KR100374308B1 - PURIFICATION METHOD OF gpI GLYCOPROTEIN OF VZV FROM RECOMBINANT CHO CELL - Google Patents

Abstract

PURPOSE: Provided is a method for purifying gp I glycoprotein of Varicella-Zoster virus(VZV) expressed from the recombinant chinese hamster ovary(CHO) economically and stablely. CONSTITUTION: A method for purifying gp I glycoprotein of Varicella-Zoster virus(VZV) expressed from recombinant chinese hamster ovary(CHO) comprises the steps of: culturing a recombinant CHO cell; centrifuging the cultured medium; dialyzing the supernatant; and preforming anion exchange chromatography with the dialysate.

Description

Purification method of gpI glycoprotein of Varicella-Zoster Virus expressed from recombinant Hamster Ovary Cell {PURIFICATION METHOD OF gpI GLYCOPROTEIN OF VZV FROM RECOMBINANT CHO CELL}

The present invention relates to a method for purifying recombinant gpI glycoprotein of chickenpox virus expressed in secreted form in genetically modified CHO (Chinese Hamster Ovary) cells.

Varicella-Zoster Virus (VZV) is a DNA virus belonging to the herpes family and causes chicken pox. Chickenpox caused by VZV is a highly contagious disease that is susceptible to development in children, the elderly, and weakened immune systems. VZV infection causes a fever, similar to the symptoms of a cold, about 14 days after infection, and creates a spotty rash throughout the body that develops acne, leaving a fatal scar in severe cases.

VZV mainly begins proliferation in the respiratory tract, the site of primary infection, flows out of the respiratory tract, and then spreads and spreads to the lymph glands to indicate primary viremia. When the cellular immune response is weakened or deficient, the next step is to move to the reticuloendothelial system, where a full-fledged multiplication is performed in the skin, extending to the lungs and liver to indicate secondary viremia. In general, secondary viremia disappears within three days by normal cellular or humoral immune defense mechanisms in normal people, wherein the cellular immune response is known to play a more important role in the inhibition of chickenpox virus.

Even after the secondary viremia disappears, two weeks after infection, the chicken pox virus penetrates into the ganglion cells of the brain and escapes the immune barrier and lurks in satellite cells surrounding the nerve cells. Then, the cellular immune response of the host inhibits proliferation of the virus for a long time and enters a full-scale incubation period. This reactivation of the latent virus cannot be prevented only by the humoral immune response, but only by the cellular immune response. That is, the proliferation of viruses can be suppressed by various kinds of cytokines produced by immune mechanisms.

However, in older people and patients whose immunity is significantly less immune than the proliferation of the virus, even if a specific antibody against VZV exists, the virus is reactivated to proliferate in ganglion cells. This causes the chickenpox virus to migrate and spread back to the skin in the reverse order of 'latency', causing herpes zoster [H. John et al., Virology , 5, 241 (1994); DG Lawrence et al., Virology , 2, 2011-2054 (1990)]. This reactivation of ganglion cells causes more severe symptoms in patients with hydrocephalus, such as primary hydrocephalus pneumonia. Abnormalities begin in the central nervous system, such as primary muscle tremor, hypotonia (paralysis), and acute degeneration, and in some cases worsen by cerebral hemorrhage, peripheral blood cell infiltration, and eventually develop neuritis or encephalitis. It can also be a fatal disease.

In the United States, about 4 million chickenpox patients and 1 million shingles develop in a year, and more than 100 people die from hydrocephalus, especially when immunosuppressive drugs, such as leukemia patients, are required. More lethal [G. Fleisher et al., Am. J. Dis. Child, 135 . 896 (1981); RP Stephen et al., Pediatrics, 68 , 14 (1981); PA Patel et al., Pediatrics, 64 , 223 (1979)].

Vidarabine (adenosine arabinoside), acyclovir (Acyclovir), etc. have been developed to treat VZV, but these treatments are very limited because they cause severe side effects on the central nervous system of the kidney and brain [TH Weller et al., New England J. Med. 309 , 1434 (1983). Therefore, development of preventive chickenpox vaccine is essential.

