CN108570098A - A kind of isolation and purification method of a variety of antigenic components of pertussis - Google Patents

Abstract

The present invention relates to a kind of isolation and purification methods of a variety of antigenic components of pertussis, a variety of antigens are that pertussis toxin (PT) antigen, filamentous hemagglutinin (FHA) antigen and pertussis adhesin (PRN) antigen, the method include the following steps：(1) pertussis bacterial strain fermented and cultured harvests culture solution supernatant bacterial sediment respectively；(2) pertussis toxin is isolated and purified from culture solution supernatant, PRN antigens and FHA antigens is isolated and purified from bacterial sediment.

Description

A kind of isolation and purification method of a variety of antigenic components of pertussis

Technical field

The present invention relates to biomedicine technical fields, in particular to a kind of separation of a variety of antigenic components of pertussis Purification process.

Background technology

Pertussis is to seriously endanger the deadly infectious disease of infantile health, and vaccine inoculation is optimal means of prevention.Nothing Cell DPT vaccine (DTaP) is second generation DTP vaccine, because its side effect is substantially less than first generation whole cell hundred in vain Broken vaccine (DTwP), has been widely applied, and immune into state plan, the annual market demand is at 60,000,000 or more.

DTaP vaccines can be divided into two kinds of copurification seedling and group seedling separation (DTacP) according to the difference of production method：The former It is saltoutd and ultracentrifugation processes extraction Effective Antigens pertussis toxin (PT) and filamentous hemagglutinin (FHA) using what is relatively fallen behind, though Right yield is high, production cost is low, but antigen ratio is unstable and purity is relatively low, and domestic enterprise is all made of this technique at present；The latter It is mainly produced by American-European enterprise, using column chromatography technique, extracts effective group of PT, FHA and pertussis adhesin (PRN) etc. respectively Point, antigen purity is high and ratio is stable, Vaccine effectiveness is high and side effect is lower.DTacP vaccines its one hundred days listed in the world Cough Effective Antigens ingredient production technology is shaped the nineties in last century more, and the isolation and purification method used has hydroxyapatite-knot Close globin column chromatography, gel filtration, blue glue affinity chromatography and myosin affinity chromatography, although process stabilizing, institute It is mostly out-of-date with chromatographic stuffing, globin or ox of the affinity ligand from people in the affinity chromatography used such as certain European company Myosin, there are the biological safety risks in aglucon source.

Since 21 century, with the development of chromatographic technique, more safe and efficient new fillers are used for pertussis antigen Purifying, achieves preferable effect.These schemes mostly use the method for cross-flow ultrafiltration concentration or ammonium sulfate precipitation by bacterium solution Supernatant concentrates, then ultrafiltration is replaced into the low pH low salt buffers of the urea containing 2M, after being enriched with by cation-exchange chromatography medium, leads to It crosses change elution requirement and harvests PT and FHA respectively.Then more knots are carried out to the liquid that flows through obtained in above-mentioned cation-exchange chromatography Conjunction property anion-exchange chromatography can also use urea or high-temperature process to dissolve Bordetella pertussis thalline, and it is heavy to carry out ammonium sulfate It forms sediment and more associativity anion-exchange chromatographies, isolates PRN.PT and FHA has very strong hydrophobicity, it is therefore desirable to 2M urea and High level salt solution keeps stablizing, and needs low conductivity loading using cation-exchange chromatography, therefore during ultrafiltration is replaced, PT and FHA has larger loss.

Furthermore PT and FHA is positively charged under the lower solution environmental of pH value and all has certain hydrophobicity, therefore pure The PT in FHA after change is difficult removal, needs to carry out carry out detoxification treatment to FHA, can destroy the immunogenicity of FHA in this way.

Invention content

The present invention relates to a kind of, and to isolate and purify a variety of pertussis from the Bordetella pertussis culture once cultivated effectively anti- Former method, a variety of Effective Antigens are that pertussis toxin (PT) antigen, filamentous hemagglutinin (FHA) antigen and pertussis are glutinous Attached element (PRN) antigen；

The method includes the following steps：

(1) pertussis bacterial strain fermented and cultured harvests culture solution supernatant bacterial sediment respectively；

(2) pertussis toxin is isolated and purified from culture solution supernatant, PRN antigens and FHA antigens is isolated and purified from bacterial sediment.

The method that PRN antigens and FHA antigens are isolated and purified in the slave bacterial sediment is：

The sodium chloride containing 1M of the pH=8.0 of precipitation capacity (weight in wet base) 10~15 times of volumes is added into the bacterial sediment, The buffer solution of 50mMPB stirs evenly, and 1-5 hours (preferably 1.5 hours) are extracted in 2~8 DEG C；

Extraction supernatant and extraction precipitation are collected after centrifugation respectively, FHA antigens are isolated and purified from the extraction supernatant, from institute It states in extraction precipitation and isolates and purifies PRN antigens.

