JPH0574599B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides nuclear antigen of hepatitis B virus (hereinafter referred to as
The present invention relates to a method for purifying HBc antigen (abbreviated as HBc antigen), and in particular to a method for purifying HBc antigen produced by a recombinant using recombinant DNA technology. Technical Background and Industrial Application Fields of the Invention Hepatitis B is caused by the hepatitis B virus (hereinafter referred to as HBV).
It is a disease caused by the virus (hereinafter referred to as "abbreviation"), and it involves very serious problems both epidemiologically and clinically, but no effective treatment has yet been found. Although this disease is widely distributed throughout the world, it is particularly prevalent in Asia and Africa, where it is becoming a problem. Early detection of HBV infection is important for effective prevention of hepatitis B, and the current clinical diagnostic methods include HBs antigen, HBs antibody, HBc antibody,
Methods for detecting HBe antigens, HBe antibodies, etc. have been used, and the r-PHA method, PHA method, EIA method, RIA method, etc. are known, and diagnostic reagents used for these methods are also commercially available. However, the antigens necessary to manufacture these diagnostic reagents and their preventive vaccines must be obtained from the blood and liver of people with latent HBV infection, known as carriers, and there are many manufacturing constraints. . In particular, HBc antigen must be prepared directly from an infectious virus, and its preparation is extremely dangerous. The present inventors have previously succeeded in introducing and expressing HBV DNA encoding the HBs antigen protein into Escherichia coli and yeast using recombinant DNA technology (see Japanese Patent Application No. 142,460/1986). ), and also developed an industrial purification method for HBs antigen, which is a raw material for vaccines (see Japanese Patent Application No. 186187/1987). The present inventors further found that by using recombinant
Attempting to produce HBc (Cell Engineering, Vol. 3,
4, pp. 367-370, 1984), succeeded in highly purifying the HBc antigen produced by this method to the extent that it could be used as a diagnostic reagent material. Since the technology that can be provided in large quantities and at low cost has been established,
It is expected that it will be applied as a diagnostic reagent for hepatitis B. Prior Art As a purification method for liver-derived HBc antigen, a method combining density gradient centrifugation and gel filtration has been proposed by Budkowaska et al., J. Immunology. 118, 1300
(1977) and Ohori, H. et al., Inter-virology, 13, 74.
(1980)], density gradient centrifugation [Feitelson, MA et al., J. Virology, 43, 687 (1982)], etc. have been reported. As a purification method for blood-derived HBc antigen, HBV
A method has been reported in which the infectious virus particles, which are the main body of the virus, are purified by centrifugation, then a surfactant is added to break up the virus particles, and the virus particles are further purified by density gradient centrifugation [Takahashi et al. et al.), J. Immunology, 122, 275 (1979), etc.]. Furthermore, density gradient centrifugation is a method for purifying HBc antigen expressed in E. coli using recombinant DNA technology [Roggendorf et al.
et al.), J.
Virological Methods, 6, 61 (1983)]. However, these methods have the disadvantage that various problems in purifying recombinant-derived HBc antigens cannot be adequately addressed. In other words, an important issue in purifying recombinant-derived HBc antigens is that contaminant components in the raw materials, such as host-derived proteins and lipids, are qualitatively and quantitatively higher than liver-derived or blood-derived HBc antigens.
Since they are completely different from antigens, sufficient purification cannot be achieved using the above-mentioned conventional techniques. In addition, the above-mentioned attempt was made to purify recombinant-derived HBc antigen.
Roggendorf et al.'s method simply purifies it to a level that can be analyzed, and the yield has not been studied, so it cannot be applied as an industrial method for purifying HBc antigen. Purpose of the Invention The present inventors have conducted various studies in order to find a safe and practical purification method for HBc antigen derived from recombinant organisms, and as a result, the present inventors have discovered that host-derived components obtained from recombinant culture By adding acid to the HBc antigen raw material solution containing HBc antigen to bring the pH to the acidic side, most of the lipids and contaminant proteins that interfere with chromatography are separated and removed from the HBc antigen as a precipitate. The present inventors have discovered that HBc antigen can be purified to a high degree by performing ion exchange chromatography using an anion exchanger and density gradient centrifugation in the next step, leading to the completion of the present invention. That is, the purpose of the present invention is to create a safe and inexpensive
The purpose of the present invention is to make it possible to supply HBc antigen stably and in large quantities, and the present invention is particularly directed to recombinant HBc antigens.
