CN108047316A - The isolation and purification method of recombination hepatitis B core antigen - Google Patents

The isolation and purification method of recombination hepatitis B core antigen Download PDF

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CN108047316A
CN108047316A CN201810006942.9A CN201810006942A CN108047316A CN 108047316 A CN108047316 A CN 108047316A CN 201810006942 A CN201810006942 A CN 201810006942A CN 108047316 A CN108047316 A CN 108047316A
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filter wash
buffer solution
ultrafiltration
obtains
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CN108047316B (en
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李建强
任苏林
顾月
周童
孙洪林
葛君
戚凤春
鲍梦汝
韩杰
陈晓晓
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Nanjing Weixin Biological Medicine Co Ltd
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    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
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Abstract

The present invention relates to a kind of isolation and purification method of recombination hepatitis B core antigen, this method changes liquid comprising thermal denaturation clarification, ammonium sulfate precipitation, ultrafiltration concentration and filter wash, depolymerizes, first step sieve chromatography, ultrafiltration concentration, filter wash and changes liquid, multiple poly- and second step sieve chromatography.High-purity, low host residual, the recombination hepatitis B core antigen that grain structure is homogeneous, highly stable obtained by this method, i.e. this method have the characteristics that antigen purity is high, host protein and host nucleic acids residual are low, antigen particles are homogeneous and are easy to industrialization amplification.

Description

The isolation and purification method of recombination hepatitis B core antigen
Technical field
The invention belongs to protein separation fields, and in particular to a kind of side of isolating and purifying of recombination hepatitis B core antigen Method.
Background technology
Hepatitis B (Hepatitis B virus, HBV) is a kind of double-stranded DNA togavirus, it can cause all kinds of urgency slow Property hepatitis even liver cancer generation.According to World Health Organization, global about 2,000,000,000 people once infect HBV, and there are about 650,000 people every year Die of hepatic failure caused by HBV infection, hepatic sclerosis and liver cancer (Rehermann et al., Nat Rev Immunol, 2005； Michel et al.,Vaccine,2002；Jung et al.,Lancet Infect Dis,2002).
Nucleocapsid (nucleocapsid) and external cyst membrane (envelop) two of the HBV virions by center package nucleic acid Part forms.Nucleocapsid is made of core protein (HBcAg), and core protein shares 183 amino acid, can be divided into according to function 3 structural domains：N-terminal is used to build the package assembly domain (1~140aa) of capsid, for combine the C tails of RNA/DNA (150~ 183aa) with a bonding pad (141~149aa) being clipped in therebetween, wherein C tails, can due to being rich in arginine sequence Nonspecific a large amount of host nucleic acids of combination (Sominskaya et al., Plos one, 2013；Birnbaum et al., Jonrnal of Virology,1990).Core protein has been used for hepatitis diagnostics or therapeutic vaccine research and opens Among hair.
Host cell residual DNA has potential tumorigenesis and infection risk in biological products, so Drug Administration department of various countries It is very strict to the limitation requirement of DNA impurity, it is always the emphasis of domestic and international Drug Administration mechanism concern.
The detection of exogenous DNA residual volumes evaluates big event as vaccine safety, is subject to during vaccine assay Pay much attention to.European Union suggests in the purified product in passage cell source that DNA residual quantities limit is 10ng/ person-portions, version in 2010《In State's pharmacopeia》Regulation Hepatitis B Vaccine Prepared From Yeast Recombinanted, the DNA residual quantities of cytokine class product are that (Qiu is few for 10ng/ agent in (three) Brightness, Fang Xin, Zhao Ran etc., assessment technique and method, 2014).Core protein is in hepatitis diagnostics or therapeutic vaccine research Effect be increasingly taken seriously, the HBcAg of overall length or truncated-type is in Escherichia coli, Pichia pastoris, Hansenula yeast and elder brother at present It is overexpressed in worm cell, but due to its preparation process, obtained HBcAg is difficult to reach medicine in terms of residual substance Use quality standard.
The content of the invention
It is an object of the invention to overcome defect present in the production of recombinant core antigen well known in the prior art, A kind of isolation and purification method of recombination hepatitis B core antigen is provided, this method comprises the following steps：
A kind of isolation and purification method of recombination hepatitis B core antigen, this method comprises the following steps：
1) somatic cells after fermentation are taken, is resuspended with buffer solution after thalline and obtains starting bacteria suspension, smudge cells centrifuges To supernatant；
2) supernatant for obtaining step 1) is placed in 40~60 DEG C of water-baths, when concussion 0.5~2 is small under 50~200rpm, It is then centrifuged for obtaining supernatant；
3) ammonium sulfate is added dropwise in the supernatant obtained to step 2) so that the final concentration of ammonium sulfate maintains 10% ~30% saturation degree centrifuges after standing, precipitates 50~100mM Tris-HCl buffer solutions of the pH 8.0 with the NaCl containing 150mM It is resuspended, centrifugation obtains supernatant；
4) supernatant for obtaining step 3) is concentrated by ultrafiltration, and then carries out ultrafiltration filter wash and changes liquid, filter wash buffer solution is The 0.1M NaHCO of pH 9.5~10.83-Na2CO3Buffer solution, harvest reflux end sample, obtains filter wash and changes liquid sample；
5) filter wash obtained to step 4) changes 6~8M urea liquids that the addition of liquid sample includes 0~2mM DTT, makes antigen Concentration is maintained at 0.25~1.0mg/mL, and pH maintains 9.5~10.8, stirs depolymerization under 50~200rpm, obtain depolymerized sample；
6) depolymerized sample for obtaining step 5) carries out sieve chromatography, and the sieve chromatography resin is Sepharose 6FF or Sephacryl S-300, eluent are the 0.1M NaHCO of pH9.5~10.8 comprising 0~2mM DTT3-Na2CO3It is slow Fliud flushing collects dimer peak sample；
7) the dimer peak sample that step 6) obtains is concentrated by ultrafiltration, concentrate first uses the 0.1M of pH9.5~10.8 NaHCO3-Na2CO3Buffer solution carries out ultrafiltration filter wash, then to contain the 0.1M phosphorus of the pH 6.5~7.8 of 0.65~1.25M NaCl Phthalate buffer carries out ultrafiltration filter wash and changes liquid, and harvest reflux end sample obtains filter wash and changes liquid sample；
8) whipping step 7 under 50~200rpm) obtained filter wash changes liquid sample, obtain again poly- sample；
9) the multiple poly- sample for obtaining step 8) carries out sieve chromatography, and the sieve chromatography resin is Toyopearl HW65S or Toyopearl HW65F, eluent are 20~50mM phosphate-buffereds of the pH 7.2 of the NaCl containing 0.05~0.15M Liquid collects purpose antigen peak, obtains the recombination hepatitis B core antigen of purifying；
Preferably, the hepatitis B core antigen is overall length or truncated core antigen；
Preferably, the somatic cells are prokaryotic cell or eukaryocyte, such as the inferior ferment of Escherichia coli, Pichia pastoris, the Chinese Mother, saccharomyces cerevisiae, insect cell or Chinese Hamster egg mother cell.
