method (1mol/LNaCl, 60 ℃ incubation 1 hour) obtains, make ammonium sulfate concentrations reach 50% weight/volume, mild stirring, fully after the dissolving, 4 ℃ of hold over night.After spending the night, centrifugal 30 minutes with 10000r/ minute in 4 ℃.The centrifugal deposition that obtains is dissolved with the Tris-HCl damping fluid of the 0.025mol/L of the pH 8.8 that contains 0.035mol/L sodium-chlor, 1mmol/L YD 30 (EDTA), 1mmol/L PMSF (PMSF), and with this damping fluid dialysed overnight.
There is insoluble white mass to produce behind the ethanol sedimentation, infers that ethanol makes protein denaturation, and target protein unchangeability behind the ammonium sulfate precipitation, so select ammonium sulfate precipitation.
2.3 the optimization of ammonium sulfate precipitation condition:
With aforesaid method (1mol/LNaCl; 60 ℃ of incubations 1 hour) the dense saline solution vat liquor that obtains is divided into 4 equal portions and slowly adds ammonium sulfate; Make ammonium sulfate concentrations reach 20 weight/volume %, 30 weight/volume %, 50 weight/volume % and 60 weight/volume % respectively; Mild stirring treats to dissolve fully back 4 ℃ of hold over night.After spending the night; 10000r/ minute centrifugal 30 minutes; The deposition of centrifugal acquisition is used the Tris-HCl damping fluid dissolution precipitation of the 0.025mol/L of the pH 8.8 that contains 0.035mol/L sodium-chlor, 1mmol/L YD 30 (EDTA), 1mmol/L PMSF (PMSF), and with this damping fluid dialysed overnight.
SDS-PAGE electrophoresis result such as Fig. 5 of (that is, each concentration) at different levels ammonium sulfate precipitation thing, wherein A:20% ammonium sulfate supernatant; The B:20% ammonium sulfate precipitation, C:33% ammonium sulfate supernatant, D:33% ammonium sulfate precipitation; E:50% ammonium sulfate supernatant, F:50% ammonium sulfate precipitation, G:60% ammonium sulfate supernatant; The H:60% ammonium sulfate precipitation, I: lower molecular weight standard.With analyzing; Can know that foreign protein in the ammonium sulfate precipitation of 20 weight/volume % is more and target protein is less; Most target protein is stayed in the supernatant, and the ammonium sulfate of 33 weight % can precipitate most target protein, and the ammonium sulfate of 50 weight % and 60 weight % can precipitate nearly all target protein; Simultaneously also precipitated a large amount of impurity albumen, so the ammonium sulfate of selecting 20 weight/volume % and 33 weight/volume % is as suitable fractionation precipitation concentration.That is, sample solution is carried out centrifuging and taking supernatant behind the incubation, this supernatant is carried out centrifuging and taking deposition behind the incubation with the ammonium sulfate of 33 weight/volume % with the ammonium sulfate of 20 weight/volume %.
Embodiment 3: the optimization of confirming the condition that reaches of the column chromatography purification method that essence is carried
3.1 blue (AGB) column chromatography of Affi-Gel:
With obtaining the throw out dissolving with dense salt lixiviate and the ammonium sulfate precipitation through 20 weight/volume % and 33 weight/volume % under the above-mentioned condition; The protein crude extract that obtains is dialysed in the damping fluid of the Tris-HCl of 20mmol/L (pH7.4); 4 ℃ of stirrings, last appearance balance.Use the concentration of pH7.5 to remove impurity albumen as the potassium dihydrogen phosphate aqueous solution washing chromatography column of 0.5mmol/L; Contain 4mol/L urea, 0.15mol/L sodium-chlor, 1mmol/LEDTA, 1mmol/L PMSF and 10mmol/L Tris-HCl buffer solution elution target protein with pH8.0.
The result is as shown in Figure 6 behind the sample electrophoresis of collecting behind the AGB post; A～D swimming lane is the phosphate buffered saline buffer wash-out impurity albumen stage; E～J swimming lane is for containing urea, the Tris-HCl buffer solution elution of sodium-chlor, the concentration and the type of elution of change elutriant; The appearance of foreign protein is all arranged in the target protein, is 82.6 weight % through the high-content (swimming lane J) of image scan analysis (seeing the following form 4) target protein.