Produced by recombinants using DNA technology
The object of the present invention is to provide a method for industrially efficiently purifying HBc antigen in large quantities and with extremely high purity. Structure and Effects of the Invention The present invention relates to purification of HBc antigen, particularly recombinant
Produced by recombinants using DNA technology
When purifying HBc antigen, acid is added to the raw material solution containing HBc antigen and host-derived components obtained from a recombinant culture to shift the pH to the acidic side, which interferes with the chromatography method. It is characterized in that most of the lipids and contaminant proteins are removed as precipitates, and then purified and concentrated directly or by salting out ammonium sulfate, followed by ion exchange chromatography using an anion exchanger. The HBc antigen material solution in the present invention refers to recombinant
Using DNA technology, a recombinant transformed to produce HBc antigen, such as Escherichia coli or yeast, is cultured in an appropriate medium and culture conditions to produce and accumulate HBc antigen. The HBc antigen is obtained by crudely extracting the HBc antigen from this culture using an appropriate method. An example of a recombinant that can produce such an HBc antigen has already been published by the present inventors (Cell Engineering, Vol. 3, No. 4, pp. 367-370, 1984 and at the 31st Annual Meeting of the Japanese Society of Virology). General Assembly, Osaka, 1983, Abstract No. 2036), for example, prepared as follows. Plasmid pHBV containing the entire DNA sequence of HBV
(see Japanese Patent Application No. 57-145093) containing the HBc gene obtained by digesting with a restriction enzyme such as RsaI.
The DNA sequence is integrated into the XhoI site of plasmid pACYC177 to obtain recombinant plasmid pAHBc. Obtained by digesting this pAHBC with the restriction enzyme XhoI.
Shuttle vector DNA sequence containing HBc gene
It is inserted into the Xho site of pAM82 (see Japanese Patent Application No. 145093/1982) to obtain HBc expression plasmid pAC301. Alternatively, the above pHBc was partially digested with the restriction enzyme Xho to remove the 5' pre-C region, and _T4
After turning the cohesive end into a flush end using DNA synthase, attach an EcoRI linker and add the Xho site to the EcoRI site. Next, the Xho site on the 5′ side of the HBc gene
Select the plasmid that replaced the EcoR site. The plasmid pHBcl thus obtained was incubated with EcoRI
After further digestion with BAL3l, a SalI linker is attached, and this is digested with restriction enzymes XhoI and SalI to obtain an HBc gene fragment from which the pre-C region of approximately 600 bp has been removed. This is inserted into the Sal site of shuttle vector pAM81 (see Japanese Patent Application No. 145093/1983) to obtain HBc expression vector pAC701. The above expression vector was transferred to the yeast fungus Saccharomyces
Cerevisiae AH22 [a leu2 his4 canl (Cir ⁺ )]
(Feikoken Jokyo No. 312) by transforming it in the same manner as described in the above Japanese patent application.
Transformed yeast Saccharomyces cerevisiae pAC301 and Saccharomyces cerevisiae pA701 capable of producing HBc antigen are obtained. A desired recombinant culture can be obtained by culturing these transformed yeast according to a conventional method. As a method for extracting HBc antigen from the above culture, first, bacterial cells are separated from the culture by centrifugation, and then disrupted in an appropriate buffer. The disruption methods include ultrasonic disruption, glass bead disruption, Manton-Gorlin disruption, or enzymatic dissolution of cell walls to form spheroplasts.