Preferably, step 1) is as follows：The thalline after fermentation is taken, with the 50 of the pH7.5-8.5 of the NaCl containing 100~150mM~ 100mM Tris-HCl buffer solutions are resuspended, and solid content are made to maintain 10%-30%, acquisition starter bacteria is hanged after thalline is resuspended in buffer solution Liquid, in 1000~1800 bars of lower broken cell homogenates 4~6 times, until cell crashing ratio reaches 80~90%, then in 12000g, 4 30min, removal precipitation are centrifuged at DEG C, centrifugation obtains supernatant；Preferably, the Tris-HCl buffer solutions are NaCl containing 150mM PH8.0 50 or 100mM Tris-HCl buffer solutions；Preferably, solid content is made to maintain 20%.
Preferably, step 2) is as follows：The supernatant that step 1) is obtained is placed in 40~60 DEG C of water-baths, under 50~200rpm When concussion 0.5~2 is small, 30min is then centrifuged at 12000g, 4 DEG C and obtains supernatant；Preferably, step 1) is obtained upper Clear liquid is placed in 60 DEG C of water-baths；Preferably, when concussion 0.5 is small under 50~200rpm.
Preferably, step 3) is as follows：Ammonium sulfate is added dropwise in the supernatant obtained to step 2) so that ammonium sulfate Final concentration maintains 10%~30% saturation degree, stands 30min after stirring evenly at room temperature, is then centrifuged at 12000g, 4 DEG C 30min, 50~100mM Tris-HCl of the precipitation pH 8.0 of the NaCl containing 150mM of 1/5~1/3 starter bacteria suspension volume Buffer solution is resuspended, and 30min is centrifuged at 12000g, 4 DEG C and obtains supernatant；Preferably, maintain the final concentration of ammonium sulfate 20% saturation degree.
Preferably, step 4) is as follows：The supernatant molecular cut off that step 3) is obtained for 100~500kDa films bag into Row is concentrated by ultrafiltration 5-10 times, then continuously changes liquid with 3~5 times of volume filter wash buffer solution ultrafiltration filter washes, and filter wash buffer solution is pH 9.5~10.8 0.1M NaHCO3-Na2CO3Buffer solution, harvest reflux end sample, obtains filter wash and changes liquid sample；Preferably, institute The system inlets end pressure for stating ultrafiltration use is maintained at 10~30psi, and reflux end pressure is maintained at 5~10psi, transmembrane pressure control In 15~20psi；Preferably, it is that 300kDa film bags are concentrated by ultrafiltration with molecular cut off, and the film packaging material matter is polyethers Sulfone or regenerated cellulose；Preferably, the filter wash buffer solution is the 0.1M NaHCO of pH 9.53-Na2CO3Buffer solution.
Preferably, step 5) is as follows：The filter wash obtained to step 4) changes 6~8M that the addition of liquid sample includes 0~2mM DTT Urea liquid, makes antigen concentration be maintained at 0.25~1.0mg/mL, and pH maintains 9.5~10.8, solution is stirred under 50~200rpm It is poly- 3~16 it is small when, obtain the sample that depolymerizes.
Preferably, step 6) is as follows：The sample that depolymerizes that step 5) is obtained carries out sieve chromatography, the molecular sieve Chromatographic resin is Sepharose 6FF or Sephacryl S-300, and sample feeding volume is the 2%~5% of column volume, is eluted Liquid is the 0.1M NaHCO of the pH 9.5~10.8 comprising 0~2mM DTT3-Na2CO3Buffer solution collects dimer peak sample；It is excellent Selection of land, the sieve chromatography resin is Sepharose 6FF, and the eluent is the pH's 9.5 comprising 0~2mM DTT 0.1MNaHCO3-Na2CO3Buffer solution.
Preferably, step 7) is as follows：With molecular cut off it is 3-5kDa film bags by dimer peak sample that step 6) obtains It is concentrated by ultrafiltration, concentrate is first 300-500kDa films bag continuously with the volume of 10~20 times of concentrates with molecular cut off Filter wash buffer solution ultrafiltration filter wash, filter wash buffer solution are the 0.1M NaHCO of pH 9.5~10.83-Na2CO3Buffer solution, then to contain The 0.1M phosphate buffers of the pH 6.5~7.8 of 0.65~1.25M NaCl carry out ultrafiltration filter wash and change liquid, harvest reflux end sample Product obtain filter wash and change liquid sample；Preferably, the system inlets end pressure that the ultrafiltration uses is maintained at 10~30psi, and flow back side pressure Power is maintained at 5~10psi, and transmembrane pressure is controlled in 15~20psi；Preferably, the film packaging material matter is polyether sulfone or regenerated fiber Element；Preferably, it is that 3-5kDa films bag carries out ultrafiltration concentration 5-10 with molecular cut off by dimer peak sample that step 6) obtains Times；Preferably, ultrafiltration is carried out with the 0.1M phosphate buffers of the pH6.5 of the NaCl containing 1.25M and changes liquid.
Preferably, step 8) is as follows：In 4 DEG C~whipping step 7 under 50~200rpm at room temperature) obtained filter wash changes liquid sample Product are stayed overnight, and obtain again poly- sample；It is highly preferred that whipping step 7 under 50~200rpm at room temperature) obtained filter wash changes liquid sample mistake Night obtains again poly- sample.
Preferably, step 9) is as follows：The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography, the molecular sieve layer Analysis resin is Toyopearl HW65S or Toyopearl HW65F, and sample feeding volume is the 2%~5% of column volume, is eluted Liquid is 20~50mM phosphate buffers of the pH 7.2 of the NaCl containing 0.05~0.15M, collects purpose antigen peak, obtains purifying Recombination hepatitis B core antigen；Preferably, the sieve chromatography resin is Toyopearl HW65S.