The J swimming lane image scan that table 4 pair protein content is the highest is analyzed, and the target protein parameter is following:
3.2EDAE-Sepharose CL-6B column chromatography:
Obtain throw out in dissolving of the Tris-HCl of the 0.025mol/L of the pH that contains 0.035mol/L sodium-chlor 8.8 damping fluid and dialysis with carrying out fractionation precipitation with dense salt lixiviate and the ammonium sulfate through 20 weight/volume % and 33 weight/volume % under the above-mentioned condition.With appearance on the sample of dialysing, with the Tris-HCl damping fluid balance of the 0.025mol/L of pH 8.8.The A pump is pH 8.8; Contain the damping fluid of 0.035mol/L sodium-chlor, 0.025mmol/L Tris-HCl, the B pump is pH 8.8, contains the damping fluid of 1mol/L sodium-chlor, 0.025mmol/L Tris-HCl; A pump and B pump solution linear gradient are mixed for wash-out, and the peak of collecting each stage detects.
DEAE-Sepharose CL-6B post linear gradient elution; Like Fig. 7 and shown in Figure 8,10 pipes are collected altogether with peak 2 in peak 1, at the peak during 1 beginning first pipe to collect sample be the A swimming lane; Obtain purer target protein this moment; The back is followed successively by B～J swimming lane, and the sample of collecting since second pipe just contains foreign protein, does not have target protein basically to the J swimming lane.Because can obtain the purer sample of A swimming lane, select this method further to optimize.
3.3EDAE-Sepharose the optimization of chromatography method:
Change the factors such as pH, elution flow rate and linear gradient of damping fluid in the EDAE-Sepharose CL-6B post, confirm the condition when the target protein separating effect is best.
Through changing pH of buffer (each condition is respectively pH=8.0,8.5,8.8,9.0,9.5); Elution flow rate (be respectively 0.3ml/ minute, 0.4ml/ minute, 0.5ml/ minute, 0.6ml/ minute, 0.7ml/ minute); The result changes obviously; PH8.0 and can not effectively tell each peak at 9.5 o'clock wherein, flow velocity can not effectively separate each peak (data not shown) during with 0.7ml/ minute in 0.3ml/ minute.In elution buffer pH value is 8.5 to 9.0, and flow velocity is when being 0.4ml/ minute to 0.6ml/ minute, and above-mentioned peak 1 separates with peak 2 fully.The only exemplary elution requirement that provided is pH of buffer=8.8, elution flow rate=0.5ml/ minute color atlas, color atlas promptly shown in Figure 9.
As shown in Figure 9, get the percolation peak and two elution peaks are SDS-PAGE, experimental result is shown in figure 10; A～E swimming lane is that sample is collected at peak 2; F～H swimming lane is that sample is collected at peak 1, and the I swimming lane is collected sample for the percolation peak, and the J swimming lane is standard substance (the PRN standard substance are available from Britain NIBSC companies).Can know by figure, obtain purer target protein sample, so select DEAE-Sepharose CL-6B column chromatography to put forward mode as essence through DEAE-Sepharose CL-6B column chromatography.
Embodiment 4
Through above-mentioned research, the purification process of the 69KD outer membrane protein of the bordetella pertussis of the present invention shown in the inventor.Present embodiment has exemplarily been described the purification process of the 69KD outer membrane protein of complete bordetella pertussis of the present invention.
The thick extraction:
Fermentation to CS strain (CMCC58003) the Whooping cough Bao Te Salmonella liquid of 22,000,000,000/ml in the S-S substratum of 10L is obtained thalline in centrifugal 10 minutes at 8000r/ minute, get the bacterial sediment part.The bacterial sediment of collecting is resuspended in the Tris-HCl damping fluid of the pH 8.8 of 1mol/L concentration sodium-chlor, and making cell concentration is 0.05g/ml, and hot dipping is carried 1 hour in 60 ℃ of temperature water-baths, and the lixiviate mixed solution was collected supernatant in 8500r/ minute centrifugal 15 minutes.With supernatant hold over night (8 hours) under the low-concentration sulfuric acid ammonium.After spending the night,, abandon deposition, in supernatant, add ammonium sulfate again it was left standstill under the high-concentration sulfuric acid ammonium 8 hours its 9500r/ minute centrifugal 30 minutes.Afterwards; Centrifugal 30 minutes with its 9500r/ minute; Abandon supernatant; With the Tris-HCl damping fluid dissolving (deposition concentration be 1.35g/ml) of deposition, and dialyse with the Tris-HCl damping fluid of the 0.025mol/L of the pH that contains 0.035mol/L sodium-chlor 8.8 of 200 times of volumes and to remove unnecessary ammonium sulfate in 12 hours with the 0.025mol/L of the pH 8.8 that contains 0.035mol/L sodium-chlor.Thereby what obtain being rich in target protein slightly carries product.