The method for isolating and purifying pertussis toxin is：

(1) concentration and ultrafiltration are carried out to supernatant of bacteria solution, the cycles of concentration is 5~16 times, and the ultrafiltration uses pH5.9 ~6.1 urea containing 2M, the 10mM sodium citrate buffers of 0.5M sodium chloride, filter wash multiple >=4；

(2) using cation exchange chromatography pertussis toxin is prepared from supernatant of bacteria solution ultrafiltrate；

The method of cation-exchange chromatography described in step (2) is：

1) the thick pure preparation of pertussis toxin：

With the PBS buffer solution of the urea containing 2M containing pH5.9~6.1, the preferably PBS buffer solution of 20mM PB and 1M sodium chloride Loading after mixed mode cation-exchange chromatography post is balanced, with same buffer solution balance chromatographic column after loading；

It is eluted with the 50mMTris-HCl buffer solutions of the urea containing 2M of pH8.4~8.6,0.5M sodium chloride, collects thick pure PT Antigenic solution；

2) the consummate preparation of pertussis toxin：

With the 10mM sodium citrate buffer ion balance displacement chromatography columns of pH5.9~6.1 urea containing 2M, loading slightly pure PT Antigenic solution, chromatographic column is balanced to pH5.9~6.1, conductivity≤10mS/cm with same buffer solution after loading；

8-12 column is eluted with the 10mM PB buffer solutions of the urea containing 2M of pH 7.3~7.5,0.5%Triton X-100 Volume；

The 10mM PB buffer solutions elution for using the urea containing 2M of pH 7.3~7.5 and the sodium chloride of 0.05~0.5M again, is collected Consummate pertussis toxin solution.

The method for isolating and purifying FHA antigens is：

(1) it saltouts：The ammonium sulfate of 30% (w/v) is added into the extraction supernatant, stirs evenly, 2~8 DEG C of standings 18~ 24 hours；

(2) secondary extraction centrifuges under the conditions of going supernatant, residue to be deposited in 14000~16000g of centrifugal force after saltouing 40~precipitation is collected after sixty minutes, the urea containing 2M of the pH=6.0 of precipitation capacity (weight in wet base) 10~15 times of volumes is added in sediment 10mM sodium citrate buffers, stir evenly, be placed in 2~8 DEG C extract 1~2 hour, leaching liquor centrifugal force 14000~ Under the conditions of 16000g centrifugation 40~collect supernatant after sixty minutes, after supernatant aseptic filtration the secondary extraction supernatant of FHA antigens；

(3) chromatography obtains the FHA antigens of purifying；

The method of chromatography described in step (3) is：

1) the thick pure preparation of FHA：

With 10mM sodium citrate buffers, urea containing 2M (pH5.9~6.1) ion balance displacement chromatography column, loading FHA resists Former secondary extraction supernatant；

With 10mM PB buffer solutions, urea containing 2M and 0.5% Triton X-100 (pH7.3~7.5) elute 8~10 Column volume；

10mM PB buffer solutions, urea containing 2M and 0.2~1.0M sodium chloride (pH7.3~7.5) elution are used again, are collected thick pure FHA antigenic solutions；

2) the consummate preparation of FHA：

With 10mM PB buffer solutions, urea containing 2M (pH6.9~7.1) balances hydroxyapatite (HA) chromatographic column；Loading walks The rapid thick pure FHA antigenic solutions, are adjusted to pH6.9~7.1, conductivity≤20mS/cm；Optimal conductivity≤15mS/ cm

With 30mM PB buffer solutions, urea containing 2M and 0.5%Triton X-100 (pH6.9~7.1) elute 8~10 columns Volume；

100-200mM PB buffer solutions, urea containing 2M and 0.2~0.5M sodium chloride (pH6.9~7.1) elution are used again, are collected Consummate FHA antigenic solutions.

The isolation and purification method of the PRN antigens is：

(1) it extracts：5 times of volume (v/w) waters for injection are added in precipitating to the extraction to redissolve, 8M urea (w/v) is added Stirring 2 to 3 hours, mixing speed are 20~30rpm, and rear addition water for injection is diluted to the stirring of 2M urea and is taken out for 2 to 3 hours It carries；

(2) it saltouts, supernatant is collected by centrifugation after extracting, by the 30% of supernatant volume, be added into supernatant solid Body ammonium sulfate, mixing speed are 20~30rpm, and 2~8 DEG C stand 18~24 hours；

(3) secondary extraction：Go supernatant, precipitation that precipitation is collected after centrifugation after saltouing；It is (wet that precipitation capacity is added in sediment The 10mM PB buffer solutions (pH 6.9~7.1) of 10~15 times of volumes again), mixing speed are 20~30rpm, are extracted 1 hour；Leaching It carries and finishing, secondary extraction supernatant is collected by centrifugation, the secondary extraction supernatant of PRN antigens is obtained after aseptic filtration；

(4) chromatography obtains the PRN antigens of purifying；

The method of chromatography described in step (4) is：

1) the thick pure preparation of PRN antigens：

With 10mM PB buffer solutions sodium chloride containing 1M (pH 6.9~7.1), hydrophobic chromatography column (6~8 CV) is balanced,

The secondary extraction supernatant of loading PRN antigens contains 0.05~0.2M (preferably with 10mM sodium-acetate buffers afterwards 0.2M) sodium chloride (pH 4.4~4.6) buffer solution elutes, and collects thick pure PRN antigenic solutions.