A specific embodiment according to the present invention, the isolation and purification method of the recombination hepatitis B core antigen are included such as Lower step：
1) bacterial cell disruption
The thalline after fermentation is taken, with 50~100mM Tris-HCl, the pH7.5-8.5 bufferings of the NaCl containing 100~150mM Liquid is resuspended, and solid content is made to maintain 10%-30%, and buffer solution obtains starting bacteria suspension after thalline is resuspended, uses high pressure homogenizer 1000~1800 bars crush starting bacteria suspension 4~6 times repeatedly, until cell crashing ratio reaches 80~90%, 12000g, 4 DEG C centrifugation 30min, removal precipitation, obtains centrifuged supernatant.Wherein, Tris-HCl is preferably 50 or 100mM, NaCl are preferably 150mM, pH Preferably 8.0；Solid content is preferably 20%；
2) thermal denaturation is clarified
The supernatant that step 1) is obtained is placed in 40~60 DEG C of water-baths, when 50~200rpm concussions holding 0.5~2 is small. Then 12000g 4 DEG C, centrifuges 30min, collects supernatant, obtain centrifuged supernatant；It is preferred that in 60 DEG C of water-baths, 50~200rpm concussions Keep 0.5 it is small when；
3) ammonium sulfate precipitation is handled
Ammonium sulfate is added dropwise into the supernatant clarified solution that step 2) obtains so that the final concentration of ammonium sulfate maintains In 10%~30% saturation degree, 30min is stored at room temperature after stirring evenly, then 12000g, 4 DEG C of centrifugation 30min.It precipitates with 1/5 50~100mM Tris-HCl buffer solutions of the pH 8.0 of the NaCl containing 150mM of~1/3 starter bacteria suspension volume are resuspended, 12000g, 4 DEG C of centrifugation 30min, removal precipitation obtain supernatant；
4) it is concentrated by ultrafiltration and filter wash changes liquid
The supernatant that step 3) obtains is concentrated by ultrafiltration with molecular cut off for 100~500kDa film bags, film packaging material Matter is polyether sulfone or regenerated cellulose.After 5-10 times of concentration, continuously with the filter wash buffer solution ultrafiltration of the volume of 3~5 times of concentrates Filter wash changes liquid, and filter wash buffer solution is the 0.1M NaHCO of pH 9.5~10.83-Na2CO3Buffer solution, ultrafiltration system are protected into end pressure It holds in 10~30psi, reflux end pressure is maintained at 5~10psi, transmembrane pressure (Transmembrane Pressure, TMP) control In 15~20psi, harvest reflux end sample obtains filter wash and changes liquid sample；
5) depolymerize
The filter wash obtained to step 4) changes in liquid sample the 6~8M urea liquids for adding in final concentration of 0~2mM DTT, makes Antigen concentration is maintained at 0.25~1.0mg/mL, and pH maintains 9.5~10.8, stirred under 50~200rpm depolymerization 3~16 it is small when, Obtain the sample that depolymerizes；
6) first step sieve chromatography
The sample that depolymerizes that step 5) is obtained carries out sieve chromatography, and the sieve chromatography resin is Sepharose 6FF or Sephacryl S-300, sample feeding volume are the 2%~5% of column volume, eluent be comprising 0~ The 0.1M NaHCO of the pH 9.5~10.8 of 2mM DTT3-Na2CO3Buffer solution collects dimer peak, obtains dimer peak sample；
7) it is concentrated by ultrafiltration, filter wash and changes liquid
With molecular cut off it is that 3KDa 5kDa film bags are concentrated by ultrafiltration by dimer peak sample that step 6) obtains, Film packaging material matter is polyether sulfone or regenerated cellulose.Concentrate is 300kD 500kDa films bag continuously with 10 with molecular cut off The filter wash buffer solution ultrafiltration filter wash of the volume of~20 times of concentrates, filter wash buffer solution are the 0.1M NaHCO of pH 9.5~10.83- Na2CO3Then buffer solution is washed with containing the progress ultrafiltration of the 0.1M phosphate buffers of the pH 6.5~7.8 of 0.65~1.25M NaCl Liquid is changed in filter, and ultrafiltration system is into end pressure PfIt is maintained at 10~30psi, reflux end pressure PrIt is maintained at 5~10psi, transmembrane pressure (Transmembrane Pressure, TMP) control obtains filter wash and changes liquid sample in 15~20psi, harvest reflux end sample；
8) it is multiple poly-
The filter wash that step 7) is obtained changes liquid sample and is placed on 4 DEG C~room temperature, is stirred under 50~200rpm, multiple overnight poly-, Obtain again poly- sample；
9) second step sieve chromatography
The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography, and the sieve chromatography resin is Toyopearl HW65S or Toyopearl HW65F, sample feeding volume be column volume 2%~5%, eluent be containing The 20mM phosphate buffers (PB) of the pH 7.2 of 0.05~0.15M NaCl collect purpose antigen peak, the restructuring purified Hepatitis B core antigen.
Above steps carries out in 2~30 DEG C of temperature ranges.
Wherein, the first step sieve chromatography is used to separate dimer；The second step sieve chromatography is used to separate Virus-like particle after gathering again.
In some embodiments of the present invention, the hepatitis B core antigen is the recombinant hepatitis B virus core antigen class of overall length Virion or truncated recombinant hepatitis B virus core antigen viruslike particle and using hepatitis B core antigen as carrier platform Other restructuring viruslike particles.Hepatitis B virus core antigen for example can be the core antigen of 183 amino acid of overall length, also may be used To be truncated core antigen, such as complete virus-like particle, length can be assembled into and resisted for the core of 143 amino acid It is former.
In some embodiments of the present invention, the recombination hepatitis B core antigen expression host cell is prokaryotic cell or true Nucleus, such as Escherichia coli, Pichia pastoris, Hansenula yeast, saccharomyces cerevisiae, insect cell, Chinese Hamster egg mother cell (CHO) Deng.
The recombinant hepatitis B virus core antigen viruslike particle obtained using method provided by the present invention has following special Point：
1) high-purity：SDS-PAGE purity >=98%, CE-SDS purity >=99%, HPLC purity >=99%, host protein Residual quantity≤0.01%；
2) low nucleic acid residual：Dot hybridization shows host nucleic acids residual≤2.5ng/100 μ g antigens；Residual quantity complies fully with 《Chinese Pharmacopoeia》In similar biological products requirement；
3) virus-like particle homogeneity is high：Analytic type ultracentrifugation, dynamic light scattering show the antigen between different batches Grain sedimentation coefficient and grain diameter are stablized, and homogeneity is high；
4) virus-like particle thermal stability is high：Virus-like particle polymerize compact stabilization, the analysis disintegration of differential calorimetric scan instrument For temperature at 90 DEG C or so, antigen particles thermal stability is high；
5) purification process step is few, is connected compact, simple production process, amplifies suitable for industrialization.
The present invention provides it is a kind of it is easy, quick, be easy to isolating and purifying overall length or truncating recombinant hepatitis B virus for amplification Core antigen viruslike particle or the method for recombinating viruslike particles using other of hepatitis B core antigen as carrier platform, should Method can obtain high-purity, low host residual, the recombination hepatitis B core antigen that grain structure is homogeneous, highly stable, have antigen Purity is high, host protein and host nucleic acids residual are low, antigen particles are homogeneous and the features such as being easy to industrialization amplification, has larger reality Border application value.