Various methods can be used, such as destroying it by acting on it with a surfactant, or a combination thereof. This cell destruction product is subjected to low-speed centrifugation to separate cell wall debris, and if necessary, is further filtered through a membrane filter to provide the HBc antigen raw material. In the present invention, the acids used in the acid addition treatment include inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid;
Examples include organic acids such as acetic acid and oxalic acid. In the acid addition treatment, the pH of the raw material liquid is adjusted by adding an acid so that the pH is 6 or less, preferably within the range of 5.0 to 5.5. Acid treatment temperature is 20℃
Hereinafter, the temperature is preferably 4 to 10°C. After adjusting the pH, the precipitate is centrifuged to obtain a supernatant containing the HBc antigen. Next, if necessary, purification and concentration operations are performed by salting out ammonium sulfate. Add aqueous ammonia at an appropriate concentration to the HBc antigen-containing supernatant obtained by acid addition treatment, and add ammonium sulfate while maintaining the pH of the supernatant in the range of 6.0 to 8.0 to precipitate the HBc antigen. ,
This is centrifuged to separate it from the supernatant. The obtained precipitate is dissolved in a suitable neutral buffer solution with an ionic strength of about 0.001 to 1.0, such as 0.1M phosphate buffer, and dialyzed against the same buffer solution using an anion exchanger. Use as material for chromatography. If ammonium sulfate precipitation is not performed, add aqueous ammonia to the HBc antigen-containing supernatant to adjust the pH to approximately 7.0, then dilute or dialyze with the same buffer as above to use as a material for chromatography. . As the anion exchanger used in the present invention, anion exchangers having diethylaminoethyl as a functional group are most effective, including DEAE-Sephadex A-25, DEAE-Sephadex A-50, DEAE
- Commercially available under the names of Cepharose, DEAE-Sefacel (manufactured by Pharmacia Fine Chemicals), DEAE Bio-Gel A, Selex D (manufactured by Bio-Rad), and DEAE-Cellofine (manufactured by Biochemical Engineering Co., Ltd.). , all of these are used. Ion exchange chromatography using this anion exchanger is performed by the following method. Adsorption of HBc antigen to an anion exchanger can be carried out by both a column method and a batch method.
In the column method, equilibration is performed by passing the same buffer solution used in preparing the chromatography material through a column packed with an anion exchanger.
Contaminant substances derived from the host are separated by passing liquid through the material and adsorbing the HBc antigen. After the completion of the liquid passage, the equilibration buffer is passed through the column, and the column is washed to wash out impurities, and then subjected to the elution procedure described below. In addition, in the batch method, an anion exchanger equilibrated with a buffer solution is added to the material solution, and the anion exchanger is added at a slow rate.
Stir for about 0.5 to 2 hours to adsorb HBc antigen.
After adsorption, the anion exchanger is collected on a filter, an equilibration buffer is added to the gel, and washing and filtration separation are repeated several times, followed by an elution operation. In addition, even in the case of adsorption by this batch method, it is preferable to perform elution using a column filled with an anion exchanger that has adsorbed the HBc antigen. For elution, the ionic strength is about 0.01 to 0.5,
Then, step elution or concentration gradient elution is performed using a near-neutral buffer solution with higher ionic strength than the equilibration buffer solution to separate and elute adsorbed contaminants and HBc antigen, and to obtain a fraction containing HBc antigen. Separate. Concentration gradient elution is, for example, 0.01M→
A 0.5M phosphate buffer concentration gradient may be used;
A 0.01 to 0.5M concentration gradient of 0.01M phosphate buffer with saline added may be used, or other suitable concentration gradient buffers that can effectively decompose the HBc antigen and contaminants may also be used. In the case of stepwise elution, the HBc antigen or contaminants are highly purified by passing through a buffer with an ionic strength that elutes only one, followed by a buffer with a higher ionic strength. Ta
Fractions containing HBc antigen are collected. The HBc antigen obtained in this way is further
Highly purified by sucrose step gradient centrifugation or sucrose linear gradient centrifugation, followed by sedimentation equilibrium centrifugation or linear gradient centrifugation using cesium chloride.