2) the consummate preparation of PRN antigens：

With 10mM sodium-acetate buffers (pH4.9~5.1) ion balance displacement chromatography column (5~8 CV), loading is slightly pure PRN antigenic solutions are adjusted to pH4.9~5.1, conductivity≤5mS/cm；

With 10mM sodium-acetate buffers containing 0.02~1M (preferably 0.1M) sodium chloride (pH 4.9~5.1) elution, collect Consummate PRN antigenic solutions.

It is an advantage of the invention that：(1) it is high-salt buffer to be pre-processed from big tank culture to thick pure overall process, is conducive to protect The stability of pertussis toxin in purification process is demonstrate,proved, the rate of recovery is improved.(2) in bacillus pertussis late stage of culture, the release of part bacterial cell disruption Going out protease, therefore the FHA contained in supernatant of bacteria solution is easy by protease hydrolytic, this technique only extracts the FHA of phage surface, because This FHA protein integrity is more preferable.(3) foreign proteins such as PT are few in thalline extraction supernatant, and thus obtained FHA purity higher reduces The risk of finished product toxicity containing PT, thus obtained FHA antigens are not necessarily to detoxification, therefore immunogenicity is more preferable.(4) PRN albumen holds It is easily hydrolyzed to the albumen of 65KDa, the PRN antigen purities obtained from thalline are high and integrality is good, are not necessarily to detoxification, therefore immune Originality is more preferable.

Description of the drawings

Fig. 1, pertussis toxin slightly pure electrophoretogram.

The thick pure color spectrogram of Fig. 2, pertussis toxin.

The consummate electrophoretogram of Fig. 3, pertussis toxin.

The consummate chromatogram of Fig. 4, pertussis toxin.

Fig. 5, FHA antigen slightly pure electrophoretogram.

The thick pure color spectrogram of Fig. 6, FHA antigen.

The consummate electrophoretogram of Fig. 7, FHA antigen.

The consummate chromatogram of Fig. 8, FHA antigen.

Fig. 9, PRN antigen slightly pure electrophoretogram.

The thick pure color spectrogram of Figure 10, PRN antigen.

The consummate electrophoretogram of Figure 11, PRN antigen.

The consummate chromatogram of Figure 12, PRN antigen.

Specific implementation mode

Embodiment 1, the fermented and cultured of Bordetella pertussis

Test material：

Strain：CS plants of pertussis I phases, CMCC58003.From National Institute for Food and Drugs Control.Pertussis I phases CS Strain, CMCC58003.From National Institute for Food and Drugs Control.

Culture medium：The full synthetic mediums of acellular pertussis SSM

Capital equipment：Fermentation system (50L, 500L, 5000L) (Bioengineering AG) more full-automatic than Europe

Test method：

By the working seed lots strain breakdown of bacillus pertussis, it is inoculated in and improves Bao-Jiang Shi culture mediums containing 15% sheep blood, It after 35 DEG C are cultivated 72 hours, reaches on half synthetic medium of activated carbon, 37 DEG C are cultivated 48 hours；Pass (the half comprehensive training of activated carbon of 3 generations Support base), after continuing 37 DEG C of cultures 48 hours, scrape kind in 250ml phosphate buffers (i.e. PBS buffer solution, 137mM NaCl, 2.7mM KCl、10mM Na₂HPO₄、2mM KH₂PO₄；PH7.2 in), bacteria suspension is obtained.Then by 150ml bacterial suspension inoculations in In the fermentation tank of 50L, the full synthetic medium loading amounts of acellular pertussis SSM of 50L fermentation tanks be 35L (121 DEG C, 30 minutes height Pressure sterilizing), in 35~36 DEG C of air agitation cultures.