Description of the drawings
Fig. 1 is first step sieve chromatography collection of illustrative plates in embodiment 1；
Fig. 2 is second step sieve chromatography collection of illustrative plates in embodiment 1；
Fig. 3 is first step sieve chromatography collection of illustrative plates in embodiment 4；
Fig. 4 is second step sieve chromatography collection of illustrative plates in embodiment 4；
Fig. 5 is the SDS-PAGE collection of illustrative plates of the product after purification of embodiment 3；
Fig. 6 is Capillary Electrophoresis (CE-SDS) collection of illustrative plates of the product after purification of embodiment 3；
Fig. 7 is the SEC-HPLC collection of illustrative plates of the product after purification of embodiment 3；
Fig. 8 is transmission electron microscope (TEM) collection of illustrative plates of the product after purification of embodiment 3；
Fig. 9 is dynamic light scattering (DLS) analysis result of the product after purification of embodiment 3；
Figure 10 be embodiment 3 after purification product differential calorimetric scan (DSC) analyze collection of illustrative plates；
Figure 11 be embodiment 3 after purification product analytic type ultracentrifugation (AUC) analyze collection of illustrative plates.
Specific embodiment
It is further illustrated the present invention with reference to embodiment and attached drawing, it should be understood that embodiment is only used for further illustrating It is of the invention with illustrating, it is not intended to limit the present invention.
Unless otherwise defined, in this specification in relation to technology and term and the those skilled in the art of science lead to What is understood is equivalent in meaning.Although it can be applied and the similar or identical method and material around here in experiment or practical application Material, herein still hereinafter describes material and method.In the case that conflicting, it is wherein fixed to be included with this specification Subject to justice, in addition, material, method and example only supply explanation, and it is without limitation.
The purifying of 1 recombination hepatitis B core antigen of embodiment
1) bacterial cell disruption
The thalline after fermentation is taken, is resuspended with 8.0 buffer solution of 50mM Tris-HCl, pH of the NaCl containing 150mM, solid content 10%-30% is maintained, obtains starting bacteria suspension, crushes starting bacteria suspension repeatedly using high pressure homogenizer 1500bar 6 times, until Cell crashing ratio reaches 80~90%；12000g, 4 DEG C of centrifugation 30min, removal precipitation obtain supernatant.
2) thermal denaturation is clarified
The supernatant that step 1) is obtained is placed in 60 DEG C of water-baths, when 50~200rpm concussions holding 0.5~2 is small.Then 12000g 4 DEG C, centrifuges 30min, collects supernatant, obtain supernatant；
3) ammonium sulfate precipitation is handled
Ammonium sulfate is added dropwise in the supernatant obtained to step 2), ammonium sulfate final concentration maintains 25% saturation Degree, it is to be mixed uniformly after be stored at room temperature 30min, then 12000g, 4 DEG C of centrifugation 30min.1/5~1/3 starting bacteria suspension of precipitation The 50mMTris-HCl buffer solutions of the pH 8.0 of the NaCl containing 150mM of volume are resuspended, in 12000g, 4 DEG C of centrifugation 30min, removal Precipitation, obtains supernatant；
4) it is concentrated by ultrafiltration and filter wash changes liquid
The supernatant that step 3) obtains is concentrated by ultrafiltration with molecular cut off for 100~500kDa film bags, film packaging material Matter is polyether sulfone or regenerated cellulose.After 5-10 times of concentration, continuously with the filter wash buffer solution ultrafiltration of the volume of 3~5 times of concentrates Filter wash changes liquid, and filter wash buffer solution is the 0.1MNaHCO of pH 9.53-Na2CO3Buffer solution, ultrafiltration system is into end pressure PfIt is maintained at 10~30psi, reflux end pressure Pr5~10psi is maintained at, transmembrane pressure control is obtained in 15~20psi, harvest reflux end sample Liquid sample is changed to filter wash；
5) depolymerize
The filter wash obtained to step 4), which is changed in liquid sample, adds in final concentration of 6M urea liquids, and antigen concentration is maintained at 0.25mg/mL, pH maintain 9.5, stirred under 50~200rpm depolymerization 3~16 it is small when, obtain the sample that depolymerizes；
6) first step sieve chromatography
The sample that depolymerizes that step 5) is obtained carries out sieve chromatography (GE Healthcare, XK26/100, CV= 475ml), the sieve chromatography resin is Sepharose 6FF, and sample feeding volume is the 2%~5% of column volume, is washed The 0.1M NaHCO that de- liquid is pH 9.53-Na2CO3Buffer solution collects dimer peak, obtains dimer peak sample (Fig. 1)；
7) it is concentrated by ultrafiltration, filter wash and changes liquid
The dimer peak sample that step 6) obtains is concentrated by ultrafiltration with molecular cut off 3kDa or 5kDa film bag, film Packaging material matter is polyether sulfone or regenerated cellulose.Concentrate with molecular cut off be 300kD 500kDa films bag continuously with 10~ The filter wash buffer solution ultrafiltration filter wash of the volume of 20 times of concentrates, filter wash buffer solution are the 0.1M NaHCO of pH 9.53-Na2CO3It is slow Fliud flushing.Then ultrafiltration changes liquid into the 0.1M phosphate buffers of the pH 6.5 of the NaCl containing 1.25M, ultrafiltration system import side pressure Power is maintained at 10~30psi, and reflux end pressure is maintained at 5~10psi, and transmembrane pressure control is in 15~20psi, harvest reflux end sample Product obtain filter wash and change liquid sample；
8) it is multiple poly-
The filter wash that step 7) is obtained changes liquid sample and is placed on 4 DEG C~room temperature, is stirred under 50~200rpm, multiple overnight poly-, Obtain again poly- sample；
9) second step sieve chromatography
The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography (GE Healthcare, XK26/100, CV= 460ml), the sieve chromatography resin is Toyopearl HW65S, and sample feeding volume is the 2%~5% of column volume, Eluent is the 20mM phosphate buffers of the pH 7.2 of the NaCl containing 0.15M, collects purpose antigen peak, the restructuring purified Hepatitis B core antigen (Fig. 2).
The purifying of 2 recombination hepatitis B core antigen of embodiment
1) bacterial cell disruption
The thalline after fermentation is taken, is resuspended with 8.0 buffer solution of 50mM Tris-HCl, pH of the NaCl containing 150mM, solid content 10%-30% is maintained, obtains starting bacteria suspension, crushes starting bacteria suspension repeatedly using high pressure homogenizer 1500bar 6 times, until Cell crashing ratio reaches 80~90%；12000g, 4 DEG C of centrifugation 30min, removal precipitation obtain supernatant.