HBc antigen can be obtained. In addition, a combination of ion exchange chromatography and hydroxyapatite chromatography is useful for increasing the degree of purification in ion exchange chromatography, or for improving the degree of purification when a wide range of fractions containing HBc antigens are pooled. be. Hydroxyapatite is a gel for affinity chromatography whose component is calcium phosphate.
Hydroxyapatite (Seikagaku Kogyo Co., Ltd., ALBIOCHEM)
- BEHRING), Hydroxylapatite (Bio-Rsd
It is marketed under the name of ``Company''. That is, the fraction containing the HBc antigen was dialyzed against a suitable near-neutral buffer with an ionic strength of about 0.01 to 0.2, such as 0.1M phosphate buffer, or diluted with the same buffer. When the solution is then passed through a hydroxyapatite column that has been equilibrated with the same buffer, the HBc antigen is not adsorbed and is collected in the flow-through fraction. The present inventors used chromatography gels such as CM cellulose, CM sepharose, and phosphate cellulose in place of anion exchangers under various conditions to purify recombinant-derived HBc antigen. However, the purification effect could hardly be obtained. Next, the present invention will be explained in more detail with reference to Examples. Example 1-1 Recombinant yeast Satucharomyces cerevisiae
After culturing AH22pho80/pAC.701, approximately 600 g of bacterial cells collected by centrifugation were added with 50 mM phosphate buffer (PH
7.2) Add 3 and apply this at a pressure of 600Kg/cm ² to about 3
The microbial cells are destroyed by passing through a Manton-Gorlin crusher for some time. The bacterial cell debris is centrifuged to separate coarse bacterial cell fragments to obtain a crude HBc antigen extract with an HBe antigen activity of about 30,000 cpm [400-fold dilution, RIA kit: HBeRIA kit (manufactured by Abbott)]. Next, add acetic acid dropwise to the crude extract while checking with a PH meter to adjust the PH to 5.4. Approximately at 4℃
After stirring for 30 minutes, remove the precipitate by centrifugation. Add ammonia water to the centrifuged supernatant and adjust the pH to approximately
6.5, gradually add ammonium sulfate to a final concentration of 2.5M. After stirring at 4°C for 30 minutes and standing overnight, the precipitate containing the HBc antigen was separated and collected by centrifugation. The collected precipitate was added to 10mM phosphate buffer, 50mM
Suspend in approximately 300 ml of potassium chloride (PH7.3) and dialyze against approximately 100 times the volume of the same buffer in a dialysis tube. After dialysis, the HBc antigen was diluted approximately 3 times with 10mM phosphate buffer and 50mM potassium chloride (PH7.3) and added to a DEAE-cellulose column (gel volume approximately 1) that had been equilibrated with the same buffer. to be adsorbed. After thoroughly washing the column with equilibration buffer, elution is carried out by passing a 50mM to 500mM potassium chloride concentration gradient phosphate buffer of approximately 4.0.
Collect the fraction containing HBc antigen. The collected fractions were pooled and concentrated using an ultrafiltration concentrator Amicon TCF-10 (manufactured by Amicon), and 60% sucrose solution, 20% sucrose solution, and HBc antigen concentrated solution were placed in ultracentrifuge tubes.
10ml, 35ml, 35ml layered, 19 at 30000 rpm, 4℃
Ultracentrifugation is performed for hours to concentrate and purify the HBc antigen at the sucrose liquid layer interface. The obtained HBc antigen fraction was mixed with 50mM Tris-HCl,
After dialyzing against 150mM sodium chloride and 1mMEDTA (PH7.5), cesium chloride was added to a concentration of 1.32g/ml, and the mixture was ultracentrifuged at 30,000 rpm and 4°C for 60 hours to obtain concentrated and purified HBc antigen. obtain. The recovery rate and degree of purification of HBc antigen up to each step are shown in Table 1-1.