Essence is carried: will slightly carry on the product appearance on DEAE sepharose CL-6B ion column; Through containing the Tris-HCl buffer solution for gradient elution of concentration from 0 to 1mol/L sodium-chlor; PH of buffer is 8.8; Last appearance 0.5ml/ minute, the B pump arrives 40 volume % during wash-out in 10CV, and the elution peak collection that contains the Tris-HCl damping fluid stage of 0.07mol/L sodium-chlor is target protein.
Get the elution peak that contains target protein and do the SDS-PAGE electrophoresis; The result sees Figure 11 (from left to right being respectively standard substance, sample 1, sample 2 and sample 3); Through computed in software, the purity of target protein is respectively 95 weight %, 96 weight % and 96.8 weight % (seeing the following form 5) in sample 1, sample 2 and the sample 3.
The Characteristics Detection of PRN: adopt Western blotting that article to be checked are detected.Concrete operations are by " SDS-PAGE is carried out in the requirement that Chinese pharmacopoeia version in 2005 is three ones; Isolating protein band is transferred on the nitrocellulose filter through electrotransfer (150mA, 2 hours), seals with 2 weight % bovine serum albumin solutions; Add anti-PRN monoclonal mouse antibody (E4 of National Institute for Food and Drugs Control strain) incubated overnight; The goat two anti-(Beijing Tiantan Biological Products Co.ltd's lot number 991001) that adds the anti-mouse of horseradish peroxidase target behind the thorough washing again after 1 hour, adds the colour developing of colour developing liquid in 37 ℃ of effects; The immunoblotting result sees Figure 12; The left side swimming lane is a molecular weight standard among the figure, and middle swimming lane is a standard P RN positive control, and the right side swimming lane is the isolating target protein of the inventive method.The PRN standard substance are available from Britain NIBSC company.
With article to be checked by " isoelectric focusing electrophoresis is carried out in the requirement that Chinese pharmacopoeia version in 2005 is three ones, detect the inventive method the iso-electric point (pI) of isolating target protein be 6.0, identical with standard substance.
The purity of purifying target protein under table 5 optimal conditions
| Numbering | Low sulfuric acid ammonium concentration | Ammonium persulfate concentration | Target protein purity |
| Sample 1 | 15% weight/volume | 30% weight/volume | 95 |
| Sample | |||
| 2 | 20% weight/volume | 33% weight/volume | 96 weight % |
| Sample 3 | 25% weight/volume | 38% weight/volume | 96.8 weight % |
Embodiment 5: the target protein PRN and PT, the FHA that get purifying do the proportioning immunity evaluation:
Immunity:
With reference to seedling: will be the reference seedling (lot number: #5) dissolve of 14IU/ml available from tiring of Nat'l Pharmaceutical & Biological Products Control Institute with the 14ml physiological sodium chloride; Be 1IU/ml, do 5 times then, 25 times of dilutions; With 20 of former times, 5 times and 25 times of each immune NIH mouse of three extent of dilution; Male and female half and half, 0.5ml/, metacoele was attacked in 21 days.Use with reference to known the tiring of seedling and demarcate tiring of each specimen.
Control group: raise 60 non-immune mouses and do contrast.
Trial-product: (PT and FHA are prepared by Tiantan Bio-pharmaceuticals with PRN, PT, FHA and the aluminium adjuvant of method for preparing of the present invention; Aluminium adjuvant is by the preparation of Tiantan Bio-pharmaceuticals vaccine chamber) abundant mixing; Saline water is diluted to different matched proportion density as shown in table 6 as trial-product; Use the NIH mouse (Tiantan Bio-pharmaceuticals goods limited-liability company animal housing) of the only immune body weight of mixed solution 0.5ml/, 20 of every group of abdominal cavity mouse, male and female half and half as 10g～12g.Metacoele was attacked in 21 days, judged the protection effectiveness (tiring) of trial-product through article group more to be checked with reference to the animals survived number of seedling group.
It is identical with above-mentioned trial-product experimental technique to be numbered 9 experimental group in the table 6, and difference is that the vaccine that adopts is external finished product (available from the GSK of GlaxoSmithKline PLC company).