It cultivates to bacterial concentration 1.0 × 10¹⁰~1.6 × 10¹⁰It is harvested when/ml, pH 7.6~8.0, as amplification tank culture Strain.The seed liquor is transferred to the 500L fermentation tanks containing the full synthetic mediums of 350L acellular pertussis SSM, in 35~36 DEG C air agitation culture.It cultivates to bacterial concentration 1.0 × 10¹⁰~1.6 × 10¹⁰It is harvested when/ml, pH 7.6~8.0, as big Tank culture strain.The seed liquor is transferred to the 5000L fermentation tanks containing the full synthetic mediums of 3500L acellular pertussis SSM, In 35~36 DEG C of air agitation cultures.After strain inoculation, the starting culture a concentration of 0.02 × 10 of big tank¹⁰~0.1 × 10¹⁰/ ml.Big tank culture to bacterial concentration reaches 1.6 × 10¹⁰~2.4 × 10¹⁰/ ml, hemagglutinative titer are not less than 1:128, pH 7.8~ Stop culture when 8.2, stops tank sampling and do pure bacterium inspection, having varied bacteria growing, person is discarded.

Test result：With the increase of incubation time, bacteria concentration gradually increases, when culture terminates (38 hours), bacteria concentration It is 2.2 × 10¹⁰/ ml, hemagglutinative titer 1：512.

The centrifugation of embodiment 2, bulk fermentation liquid

Experiment material and sample：Pertussis bacilli * 3500L

Capital equipment：Disk centrifugal separator, Alfa Laval company CLARA 250

Test method：

500 liters of pertussis fermentation harvest bacterium solutions are centrifuged using disk centrifugal separator, the feed rate of centrifugation is 1000- 1500L/h, deslagging time are 5-10min, by reducing feed rate and shortening the deslagging time so that supernatant of bacteria solution is turbid after centrifugation Degree≤30NTU collects bacterial sediment.

Test result：

Harvest 475 liters of supernatant of bacteria solution, turbidity value=11.4NTU.

Embodiment 3 isolates and purifies pertussis toxin from fermented liquid supernatant

1, the hyperfiltration treatment of zymotic fluid

Test material and sample：

Ultrafiltration membrane packet：Pellicon2 Biomax 30, Milipore

Tangential stream processing system：CUF200SA film packet ultrafiltration systems, Milipore

Sample：Pertussis fermentation harvest bacterium solution, is centrifuged the supernatant after thalline, and sample size is 475 liters.

Test method：

Supernatant after thalline is detached is concentrated by ultrafiltration using the poly (ether sulfone) film packet of 10KD/30KD molecular cut offs, Concentration process is kept to feed end pressure in 15psi-25psi.When volume concentration is to about 50 liters, with 3~5 times of volume displaced liquid (10mM sodium citrate+2M urea+0.5MNaCl, pH=5.9-6.1) carries out filter wash, until harvest liquid conductivity >=30mS/cm, then Rinse is carried out to system with about 10 liters of displacement liquids, it is final to harvest 60L feed liquids.

2, the chromatographic purifying of pertussis toxin (PT) antigen

Experiment material and sample

Chromatographic column：BPG-140/500 GE

Purification system：AKTA pilot

Dewatering filling：HCX Millipore

Ion-exchange packing：Capto SP GE

Sample：Treated harvest liquid 60L is concentrated by ultrafiltration in 3 previous step of embodiment

2.1, the thick pure preparation of pertussis toxin：

(1) by 3LHCX media are packed into BPG-140/500 chromatographic columns, with pH 6.0,2M urea+20mMPB + 1M chlorinations 4-5 column volume of sodium balance.

(2) the supernatant 60L that hyperfiltration treatment obtains is taken, with the flow velocity of 120-200ml/min the liquid by chromatographic column, Absorption value at ultraviolet monitoring protein UV280.

(3) buffer solution of the 2M urea+20mMPB+1M sodium chloride of pH6.0 is used to balance chromatographic column after end of the sample again, until Show that absorption value is steadily to≤10mAU at A280 on AKTA.

(4) it is eluted with the 2M urea+50mMTris-HCl+0.5M sodium chloride of pH 8.5, flow velocity 120-200ml/ Min elutes 5 to 8 column volumes and collects eluting peak, which is thick pure pertussis toxin solution.

(5) buffer solution that the 2M urea+10mM sodium citrates of pH6.0 are added into the thick pure pertussis toxin solution of collection carries out Dilution, until for use between conductivity≤10mS/cm, pH 5.9-6.1.

Experimental result：Fig. 1 is PT slightly pure electrophoretograms, and Fig. 2 is the thick pure color spectrograms of PT

2.2, the consummate preparation of pertussis toxin：

(6) 3L Capto SP media are packed into BPG-140/500 chromatographic columns, with pH6.0 2M urea+10mM citric acids The buffer solution of sodium balances 4-5 column volume.It is ultraviolet by the sample after dilution with the flow velocity of 120-200ml/min by chromatographic column Monitor the absorption value at albumin A 280.

(7) pH6.0, the buffer solution rebalancing of 2M urea+10mM sodium citrates to ultraviolet monitoring egg are used after end of the sample White A280 is steadily to≤10mAU.

(8) pH7.4, the buffer solution of 2M urea+10mM PB+0.5%tritonX-100 is used to elute 8-10 column volume.