2) thermal denaturation is clarified
The supernatant that step 1) is obtained is placed in 60 DEG C of water-baths, when 50~200rpm concussions holding 0.5~2 is small.Then 12000g 4 DEG C, centrifuges 30min, collects supernatant, obtain supernatant；
3) ammonium sulfate precipitation is handled
Ammonium sulfate is added dropwise in the supernatant obtained to step 2), ammonium sulfate final concentration maintains 20% saturation Degree, it is to be mixed uniformly after be stored at room temperature 30min, then 12000g, 4 DEG C of centrifugation 30min.1/5~1/3 starting bacteria suspension of precipitation The 100mMTris-HCl buffer solutions of the pH 8.0 of the NaCl containing 150mM of volume are resuspended, and are gone in 12000g, 4 DEG C of centrifugation 30min Except precipitation, supernatant is obtained；
4) it is concentrated by ultrafiltration and filter wash changes liquid
The supernatant that step 3) obtains is concentrated by ultrafiltration with molecular cut off for 100~500kDa film bags, film packaging material Matter is polyether sulfone or regenerated cellulose.After 5-10 times of concentration, continuously with the filter wash buffer solution ultrafiltration of the volume of 3~5 times of concentrates Filter wash changes liquid, and filter wash buffer solution is the 0.1MNaHCO of pH 9.53-Na2CO3Buffer solution, ultrafiltration system is into end pressure PfIt is maintained at 10~30psi, reflux end pressure Pr5~10psi is maintained at, transmembrane pressure is controlled in 15~20psi；Harvest reflux end sample, obtains Liquid sample is changed to filter wash；
5) depolymerize
The filter wash obtained to step 4), which is changed in liquid sample, adds in final concentration of 6M urea liquids, and antigen concentration is maintained at 0.5mg/mL, pH maintain 9.5, stirred under 50~200rpm depolymerization 3~16 it is small when, obtain the sample that depolymerizes；
6) first step sieve chromatography
The sample that depolymerizes that step 5) is obtained carries out sieve chromatography (GE Healthcare, XK26/100, CV= 475ml), the sieve chromatography resin is Sepharose 6FF, and sample feeding volume is the 2%~5% of column volume, is washed The 0.1M NaHCO that de- liquid is pH 9.53-Na2CO3Buffer solution collects dimer peak, obtains dimer peak sample；
7) it is concentrated by ultrafiltration, filter wash and changes liquid
The dimer peak sample that step 6) obtains is concentrated by ultrafiltration with molecular cut off 3kDa or 5kDa film bag, film Packaging material matter is polyether sulfone or regenerated cellulose.Concentrate with molecular cut off be 300kD 500kDa films bag continuously with 10~ The filter wash buffer solution ultrafiltration filter wash of the volume of 20 times of concentrates, filter wash buffer solution are the 0.1M NaHCO of pH 9.53-Na2CO3It is slow Fliud flushing.Then ultrafiltration changes liquid into the 0.1M phosphate buffers of the pH 6.5 of the NaCl containing 1.25M, ultrafiltration system import side pressure Power is maintained at 10~30psi, and reflux end pressure is maintained at 5~10psi, and transmembrane pressure control is in 15~20psi, harvest reflux end sample Product obtain filter wash and change liquid sample；
8) it is multiple poly-
The filter wash that step 7) is obtained changes liquid sample and is placed on 4 DEG C~room temperature, is stirred under 50~200rpm, multiple overnight poly-, Obtain again poly- sample；
9) second step sieve chromatography
The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography (GE Healthcare, XK26/100, CV= 460ml), the sieve chromatography resin is Toyopearl HW65S, and sample feeding volume is the 2%~5% of column volume, Eluent is the 20mM phosphate buffers of the pH 7.2 of the NaCl containing 0.05M, collects purpose antigen peak, obtains the restructuring second of purifying Liver core antigen.
The purifying of 3 recombination hepatitis B core antigen of embodiment
1) bacterial cell disruption
The thalline after fermentation is taken, is resuspended with 8.0 buffer solution of 50mM Tris-HCl, pH of the NaCl containing 150mM, solid content 10%-30% is maintained, obtains starting bacteria suspension, is crushed repeatedly using high pressure homogenizer 1800bar and obtains starting bacteria suspension 4 It is secondary, until cell crashing ratio reaches 80~90%；12000g, 4 DEG C of centrifugation 30min, removal precipitation obtain supernatant.
2) thermal denaturation is clarified
The supernatant that step 1) is obtained is placed in 50 DEG C of water-baths, when 50~200rpm concussions holding 0.5~2 is small.Then 12000g 4 DEG C, centrifuges 30min, collects supernatant, obtain supernatant；
3) ammonium sulfate precipitation is handled
Ammonium sulfate is added dropwise in the supernatant obtained to step 2), ammonium sulfate final concentration maintains 30% saturation Degree, it is to be mixed uniformly after be stored at room temperature 30min, then 12000g, 4 DEG C of centrifugation 30min.1/5~1/3 starting bacteria suspension of precipitation The 50mMTris-HCl buffer solutions of the pH 8.0 of the NaCl containing 150mM of volume are resuspended, in 12000g, 4 DEG C of centrifugation 30min, removal Precipitation, obtains supernatant；
4) it is concentrated by ultrafiltration and filter wash changes liquid
The supernatant that step 3) obtains is concentrated by ultrafiltration with molecular cut off for 100~500kDa film bags, film packaging material Matter is polyether sulfone or regenerated cellulose.After 5-10 times of concentration, continuously with the filter wash buffer solution ultrafiltration of the volume of 3~5 times of concentrates Filter wash changes liquid, and filter wash buffer solution is the 0.1MNaHCO of pH 9.53-Na2CO3Buffer solution, ultrafiltration system is into end pressure PfIt is maintained at 10~30psi, reflux end pressure Pr5~10psi is maintained at, transmembrane pressure is controlled in 15~20psi；Harvest reflux end sample, obtains Liquid sample is changed to filter wash；
5) depolymerize
The filter wash obtained to step 4), which is changed in liquid sample, adds in final concentration of 8M urea liquids, and antigen concentration is maintained at 1mg/ ML, pH maintain 9.5, stirred under 50~200rpm depolymerization 3~16 it is small when, must depolymerize sample；
6) first step sieve chromatography
By step 5) depolymerize sample carry out sieve chromatography (GE Healthcare, XK26/100, CV=475ml), institute The sieve chromatography resin stated is Sepharose 6FF, and sample feeding volume is the 2%~5% of column volume, eluent pH 9.5 0.1M NaHCO3-Na2CO3Buffer solution collects dimer peak, obtains dimer peak sample；
7) it is concentrated by ultrafiltration, filter wash and changes liquid
The dimer peak sample that step 6) obtains is concentrated by ultrafiltration with molecular cut off 3kDa or 5kDa film bag, film Packaging material matter is polyether sulfone or regenerated cellulose.Concentrate with molecular cut off be 300kD 500kDa films bag continuously with 10~ The filter wash buffer solution ultrafiltration filter wash of the volume of 20 times of concentrates, filter wash buffer solution are the 0.1M NaHCO of pH 9.53-Na2CO3It is slow Fliud flushing.Then ultrafiltration changes liquid into the 0.1M phosphate buffers of the pH 7.8 of the NaCl containing 0.65M, ultrafiltration system import side pressure Power is maintained at 10~30psi, and reflux end pressure is maintained at 5~10psi, and transmembrane pressure control is in 15~20psi, harvest reflux end sample Product obtain filter wash and change liquid sample；
8) it is multiple poly-
Product are varied into the filter wash filter that step 7) obtains and are placed on 4 DEG C~room temperature, are stirred under 50~200rpm, it is multiple overnight poly-, Obtain again poly- sample；
9) second step sieve chromatography
The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography (GE Healthcare, XK26/100, CV= 460ml), the sieve chromatography resin is Toyopearl HW65S, and sample feeding volume is the 2%~5% of column volume, Eluent is the 20mM phosphate buffers of the pH 7.2 of the NaCl containing 0.05M, collects purpose antigen peak, the restructuring purified Hepatitis B core antigen.