Many studies have been done to prevent chickenpox disease, but this is based on the classic live attenuate vaccine, not a high level genetic engineering recombinant vaccine. The fresh chickenpox vaccine was first developed in 1975 by the Takahashi Group of Osaka University, Osaka, and produced as a product by BIKEN. Although this vaccine has a good protective effect against VZV, there are some problems with the post-immune preventable period [Johnson, C., Rome, et al., Pediatrcs 1989, 84 , 418-421], furthermore As with infections, there has been a reported problem that the vaccine can lie in ganglion cells and then reactivate and cause chickenpox [Hardy, I., gershon, AA, Steinberg, SP, and LaRussa, N. Engl. J. Med. 1991, 325 , 1545-1550; Plotkin, SA, Starr, S., Connor, K and Morton, D., J. Infect. Dis. 1989, 159 , 1000]. Accordingly, there is a need for the development of new vaccines that can prevent viral infections, such as subunit vaccines, but do not have latent potential.

In the Herpesvirus family, subunit vaccines against several viruses belonging to this family have been developed [Collett, MS, Adv. Vet. Sci. Comp. Med. 1989, 33 , 109-172. Vaccines against these viruses are centered on virus glycoproteins, which not only produce highly effective immune mechanisms, but also cause the same cellular and humoral immune responses that occur during viral infection. [Collett, MS, Adv. Vet. Sci. Comp. Med . 1989, 33 , 109-172.

On the other hand, VZV was A. 1986. second. After DNA sequencing was fully revealed by AJ Davison [AJ Davison et al., J. Gen. Virol. 67, 1759 (1986)]. Five glycoproteins known as gpI, gpII, gpIII, gpIV, gpV are known to exist on the surface of VZV (AJ Davison et al., Journal of Virology 1986, 57, 1195-1197). Of these five glycoproteins, gpI, which can produce neutralizing antibodies, has become one of the most important research targets [Stanley A. Plotkin, Science 1994, 265 , 1383-1385; In C. Sabella et al., J. Virol. 67 , 7673 (1993). In particular, the Vafai group at the University of Illinois, expressed genetically engineered gpI glycoproteins in vaccinia and purified them to carry out immunoneutralization of VZV in rabbits. It can be used (Abbas Vafai, Vaccine, 11, 1993, 937-940: Abbas Vafai, Vaccine, 12 , 1994, 1265-1269). However, in order to commercialize such a gpI glycoprotein into a vaccine, an economical method of expressing and purifying the gpI glycoprotein is required. Thus, purity and yield in purification processes, post-translational modification of recombinant gpI glycoproteins, immunogenicity, clearance rate in vivo, and Given the enduring production aspects, the use of CHO cells as hosts provides optimal conditions [Charles F. Goochee, Michael J. Gramer, Dada C. andersen, Jennifer B. and James R. Rasmussen, Bio / Technology -1991, 9 , 1347-1355; Korean patent application filed as the same applicant.

Existing methods for purifying glycoproteins of VZV consist of methods using immunoaffinity chromatography using monoclonal antibodies [Pual M. Keller et al., US patent 5, 306, 635, 1994; Abbas Vafai, Vaccine, 12 , 1994, 1265-1269. These methods are not economical and efficient due to the yield and safety of the affinity gel matrix to be used for purification and the need to prepare excess monoclonal antibodies.

Therefore, the present inventors began to study the purification method of gpI glycoprotein expressed in CHO cells in the form of secretion, focusing on economics and efficiency, and developed a purification method using ion exchange resin, which is well known for its safety and properties. .

That is, it is an object of the present invention to provide a method for high-purity, high-purity purification of the gpI glycoprotein of VZV, which can be used as a subunit vaccine to prevent infection of VZV.

In order to achieve the above object, in the present invention, in purifying the gpI glycoprotein of VZV expressed in recombinant CHO cells, culture of recombinant CHO cells, centrifugation of the medium, dialysis of the supernatant, and anion exchange chromatography of the dialysate It provides a purification method comprising the step.

Hereinafter, the present invention will be described in more detail.

First, a primary culture medium containing 10% FBS is placed in an animal cell culture container, followed by culturing recombinant CHO cells. If CHO cells grow uniformly on the surface of the container, change to a secondary culture medium and incubate. Culture medium containing gpI glycoprotein is collected and centrifuged to obtain supernatant. This supernatant is put in a dialysis membrane and dialyzed with distilled water. After dialysis for at least 10 hours, the dialysate is recovered and adsorbed onto an anion exchange resin. Salt washing removes most contaminating proteins and then elutes with a linear gradient of sodium chloride solution to recover the pure gpI glycoprotein.