(9) it uses the buffer solution of the 2M urea+10m MPB of pH7.4 to balance chromatographic column, is absorbed at A280 until being shown on AKTA Value is steadily to≤10mAU.

Respectively with following buffer solution 1)~3)：

Buffer solution 1) 10mM PB+2M urea+0.05M sodium chloride pH7.4；

Buffer solution 2) 10mM PB+2M urea+0.1M sodium chloride pH7.4；

Buffer solution 3) 10mM PB+2M urea+0.4M sodium chloride pH7.4,

Three kinds of buffer solutions carry out continuously elution (each effluent volume is 10L) with the flow velocity of 120-200ml/min, respectively Collect eluent.Wherein, the buffer solution (buffer solution 2) of the 10mM PB+2M urea+0.1M sodium chloride of pH7.4) collect elution Peak is consummate pertussis toxin solution, 2-8 DEG C of preservation

Experimental result：Fig. 3 is the consummate electrophoretograms of PT, and Fig. 4 is the consummate chromatograms of PT

Bacterial sediment after embodiment 4, extraction fermentation collects extraction supernatant and extraction precipitation

Test material and sample：

Desk centrifuge：J-26XP, Beckman

Sample：The pertussis fermentation harvest bacterium solution that embodiment 2 obtains is centrifuged the precipitation after thalline, and sample size is 6.0kg。

Test method：

The 50mM PB buffer solutions that big tank Pertussis somatic precipitation 6kg is added to precipitation capacity (weight in wet base) 10~15 times of volumes contain 1M sodium chloride, pH=8.0 are extracted 1~2 hour, are centrifuged (15182g, 40~60 minutes)

Extraction supernatant isolating and purifying for FHA is collected, extraction precipitation isolating and purifying for PRN is collected.

Embodiment 5, pertussis filamentous hemagglutinin (FHA) antigen isolate and purify

1, the processing of saltouing of supernatant is extracted

(1) ammonium sulfate is added in embodiment obtains extraction supernatant makes ammonium sulfate concentrations reach 30% weight/volume, stands It 18-24 hours, centrifuges (15182g, 40~60 minutes), collects precipitation.

(2) the 10mM citric acids of the urea containing 2M of the pH=6.0 of precipitation capacity (weight in wet base) 10~15 times of volumes are added in sediment Sodium buffer solution, stirs evenly, and is placed in 2~8 DEG C and extracts 1~2 hour, leaching liquor centrifuges 40~60 under the conditions of centrifugal force 15182g Minute, supernatant is collected, the secondary extraction supernatant of FHA antigens is obtained after supernatant aseptic filtration.

2, the chromatographic purifying of pertussis filamentous hemagglutinin (FHA) antigen

Experiment material and sample

Chromatographic column：BPG-140/500 GE XK-50/30 GE

Purification system：AKTA pilot

Ion-exchange packing：Capto SP GE

Ion-exchange packing：CHT Type 1 Bio-Rad

Sample：Bis- extraction supernatant 30L of pertussis filamentous hemagglutinin FHA

2.1, the thick pure preparation of FHA

(1) 3L Capto SP media are packed into BPG-140/500 chromatographic columns, with pH6.0 2M urea+10mM citric acids The buffer solution of sodium balances 4-5 column volume.

(2) the secondary extraction supernatant of pertussis filamentous hemagglutinin is passed through into chromatographic column, purple with the flow velocity of 120-200ml/min Absorption value at external monitor albumin A 280.

(3) the buffer solution rebalancing of pH6.0 2M urea+10mM sodium citrates to ultraviolet monitoring egg is used after end of the sample White A280 is steadily to≤10mAU.

(4) buffer solution of pH7.4 2M urea+10mM PB+0.5%tritonX-100 is used to elute 6-8 column volume.

(5) it uses the buffer solution of pH7.4 2M urea+10m MPB to balance chromatographic column, is absorbed at A280 until being shown on AKTA Value is steadily to≤10mAU.

(6) it uses respectively：

PH7.4 10mMPB+2M urea+0.3M sodium chloride,

PH7.4 10mMPB+2M urea+0.4M sodium chloride,

PH7.4 10mMPB+2M urea+1M sodium chloride

Three kinds of buffer solutions carry out continuously elution (each effluent volume is 10L) with the flow velocity of 120-200ml/min, respectively Collect eluent.Wherein：The eluting peak that the buffer solution of the 10mMPB+2M urea+0.4M sodium chloride of pH7.4 is collected is thick pure FHA Antigenic solution.

(7) thick pure FHA antigenic solutions are preserved for 2-8 DEG C.

Experimental result：Fig. 5 is FHA slightly pure electrophoretograms, and Fig. 6 is the thick pure color spectrograms of FHA

2.2, the consummate preparation of FHA

(8) buffer solution that pH7.0 10mM PB+2M urea is added into the thick pure FHA antigenic solutions of collection is diluted, It is for use between pH 6.9-7.1 to conductivity≤15mS/cm.