The purifying of 4 recombination hepatitis B core antigen of embodiment
1) bacterial cell disruption
The thalline after fermentation is taken, is resuspended with 8.0 buffer solution of 100mM Tris-HCl, pH of the NaCl containing 150mM, solid content 10%-30% is maintained, obtains starting bacteria suspension, is crushed repeatedly using high pressure homogenizer 1000bar and obtains starting bacteria suspension 6 It is secondary, until cell crashing ratio reaches 80~90%；12000g, 4 DEG C of centrifugation 30min, removal precipitation obtain supernatant.
2) thermal denaturation is clarified
The supernatant that step 1) is obtained is placed in 40 DEG C of water-baths, when 50~200rpm concussions holding 0.5~2 is small.Then 12000g 4 DEG C, centrifuges 30min, collects supernatant, obtain supernatant；
3) ammonium sulfate precipitation is handled
Ammonium sulfate is added dropwise in the supernatant obtained to step 2), ammonium sulfate final concentration maintains 10% saturation Degree, it is to be mixed uniformly after be stored at room temperature 30min, then 12000g, 4 DEG C of centrifugation 30min.1/5~1/3 starting bacteria suspension of precipitation The 20mMTris-HCl buffer solutions of the pH 8.0 of the NaCl containing 150mM of volume are resuspended, in 12000g, 4 DEG C of centrifugation 30min, removal Precipitation, obtains centrifuged supernatant；
4) it is concentrated by ultrafiltration and filter wash changes liquid
The supernatant that step 3) obtains is concentrated by ultrafiltration with molecular cut off for 100~500kDa film bags, film packaging material Matter is polyether sulfone or regenerated cellulose.After 5-10 times of concentration, continuously with the filter wash buffer solution ultrafiltration of the volume of 3~5 times of concentrates Filter wash changes liquid, and filter wash buffer solution is the 0.1MNaHCO of pH 10.83-Na2CO3Buffer solution, ultrafiltration system is into end pressure PfIt is maintained at 10~30psi, reflux end pressure Pr5~10psi is maintained at, transmembrane pressure control is obtained in 15~20psi, harvest reflux end sample Liquid sample is changed to filter wash；
5) depolymerize
The filter wash obtained to step 4), which is changed in liquid sample, adds in final concentration of 2mM DTT, 8M urea liquids, and antigen concentration is protected Hold in 1mg/mL, pH maintains 10.8, stirred under 50~200rpm depolymerization 3~16 it is small when, obtain the sample that depolymerizes；
6) first step sieve chromatography
The sample that depolymerizes that step 5) is obtained carries out sieve chromatography (GE Healthcare, XK16/100, CV= 185ml), the sieve chromatography resin is Sephacryl S-300, and sample feeding volume is the 2%~5% of column volume, Eluent is the 0.1M NaHCO of the pH 10.8 of the DTT containing 2mM3-Na2CO3Buffer solution collects dimer peak, obtains dimer peak Sample (Fig. 3)；
7) it is concentrated by ultrafiltration, filter wash and changes liquid
The dimer peak sample that step 6) obtains is concentrated by ultrafiltration with molecular cut off 3kDa or 5kDa film bag, film Packaging material matter is polyether sulfone or regenerated cellulose.Concentrate with molecular cut off be 300kD 500kDa films bag continuously with 10~ The filter wash buffer solution ultrafiltration filter wash of the volume of 20 times of concentrates, filter wash buffer solution are the 0.1M NaHCO of pH 10.83-Na2CO3It is slow Fliud flushing.Then ultrafiltration changes liquid into the 0.1M phosphate buffers of the pH 7.8 of the NaCl containing 0.65M, ultrafiltration system import side pressure Power is maintained at 10~30psi, and reflux end pressure is maintained at 5~10psi, and transmembrane pressure control is in 15~20psi, harvest reflux end sample Product obtain filter wash and change liquid sample；
8) it is multiple poly-
The filter wash that step 7) is obtained changes liquid sample and is placed on 4 DEG C~room temperature, is stirred under 50~200rpm, multiple overnight poly-, Obtain again poly- sample；
9) second step sieve chromatography
The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography (Millipore, Vantage-L ColVL32x 1000, CV=676ml), the sieve chromatography resin is Toyopearl HW65F, and sample feeding volume is column volume 2%~5%, eluent is the 20mM phosphate buffers (Fig. 4) of the pH 7.2 of the NaCl containing 0.15M, collects purpose antigen peak, The recombination hepatitis B core antigen that must be purified.
5 recombination hepatitis B core antigen purity analysis of embodiment
1) SDS-PAGE electrophoresis
Obtained expressed by Hansenula yeast recombination hepatitis B core antigen is purified to embodiment 3 and carries out SDS-PAGE electrophoretic analysis, As shown in figure 5, SDS-PAGE electrophoresis gray analysis shows antigen purity >=98%.
2) CE-SDS is analyzed
Obtained expressed by Hansenula yeast recombination hepatitis B core antigen is purified to embodiment 3 and carries out Capillary Electrophoresis CE-SDS, Electrophoresis pattern is as shown in fig. 6, antigen purity >=99% (table 1).
The reduction CE-SDS testing results of 1 sample of table
Sample lot number Degradable component (%) Main peak (%) Aggressiveness (%) Purity (%)
P201611005 0.23 95.86 3.73 99.59
3) SEC-HPLC is detected
Efficient molecular-exclusion chromatography (SEC-HPLC, TSKG5000PWxl chromatography is carried out to the final sample that embodiment 3 purifies Column, 300mm × 7.8mm) analysis, recombination hepatitis B core antigen purity is analyzed by integrating peak areas and reaches more than 99% (Fig. 7).