Attack the preparation of bacterium liquid: scrape the peptone water solution that the third generation 18323 (CMCC58030) lawn of getting on the comprehensive nutrient agar of carbon (Tiantan Bio-pharmaceuticals limited-liability company Culture Medium Laboratory) is dissolved in 1 weight %, filter turbidimetric assay concentration with absorbent cotton; With peptone water the bacterium number is adjusted to 1,600,000,000/ml, to 80000/0.03ml, be and attack bacterium liquid through doubling dilution; Take a morsel and attack bacterium liquid doubling dilution to 8000/0.03ml; 800/0.03ml, 80/0.03ml, 8/0.03ml.
Encephalocoele is attacked: attacked in 21 days behind the mouse immune, the strong rate of depositing of every group of immune mouse of internal reference seedling and trial-product is at least 94% around here.Every mouse is with attacking 0.03ml bacterium liquid (80000 bacterium/0.03ml) in the syringe encephalocoele of 0.25ml.Control group is divided into 5 groups, and 12 every group, male and female half and half are attacked the bacterium liquid of 5 concentration respectively: 80000/0.03ml, 8000/0.03ml, 800/0.03ml, 80/0.03ml, 8/0.03ml, be used for measuring the LD that attacks bacterium
50Must not be above 2.5 hours from beginning to scrape bacterium to having injected last 1 mouse.
The result observes calculating:
Mouse is attacked the back and observed 14 days, statistical magnitude.Attacked back 3 days, at least 16 of experimental group animal per groups are observed animal day by day and are write down death toll, and dead animal was not cooked statistics in initial 3 days, to the 14th day paralysis was arranged, head swelling, and the back of a bow and the obvious animal of alarmming hair are also by dead calculating.
Calculate each compatibility group by qualitative response parallel method (2010 editions the 3rd P64 of Pharmacopoeia of People's Republic of China) and tire, 6 (except the finished product vaccine of last group for the GlaxoSmithKline PLC manufacturing, all the other used PRN are the present embodiment purifying in this table) of seeing the following form.Survival quantity through article group mouse more to be checked and with reference to the survival quantity of the animal of seedling group, (tiring) renderd a service in the protection that calculates trial-product according to tire (14IU/ml) of canonical reference seedling.This calculating is carried out through the software for calculation that calculates vaccine valence that is exclusively used in that Chinese food drug assay institute provides, and this software designs according to qualitative response parallel method (2010 editions the 3rd P64 of Pharmacopoeia of People's Republic of China).
Table 6 calculates tiring of each compatibility group by the qualitative response parallel method
Two component vaccines that obviously are superior to not containing PRN are renderd a service in the three component vaccines protection that contains PRN.
Compare with external similar vaccine, antigen type, proportioning are identical, and be suitable to mouse protection effort levels.
Along with the increase of PRN content, the protection of three component vaccines is renderd a service also and is obviously raise.
The raw data and the method for calculation of each experimental group in the table 6 (in the table 6 once being first group to the 9th group from top to bottom) are as follows.
First group: PRN50ug/ml (following first group to the 9th group, N is an experimental group animal sum in every group of form, r is the survival number)
Reference: Y=0.85X+6.26
First group: Y=0.85X+5.90 computed in software screenshotss are seen Figure 13-1.
Second group: PT 50ug/ml
Reference: Y=1.37X+7.02
Second group: Y=1.37X+6.71 computed in software screenshotss are seen Figure 13-2.
The 3rd group: FHA50ug/ml
Reference: Y=0.90X+6.22
The 3rd group: Y=0.90X+5.01 computed in software screenshotss are seen Figure 13-3.
The 4th group: PT50ug/ml+FHA50ug/ml
Reference: Y=1.27X+6.85
The 4th group: Y=1.27X+6.77 computed in software screenshotss are seen Figure 13-4.
The 5th group: PRN16ug/ml+PT50ug/ml
Reference: Y=1.33X+6.82
The 5th group: Y=1.33X+6.59 computed in software screenshotss are seen Figure 13-5.
The 6th group: PRN16ug/ml+FHA50ug/ml
Reference: Y=1.34X+6.76
The 6th group: Y=1.34X+6.80 computed in software screenshotss are seen Figure 13-6.
The 7th group: PRN16ug/ml+PT50ug/ml+FHA50ug/ml
Reference: Y=1.21X+6.62
The 7th group: Y=1.21X+6.75 computed in software screenshotss are seen Figure 13-7.
The 8th group: PRN50ug/ml+PT50ug/ml+FHA50ug/ml
Reference: Y=1.47X+7.05
The 8th group: Y=1.47X+7.49 computed in software screenshotss are seen Figure 13-8.
The 9th group: PRN16ug/ml+PT50ug/ml+FHA50ug/ml (GSK)
Reference: Y=1.36X+7.16
The 9th group: Y=1.36X+7.32 computed in software screenshotss are seen Figure 13-9.