(9) 1 media of 0.4L CHT Type are packed into XK-50/30 chromatographic columns, with pH7.0 10mM PB+2M urea Buffer solution balances 4-5 column volume.

(10) by the sample after dilution with the flow velocity of 40-60ml/min by chromatographic column, at ultraviolet monitoring albumin A 280 Absorption value.It is steady to ultraviolet monitoring albumin A 280 using the buffer solution rebalancing of pH7.0 10mM PB+2M urea after end of the sample To≤10mAU.

(11) buffer solution of pH7.0 2M urea+30mM PB+0.5%tritonX-100 is used to elute 6-8 column volume.

(12) buffer solution of the 2M urea+30m MPB of pH7.0 is used to balance chromatographic column again, until being shown at A280 on AKTA Absorption value is steadily to≤10mAU.

(13) it uses respectively：

PH7.0 100mM PB+2M urea+0.3M sodium chloride,

PH7.0 200mM PB+2M urea+0.4M sodium chloride

Buffer solution continuously elution (each effluent volume is 2L) is carried out with the flow velocity of 40-60ml/min, collect respectively Eluent.The eluting peak that the buffer solution of the 200m MPB+2M urea+0.4M sodium chloride of wherein pH7.0 is collected is consummate FHA antigens Solution.

(14) consummate FHA antigenic solutions are preserved for 2-8 DEG C.

Experimental result：Fig. 7 is FHA slightly pure electrophoretograms, and Fig. 8 is the thick pure color spectrograms of FHA

Embodiment 6, pertussis adhesin (PRN) antigen isolate and purify

1, the extracting of extraction precipitation is saltoutd

(1) extraction for harvesting embodiment 4 is precipitated according to 1:The ratio of 5 (W/V) is dissolved in the injection containing 8M urea In water, mixture is at least stirred 2 hours or is stood overnight at 2-8 DEG C.

(2) 2 hours are at least stirred after being diluted to 4 times of volumes or is stood overnight, supernatant is collected in centrifugation (12000g, 1h), is abandoned Fall precipitation.

(3) ammonium sulfate is added in supernatant makes ammonium sulfate concentrations reach 20% weight/volume to 30% weight/volume, quiet It sets 1-24 hours, centrifuges (12000g, 1h), collect precipitation.

(4) it is dissolved and is precipitated with the 10mM sodium phosphate buffers of pH6.9-7.1, supernatant is collected in centrifugation (12000g, 1h), is used in combination 0.22 μm of filter clarification, obtains pertussis adhesin crude extract.

2, the chromatographic purifying of pertussis adhesin (PRN) antigen

Experiment material and sample

Chromatographic column：BPG-140/500 GE XK-50/30 GE

Purification system：AKTA pilot

Ion-exchange packing：Capto SP GE

Ion-exchange packing：Capto adhere GE

Sample：Pertussis adhesin crude extract 30L

2.1, the thick pure preparation of PRN antigens

(1) 3L Capto adhere media are packed into BPG-140/500 chromatographic columns, with pH7.0 10mM PB+1M chlorine The buffer solution for changing sodium balances 6-8 column volume.

(2) pertussis adhesin crude extract is passed through into chromatographic column, ultraviolet monitoring albumen with the flow velocity of 120-200ml/min Absorption value at A280.

(3) the buffer solution rebalancing of pH7.0 10mM PB+1M sodium chloride to ultraviolet monitoring albumen is used after end of the sample A280 values≤25mAU.

(4) pH4.5 10mM CH are used₃The buffer solution of COONa+1M sodium chloride elutes steady extremely to ultraviolet monitoring albumin A 280 ≤10mAU。

(5) it uses respectively

pH4.5、10mMCH₃COONa+0.2M sodium chloride,

pH4.5、10mMCH₃COONa+0.1M sodium chloride,

pH4.5、10mM CH₃COONa

Buffer solution continuously elution (each effluent volume is 10L) is carried out with the flow velocity of 120-200ml/min, receive respectively Collect eluent.Wherein pH4.5,10mM CH₃The eluting peak that the buffer solution of COONa+0.2M sodium chloride is collected is thick pure PRN antigens Solution.

(6) thick pure PRN antigenic solutions are preserved for 2-8 DEG C.

Experimental result：Fig. 9 is PRN slightly pure electrophoretograms, and Figure 10 is the thick pure color spectrograms of PRN

2.2, the consummate preparation of PRN antigens

(7) pH5.0 10mM CH are added into the thick pure PRN antigenic solutions of collection₃The buffer solution of COONa is diluted, It is for use between pH 4.9-5.1 to conductivity≤5mS/cm.