6 recombination hepatitis B core antigen host's retention analysis of embodiment
1) host protein remains
Obtained different batches sample is purified by enzyme-linked immunization to embodiment 3 to be detected, host protein residual is equal ＜ 0.01% (table 2)；
The host protein residue detection result of 2 different batches sample of table
Lot number Host protein residual quantity (%, μ g/ μ g)
FC20170501P201706002 <0.0037
FC20170602P201706006 <0.0025
FC20170603P201707007 0.0024
FC20170704P201707018 0.0019
2) host nucleic acids remain
The host nucleic acids residual of 3 purification of samples of embodiment is detected by dot hybridization, the core of multiple purification batch Sour residual quantity≤2.5ng/100 μ g antigens (table 3)；
The host nucleic acids residual mark hybridization check result of 3 different batches sample of table
Lot number Nucleic acid remains (ng/100 μ g HBcAg)
FC20170806P201708017 1.0
FC20170806P201708062 2.5
FC20170807P201709004 2.5
7 recombination hepatitis B core antigen particles property analysis of embodiment
1) transmission electron microscope (TEM) is analyzed
Transmission electron microscope (Transmission Electron Microscope, TEM) is former according to electron-optical Reason, replaces light beam and optical lens with electron beam and electron lens, makes the fine structure of substance under very high amplification factor The instrument of imaging.Recombination hepatitis B core antigen is obtained to the purifying of embodiment 3 and carries out TEM (Hitachi, HT7700) analyses, such as Fig. 8 Shown, hepatitis B core antigen can form virus-like particle and particle is homogeneous.
2) dynamic scattering analysis (Dynamic Light Scattering, DLS)
Dynamic light scattering technique can be used for the size of measurement particles in solution and distribution, measurement range to be usually 1nm-1 μ M includes the size scale of virus-like particle in vaccine, therefore can be with dynamic light scattering technique to the virus-like in vaccinogen liquid Particle is detected, and can obtain the granular size in solution and distribution, so as to reflect the particle purity of vaccine.It is pure to embodiment 3 Change obtains recombination hepatitis B core antigen and carries out DLS (Malvern, Zetasizer Nano ZS) analysis shows that antigen particles are homogeneous, Assembling is completely (Fig. 9).
3) differential scanning calorimetry (Differential Scanning Calorimetry, DSC)
Differential canning calorimetry is a kind of physical property thermal change of measurement of species under temperature programmed control and the change of enthalpy Change a kind of technology varied with temperature.The stability of melting temperature reflection albumen in this state, Tm is higher, and albumen is more stable； Conversely, the easier enthalpy change that occurs of albumen becomes another state, it is also more unstable.Recombination hepatitis B core is obtained to the purifying of embodiment 3 Heart antigen carries out DSC (GE Healthcare, MicroCal VP-DSC) and analyzes, the result is shown in Figure 10, it is shown that recombination hepatitis B core The good thermal stability of heart antigen, at about 91 DEG C, depolymerization takes place in core antigen, then occurs with the rise that continues of temperature The phenomenon that aggregation.
4) analytic type ultracentrifugal analysis (Analytical Ultra-centrifugation, AUC)
Analytic type ultra centrifugation techniques are for studying a kind of technology of Proteins In Aqueous Solutions property and coherent condition.From The settling phase of large biological molecule is observed in heart gravitational field, it is analytic type to obtain property of the protein under native state The characteristics of ultra centrifugation techniques.AUC can not only provide the relative molecular mass of protein, but also can differentiate protein point The homogeneity of son.Recombination hepatitis B core antigen is obtained to the purifying of embodiment 3 and carries out AUC (Beckman, ProteomeLab XL-I) Analysis shows that antigen particles are homogeneous (Figure 11).Although present invention has been a degree of descriptions, it will be apparent that, do not departing from this Under conditions of the spirit and scope of invention, the appropriate variation of each condition can be carried out.It is appreciated that the invention is not restricted to the realities Scheme is applied, and is attributed to the scope of claim, includes the equivalent substitution of each factor.

Claims (10)

1. a kind of isolation and purification method of recombination hepatitis B core antigen, this method comprises the following steps：
1) somatic cells after fermentation are taken, is resuspended with buffer solution after thalline and obtains starting bacteria suspension, smudge cells, centrifugation obtains Clear liquid；
2) supernatant for obtaining step 1) is placed in 40~60 DEG C of water-baths, when concussion 0.5~2 is small under 50~200rpm, then Centrifugation obtains supernatant；
3) ammonium sulfate is added dropwise in the supernatant obtained to step 2) so that and the final concentration of ammonium sulfate maintains 10%~ 30% saturation degree centrifuges after standing, precipitates 50~100mM Tris-HCl buffer solution weights with the pH 8.0 of the NaCl containing 150mM Outstanding, centrifugation obtains supernatant；
4) supernatant for obtaining step 3) is concentrated by ultrafiltration, and then carries out ultrafiltration filter wash and changes liquid, filter wash buffer solution is pH 9.5~10.8 0.1M NaHCO3-Na2CO3Buffer solution, harvest reflux end sample, obtains filter wash and changes liquid sample；
5) filter wash obtained to step 4) changes 6~8M urea liquids that the addition of liquid sample includes 0~2mM DTT, makes antigen concentration 0.25~1.0mg/mL is maintained at, pH maintains 9.5~10.8, stirs depolymerization under 50~200rpm, obtain depolymerized sample；
6) depolymerized sample for obtaining step 5) carries out sieve chromatography, and the sieve chromatography resin is Sepharose 6FF Or Sephacryl S-300, eluent are the 0.1M NaHCO of the pH 9.5~10.8 comprising 0~2mM DTT3-Na2CO3Buffering Liquid collects dimer peak sample；
7) the dimer peak sample that step 6) obtains is concentrated by ultrafiltration, concentrate first uses the 0.1M of pH 9.5~10.8 NaHCO3-Na2CO3Buffer solution carries out ultrafiltration filter wash, then to contain the 0.1M phosphorus of the pH 6.5~7.8 of 0.65~1.25M NaCl Phthalate buffer carries out ultrafiltration filter wash and changes liquid, and harvest reflux end sample obtains filter wash and changes liquid sample；
8) whipping step 7 under 50~200rpm) obtained filter wash changes liquid sample, obtain again poly- sample；
9) the multiple poly- sample for obtaining step 8) carries out sieve chromatography, and the sieve chromatography resin is Toyopearl HW65S or Toyopearl HW65F, eluent are 20~50mM phosphate-buffereds of the pH 7.2 of the NaCl containing 0.05~0.15M Liquid collects purpose antigen peak, obtains the recombination hepatitis B core antigen of purifying；
Preferably, the hepatitis B core antigen is overall length or truncated core antigen；
Preferably, the somatic cells are prokaryotic cell or eukaryocyte, such as Escherichia coli, Pichia pastoris, Hansenula yeast, wine Brewer yeast, insect cell or Chinese Hamster egg mother cell.