[Table] Example 1-2 HBc antigen was concentrated and purified at the sucrose liquid layer interface under exactly the same conditions as in Example 1-1. The obtained HBc antigen fraction is dialyzed against 0.1M phosphate buffer (PH7.3), and then passed through a hydroxyapatite column equilibrated with the same buffer. After pooling the HBc antigen-containing fractions collected in the flow-through fraction, cesium chloride was added at 1.32 g/
ml concentration, 30000 rpm, 4℃
Ultracentrifuge for 60 hours to obtain concentrated and purified HBc antigen. The recovery rate and degree of purification of the HBc antigen at this time are as shown in Table 1-2.

[Table] Example 2 Recombinant yeast Satucharomyces cerevisiae
After culturing AH22pho80/pAC701, the cells (100g) collected by centrifugation were added to 50mM phosphate buffer (PH
7.2) Add 500ml and subject it to ultrasonication at 4℃ for about 90 minutes to destroy the bacterial cells. Example 1
In the same manner as above, remove bacterial cell debris by centrifugation to obtain a crude extract. This crude extract is purified in the same manner as in Example 1 to obtain HBc antigen. Table 2 shows the recovery rate and degree of purification of HBc antigen up to each step.

[Table] Example 3 Recombinant yeast Satucharomyces cerevisiae
After culturing AH22pho80/pAC701, 600ml of 50mM potassium phosphate buffer (PH7.2) containing 100μg/ml of Zymolyase (Seikagaku Corporation) was added to the bacterial cells (200g) collected by centrifugation, and incubated at 30°C for 30 minutes. Stir for a minute and centrifuge to obtain spheroplasted yeast as a precipitate. Add 200ml of 50mM phosphate buffer (PH7.2) containing 0.1% Triton . Add 40% (w/w) polyethylene glycol 6000 to this crude extract to give a final polyethylene glycol concentration of 3%. After stirring at 4°C for 1 hour, remove the supernatant by centrifugation, and suspend the precipitate by adding 80 ml of 10 mM potassium phosphate buffer and 50 mM potassium chloride (PH7.2). This suspension is solubilized by sonication for 5 minutes. This is dialyzed overnight at 4°C against 10mM potassium phosphate buffer and 50mM KCl (PH7.2), and then added to DEAE-cellulose. The HBc antigen is then purified in the same manner as in Example 1 to obtain the HBc antigen.
Table 3 shows the recovery rate and degree of purification of HBc antigen up to each step.

【table】

Claims (1)

[Claims] 1. Expressed and produced in a transformant having the ability to produce HBc antigen obtained by recombinant DNA technology
HBc antigen raw material containing HBc antigen has a pH of 6.
1. A method for purifying HBc antigen, which comprises treating with an acid to remove precipitated lipids and contaminant proteins, and then subjecting the acid-treated solution to ion exchange chromatography using an anion exchanger. 2. The method according to item 1, wherein after the acid treatment, the acid-treated solution is salted out with ammonium sulfate and then subjected to ion exchange chromatography. 3. The HBc antigen raw material containing the HBc antigen is an HBc antigen crude extract obtained by disrupting transformed cells by ultrasonic disruption, glass bead disruption, Manton-Gorlin disruption, surfactant treatment, or a combination thereof. The method described in Section 1. 4 The acid used for acid treatment is hydrochloric acid, sulfuric acid, phosphoric acid,
The method according to item 1 above, wherein at least one selected from acetic acid and oxalic acid is used. 5. The method according to item 1 above, in which an anion exchanger having a diethylaminoethyl group as a functional group is used as an anion exchanger in ion exchange chromatography. 6 Ion exchange chromatography involves contacting the HBc antigen-containing solution with an anion exchanger equilibrated with a buffer having an ionic strength of at least 0.001 in order to adsorb the HBc antigen onto the ion exchanger; 2. The method according to item 1, comprising the step of eluting with a buffer solution having an ionic strength of 0.01 to 0.5 higher than that of the buffer solution used for equilibration. 7. The method according to item 1 above, wherein the ion exchange chromatography is carried out by a batch method or a column method. 8. The method according to item 1 above, wherein ion exchange chromatography and hydroxyapatite chromatography are carried out in combination.