Claims (10)
1. the method for the 69KD outer membrane protein of the bacillus pertussis of purifying said method comprising the steps of:
B. in containing the cellular lysate thing supernatant of said 69KD outer membrane protein, add ammonium sulfate and make ammonium sulfate concentrations reach 15% weight/volume to 25% weight/volume, leave standstill 1 hour to 8 hours, centrifugal, collect supernatant;
C. in the supernatant that step b collects, add ammonium sulfate and make ammonium sulfate concentrations reach 30% weight/volume to 38% weight/volume, leave standstill 1 hour to 8 hours, centrifugal, abandon supernatant, collecting precipitation;
D. remove ammonium sulfate with the deposition and the dialysis of collecting among the Tris-HCl damping fluid dissolving step c;
E. cross DEAE sepharose ion exchange column,, collect target protein with the buffer solution elution of linear gradient sodium chloride concentration from 0mol/L to 1mol/L.
2. the method for claim 1, wherein the ammonium sulfate concentrations among the step b is 20% (weight/volume).
3. the method for claim 1, wherein the ammonium sulfate concentrations among the step c is 33% (weight/volume).
4. like each described method in the claim 1 to 3, wherein, the DEAE sepharose ion exchange column among the step e is a DEAE sepharoseCL-6B ion exchange column.
5. like each described method in the claim 1 to 3, wherein, the pH of the elution buffer among the step e is 8.5 to 9.0, and flow velocity is 0.4ml/ minute to 0.6ml/ minute, and the sodium-chlor wash-out concentration of said 69KD outer membrane protein is 0.05mol/L to 0.12mol/L.
6. method as claimed in claim 5, wherein, the sodium-chlor wash-out concentration of said 69KD outer membrane protein is 0.07mol/L.
7. like each described method in the claim 1 to 3, said method also comprises step a:
A. with bordetella pertussis CS thalline with the damping fluid of the pH8.8 that contains 1mol/L to 2mol/L sodium-chlor 40 to 60 ℃ of lixiviates 30 minutes to 3 hours, centrifugal, collect supernatant, obtain containing the cellular lysate thing supernatant of said 69KD outer membrane protein.
8. method as claimed in claim 7, wherein, the sodium chloride concentration among the step a is 1mol/L.
9. method as claimed in claim 7, wherein, the extraction temperature among the step a is 40 to 60 ℃.
10. method as claimed in claim 7, wherein, the extraction time among the step a is 1 hour to 3 hours.
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| CN105131110A (en) * | 2015-08-05 | 2015-12-09 | 山东亦度生物技术有限公司 | Immunoaffinity chromatography column and purification method for purifying Bordetella pertussis Prn protein |
| CN107478846A (en) * | 2017-08-10 | 2017-12-15 | 迈克生物股份有限公司 | A kind of immunoglobulin G detection reagent box and detection method |
| CN107478853A (en) * | 2017-08-10 | 2017-12-15 | 迈克生物股份有限公司 | A kind of apolipoprotein B detection kit and detection method |
| CN117783517A (en) * | 2024-02-23 | 2024-03-29 | 北京生物制品研究所有限责任公司 | Method for characterizing pertussis toxin imaging capillary isoelectric focusing electrophoresis and application |
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| CN104262469A (en) * | 2014-09-30 | 2015-01-07 | 成都欧林生物科技股份有限公司 | Method for separating and purifying pertactin |
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| CN107478846A (en) * | 2017-08-10 | 2017-12-15 | 迈克生物股份有限公司 | A kind of immunoglobulin G detection reagent box and detection method |
| CN107478853A (en) * | 2017-08-10 | 2017-12-15 | 迈克生物股份有限公司 | A kind of apolipoprotein B detection kit and detection method |
| CN107478853B (en) * | 2017-08-10 | 2018-10-12 | 迈克生物股份有限公司 | A kind of apolipoprotein B detection kit and detection method |
| CN107478846B (en) * | 2017-08-10 | 2018-11-23 | 迈克生物股份有限公司 | A kind of immunoglobulin G detection reagent box and detection method |
| CN117783517A (en) * | 2024-02-23 | 2024-03-29 | 北京生物制品研究所有限责任公司 | Method for characterizing pertussis toxin imaging capillary isoelectric focusing electrophoresis and application |
| CN117783517B (en) * | 2024-02-23 | 2024-04-19 | 北京生物制品研究所有限责任公司 | Method for characterizing pertussis toxin imaging capillary isoelectric focusing electrophoresis and application |
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