2. isolation and purification method as described in claim 1, wherein, step 1) is as follows：
The thalline after fermentation is taken, with 50~100mM Tris-HCl buffer solution weights of the pH7.5-8.5 of the NaCl containing 100~150mM It is outstanding, solid content is made to maintain 10%-30%, buffer solution obtains starting bacteria suspension after thalline is resuspended, even under 1000~1800 bars It starches smudge cells 4~6 times, until cell crashing ratio reaches 80~90%, 30min is then centrifuged at 12000g, 4 DEG C, removal is heavy It forms sediment, centrifugation obtains supernatant；
Preferably, the Tris-HCl buffer solutions are the 50 or 100mM Tris-HCl buffer solutions of the pH8.0 of the NaCl containing 150mM；
Preferably, solid content is made to maintain 20%.
3. isolation and purification method as claimed in claim 1 or 2, wherein, step 2) is as follows：
The supernatant that step 1) is obtained is placed in 40~60 DEG C of water-baths, when concussion 0.5~2 is small under 50~200rpm, Ran Hou 12000g, centrifugation 30min obtains supernatant at 4 DEG C；
Preferably, supernatant step 1) obtained is placed in 60 DEG C of water-baths；Preferably, when concussion 0.5 is small under 50~200rpm.
4. isolation and purification method as claimed any one in claims 1 to 3, wherein, step 3) is as follows：
Ammonium sulfate is added dropwise in the supernatant obtained to step 2) so that the final concentration of ammonium sulfate maintains 10%~30% Saturation degree stands 30min at room temperature after stirring evenly, 30min is then centrifuged at 12000g, 4 DEG C, precipitates with 1/5~1/3 50~100mM Tris-HCl buffer solutions of the pH 8.0 of the NaCl containing 150mM of beginning bacteria suspension volume are resuspended, 12000g, 4 DEG C Lower centrifugation 30min obtains supernatant；
Preferably, the final concentration of ammonium sulfate is made to maintain 20% saturation degree.
5. isolation and purification method according to any one of claims 1 to 4, wherein, step 4) is as follows：
The supernatant that step 3) obtains is subjected to 5-10 times of ultrafiltration concentration with molecular cut off for 100~500kDa films bag, then Liquid is continuously changed with the filter wash buffer solution ultrafiltration filter wash of the volume of 3~5 times of concentrates, filter wash buffer solution is pH's 9.5~10.8 0.1M NaHCO3-Na2CO3Buffer solution, harvest reflux end sample, obtains filter wash and changes liquid sample；
Preferably, the system inlets end pressure that the ultrafiltration uses is maintained at 10~30psi, and reflux end pressure is maintained at 5~ 10psi, transmembrane pressure are controlled in 15~20psi；
Preferably, it is that 300kDa film bags are concentrated by ultrafiltration with molecular cut off, and the film packaging material matter is polyether sulfone or regeneration Cellulose；
Preferably, the filter wash buffer solution is the 0.1M NaHCO of pH 9.53-Na2CO3Buffer solution.
6. the isolation and purification method as any one of claim 1 to 5, wherein, step 5) is as follows：
The filter wash obtained to step 4) changes 6~8M urea liquids that the addition of liquid sample includes 0~2mM DTT, protects antigen concentration Hold in 0.25~1.0mg/mL, pH maintains 9.5~10.8, stirred under 50~200rpm depolymerization 3~16 it is small when, depolymerized Sample.
7. such as isolation and purification method according to any one of claims 1 to 6, wherein, step 6) is as follows：
The sample that depolymerizes that step 5) is obtained carries out sieve chromatography, and the sieve chromatography resin is Sepharose 6FF or Sephacryl S-300, sample feeding volume are the 2%~5% of column volume, and eluent is to include 0~2mM DTT's The 0.1M NaHCO of pH 9.5~10.83-Na2CO3Buffer solution collects dimer peak sample；
Preferably, the sieve chromatography resin is Sepharose 6FF, and the eluent is the pH for including 0~2mM DTT 9.5 0.1MNaHCO3-Na2CO3Buffer solution.
8. the isolation and purification method as any one of claim 1 to 7, wherein, step 7) is as follows：
With molecular cut off it is that 3-5kDa film bags are concentrated by ultrafiltration by dimer peak sample that step 6) obtains, concentrate is first used Molecular cut off is 300-500kDa films bag continuously with the filter wash buffer solution ultrafiltration filter wash of the volume of 10~20 times of concentrates, filter wash Buffer solution is the 0.1M NaHCO of pH 9.5~10.83-Na2CO3Buffer solution, then to contain the pH of 0.65~1.25M NaCl 6.5~7.8 0.1M phosphate buffers carry out ultrafiltration filter wash and change liquid, and harvest reflux end sample obtains filter wash and changes liquid sample；
Preferably, the system inlets end pressure that the ultrafiltration uses is maintained at 10~30psi, and reflux end pressure is maintained at 5~ 10psi, transmembrane pressure are controlled in 15~20psi；
Preferably, the film packaging material matter is polyether sulfone or regenerated cellulose；
Preferably, it is that 3-5kDa films bag carries out ultrafiltration concentration 5-10 with molecular cut off by dimer peak sample that step 6) obtains Times；
Preferably, ultrafiltration is carried out with the 0.1M phosphate buffers of the pH6.5 of the NaCl containing 1.25M and changes liquid.
9. such as isolation and purification method described in any item of the claim 1 to 8, wherein, step 8) is as follows：
Preferably, in 4 DEG C~whipping step 7 under 50~200rpm at room temperature) obtained filter wash changes liquid sample overnight, gathered again Sample；
It is highly preferred that whipping step 7 under 50~200rpm at room temperature) obtained filter wash changes liquid sample overnight, obtain again poly- sample Product.
10. isolation and purification method as claimed in any one of claims 1-9 wherein, wherein, step 9) is as follows：
The multiple poly- sample that step 8) is obtained is carried out from sieve chromatography, and the sieve chromatography resin is Toyopearl HW65S or Toyopearl HW65F, sample feeding volume are the 2%~5% of column volume, and eluent is containing 0.05~0.15M 20~50mM phosphate buffers of the pH 7.2 of NaCl collect purpose antigen peak, the recombination hepatitis B core antigen purified；
Preferably, the sieve chromatography resin is Toyopearl HW65S.
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