(8) 0.4L Capto adhere media are packed into XK-50/30 chromatographic columns, with pH5.0 10mM CH₃COONa Buffer solution balance 3-4 column volume.

(9) by the sample after dilution with the flow velocity of 40-60ml/min by chromatographic column, the suction at ultraviolet monitoring albumin A 280 Receipts value.

(10) pH5.0 10mM CH are used after end of the sample₃The buffer solution rebalancing of COONa is to ultraviolet monitoring albumin A 280 Steadily to≤10mAU.

(11) it uses respectively：

PH5.0 10mM CH3COONa+0.05M sodium chloride,

PH5.0 10mM CH3COONa+0.1M sodium chloride,

PH5.0 10mM CH3COONa+0.5M sodium chloride

Buffer solution continuously elution (each effluent volume is 2L) is carried out with the flow velocity of 40-60ml/min, collect respectively Eluent.The eluting peak that wherein buffer solution of pH5.0 10mM CH3COONa+0.1M sodium chloride is collected is that consummate PRN antigens are molten Liquid.

(12) consummate PRN antigenic solutions are preserved for 2-8 DEG C.

Experimental result：Figure 11 is the consummate electrophoretograms of PRN, and Figure 10 is the consummate chromatograms of PRN

Embodiment 7, the rate of recovery of antigen repeatedly purified measures and quality research

The rate of recovery result of the three kinds of antigens prepared according to the method for previous embodiment 1~6, point six batches is examined and determine as follows Table 1.

The 1 purifying antigen rate of recovery of table

2 are shown in Table by three batches of pertussis toxin antigenic solution verification results of process purification of aforementioned determination, purifies three batches of one hundred days Cough Venom antigens solution verification result is shown in Table 3, and three batches of pertussis adhesin antigenic solution verification results of purifying are shown in Table 4

The verification result of 2 six batches of pertussis toxin antigenic solutions of table

The verification result of 3 six batches of pertussis filamentous hemagglutinin antigenic solutions of table

The quality inspection result of 4 six batches of pertussis adhesin antigenic solutions of table

The results show that meeting the manufacture and calibrating of this product according to the stoste of three batches of pilot-scales of determining technique productions Regulation (draft) requirement, illustrates stoste stable preparation process, quality controllable, reproducible, can be mass.

Finally, it should be noted that above example only helps skilled in the art to understand the essence of the present invention, do not have to Do limiting the scope of the present invention.

Claims (7)

1. a kind of method isolating and purifying a variety of pertussis Effective Antigens from the Bordetella pertussis culture once cultivated, described A variety of Effective Antigens be that pertussis toxin (PT) antigen, filamentous hemagglutinin (FHA) antigen and pertussis adhesin (PRN) are anti- It is former, which is characterized in that the method includes the following steps：

(1) pertussis bacterial strain fermented and cultured harvests culture solution supernatant bacterial sediment respectively；

(2) pertussis toxin is isolated and purified from culture solution supernatant, PRN antigens and FHA antigens is isolated and purified from bacterial sediment；

The method that PRN antigens and FHA antigens are isolated and purified in the slave bacterial sediment is：

The sodium chloride containing 1M of the pH=8.0 of precipitation capacity (weight in wet base) 10~15 times of volumes, 50mM PB are added into the bacterial sediment Buffer solution stir evenly, in 2~8 DEG C extract 1-5 hours (preferably 1.5 hours)；

Extraction supernatant and extraction precipitation are collected after centrifugation respectively, FHA antigens are isolated and purified from the extraction supernatant, from the leaching It carries in precipitation and isolates and purifies PRN antigens.

2. according to the method described in claim 1, it is characterized in that, the method for isolating and purifying pertussis toxin is：

(1) to supernatant of bacteria solution carry out concentration and ultrafiltration, the cycles of concentration be 5~16 times, the ultrafiltration using pH5.9~ 6.1 urea containing 2M, the 10mM sodium citrate buffers of 0.5M sodium chloride, filter wash multiple >=4；

(2) using cation exchange chromatography pertussis toxin is prepared from supernatant of bacteria solution ultrafiltrate.

3. according to the method described in claim 2, it is characterized in that, the method for the cation-exchange chromatography described in step (2) is：

(1) the thick pure preparation of pertussis toxin：

With the PBS buffer solution of the urea containing 2M containing pH5.9~6.1, the preferably PBS buffer solution of 20mM PB and 1M sodium chloride, balance Loading after mixed mode cation-exchange chromatography post balances chromatographic column after loading with same buffer solution；

It is eluted with the 50mMTris-HCl buffer solutions of the urea containing 2M of pH8.4~8.6,0.5M sodium chloride, collects thick pure pertussis toxin Solution；

(2) the consummate preparation of pertussis toxin：

With the 10mM sodium citrate buffer ion balance displacement chromatography columns of pH5.9~6.1 urea containing 2M, loading slightly pure pertussis toxin Solution balances chromatographic column to pH5.9~6.1, conductivity≤10mS/cm after loading with same buffer solution；

8-10 column volume is eluted with the 10mM PB buffer solutions of the urea containing 2M of pH 7.4~7.6,0.5%Triton X-100；

The 10mM PB buffer solutions elution for using the urea containing 2M of pH 7.4~7.6 and the sodium chloride of 0.05~0.5M again, is collected consummate Pertussis toxin solution.

4. method according to claim 1 or 2, which is characterized in that the method for isolating and purifying FHA antigens is：

(1) it saltouts：The ammonium sulfate of 30% (w/v) is added into the extraction supernatant, stirs evenly, 2~8 DEG C of standings 18~24 are small When；

(2) secondary extraction, removes supernatant after saltouing, residue be deposited in centrifugation 40 under the conditions of 14000~16000g of centrifugal force~ Precipitation is collected after sixty minutes, and the urea containing 2M of the pH=5.9-6.1 of precipitation capacity (weight in wet base) 10~15 times of volumes is added in sediment 10mM sodium citrate buffers, stir evenly, be placed in 2~8 DEG C extract 1~2 hour, leaching liquor centrifugal force 14000~ Under the conditions of 16000g centrifugation 40~collect supernatant after sixty minutes, after supernatant aseptic filtration the secondary extraction supernatant of FHA antigens；

(3) chromatography obtains the FHA antigens of purifying.

5. according to the method described in claim 4, it is characterized in that, the method for the chromatography described in step (3) is：

(1) the thick pure preparation of FHA：

With 10mM sodium citrate buffers, urea containing 2M (pH5.9~6.1) ion balance displacement chromatography column, loading FHA antigens two Secondary extraction supernatant；

With 10mM PB buffer solutions, urea containing 2M and 0.5% Triton X-100 (pH7.4~7.6) elute 6~9 cylinders Product；

10mM PB buffer solutions, urea containing 2M and 0.2~1.2M sodium chloride (pH7.4~7.6) elution are used again, and it is anti-to collect thick pure FHA Original solution；

(2) the consummate preparation of FHA：

With 10mM PB buffer solutions, urea containing 2M (pH6.9~7.1) balances hydroxyapatite (HA) chromatographic column；Loading step institute The thick pure FHA antigenic solutions stated, are balanced with same buffer solution to pH6.9~7.1, conductivity≤20mS/cm；

With 30mM PB buffer solutions, urea containing 2M and 0.5%Triton X-100 (pH6.9~7.1) elute 6~9 column volumes；

200mM PB buffer solutions, urea containing 2M and 0.2~0.5M sodium chloride (pH6.9~7.1) elution are used again, collect consummate FHA Antigenic solution.

6. method according to claim 1 or 2, which is characterized in that the isolation and purification method of the PRN antigens is：

(1) it extracts：5 times of volume (v/w) waters for injection are added in precipitating to the extraction to redissolve, 8M urea (w/v) is added and stirs 2 By 3 hours, mixing speed was 20~30rpm, and rear addition water for injection is diluted to the stirring of 2M urea and is stripped for 2 to 3 hours；

(2) it saltouts, supernatant is collected by centrifugation after extracting, by the 30% of supernatant volume, solid sulfur is added into supernatant Sour ammonium, mixing speed are 20~30rpm, and 2~8 DEG C stand 18~24 hours；

(3) secondary extraction：Go supernatant, precipitation that precipitation is collected after centrifugation after saltouing；Precipitation capacity (weight in wet base) 10 is added in sediment The 10mM PB buffer solutions (pH 6.9~7.1) of~15 times of volumes, mixing speed are 20~30rpm, are extracted 1 hour；It has extracted Finish, secondary extraction supernatant is collected by centrifugation, the secondary extraction supernatant of PRN antigens is obtained after aseptic filtration；

(4) chromatography obtains the PRN antigens of purifying.

7. according to the method described in claim 6, it is characterized in that, the method for the chromatography described in step (4) is：

(1) the thick pure preparation of PRN antigens：

With 10mM PB buffer solutions sodium chloride containing 1M (pH 6.9~7.1), hydrophobic chromatography column, the secondary extraction of loading PRN antigens are balanced Supernatant；

It is eluted containing 0~0.2M sodium chloride (pH 4.4~4.6) buffer solution with 10mM sodium-acetate buffers, collects thick pure PRN antigens Solution；

(2) the consummate preparation of PRN antigens：

With 10mM sodium-acetate buffers (pH4.9~5.1) ion balance displacement chromatography column, slightly pure PRN antigenic solutions are adjusted loading To pH4.9~5.1, conductivity≤5mS/cm；

It is eluted containing 0.02~1M sodium chloride (pH 4.9~5.1) with 10mM sodium-acetate buffers, collects consummate PRN antigenic solutions.