As the anion exchange resin used in the purification method of the present invention, Q-Sepharose or DEAE-Sepharose is preferable, and Q-Sepharose is more preferably used. When performing anion exchange chromatography, the pH of the buffer is preferably in the range of 5.9 to 9.5, more preferably pH 6.5. The buffer preferably contains sodium phosphate, bis-tris, tris or ethanolamine as buffers, and when washing, a buffer solution containing sodium chloride at a concentration of 50 mM or more is used and 0.15 M The concentration is preferable. As the eluate, it is preferable to use a sodium chloride solution having a linear concentration gradient of 0.15 M to 0.6 M.

The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following examples.

(Reference Example 1) Adsorption of gpI glycoprotein to ion exchange resin

In order to confirm the optimal conditions for the purification of the separation by investigating the relative adsorption of contaminating proteins and gpI glycoproteins to the ion exchange resins in the medium according to the pH of the buffer and the type of buffer used, the cation exchange resins were S-, CM- Sepharose, anion exchange resin using Q-, DEAE- Sepharose resin, it was examined whether the adsorption according to the following pH and buffer.

From the above results, it was found that chromatography at pH 5.9 to 9.5 using ethanolamine, tris, bis-tris or sodium phosphate as a buffer is preferred.

(Reference Example 2) Western blotting of gpI glycoprotein

Western blotting used to identify the fractions obtained by ion exchange chromatography in the purification method of the present invention was carried out as follows.

20 μl of the solution to be confirmed was added 20 μl of SDS-PAGE buffer according to the method of Lamley (Laemmli, (1970) Nature 227 , 680), and then heated for 3 minutes, followed by electrophoresis on a 10% polyacrylamide gel. Proteins isolated on gels were transferred to nitrocellulose filters according to Tobin's method (Towbin et al., (1979) Proc. Natl. Acad. Sci. USA, 76 , 4750). The filter was placed in PBS containing 0.2% gelatin and shaken for 30 minutes to block excess protein adsorption sites. Then was added a 0.25% gelatin, 1% Triton X-100, and 0.02% Ti booties turned a rabbit anti-diluted 500 times with PBS containing VZV antibody ^{(Cytimmune? Cat. No. 06361,} Cortex Biochem Inc., USA) at room temperature The reaction was shaken gently for 1 hour at.

The filter was washed four times with PBS containing 0.05% Tween 20 and then horseradish peroxidase diluted 500-fold with PBS containing 0.25% gelatin, 1% Triton X-100, and 0.02% thimerosal (Horse). Goat anti-Rabbit IgG-HRP (American Qualex, Cat. No. A102 PS) labeled with Radish Peroxidase was added and reacted with gentle shaking for 1 hour at room temperature. The filter was washed four times with PBS containing 0.05% Tween 20 and then twice with 50 mM Tris buffer (pH 8.0). The filter was developed by adding 50 mM Tris buffer (pH 8.0) containing 400 µg / ml 4-chloro-1-naphthol and 0.03% hydrogen peroxide water.

Example 1

(Step 1) CHO Cell Culture

1 ml of recombinant CHO cell stock (KCTC 0176BP) expressing the gpI glycoprotein of chickenpox virus stored in liquid nitrogen was thawed in a 37 ° C water bath, followed by 10% dialysis FBS (Gibco), antibiotics (Antibiotic, Gibco) and 1.6 10 ml of α-MEM (20% nucleoside, Gibco) medium containing μM MTX was mixed well and centrifuged at 1,00 rpm for 5 minutes. Precipitated cells were suspended in 10 ml of the same medium, poured into 100 mm culture dishes, shaken well, and cultured in an incubator containing 5% CO ₂ . After incubation for 3-4 days, the medium was removed, washed with PBS buffer, and cells were removed from the culture vessel using 0.2% trypsin. For seed culture, the detached cells were placed in a T-80 culture vessel at a cell density of 2 × 10 ⁴ / cm _{2 and} incubated in a CO ₂ incubator for 4-5 days. Cells grown in culture vessels were detached again and inoculated in T-175 culture test tubes. At this time, 30 ml of the medium was added so as to have a cell density of 2 × 10 ⁴ / cm 2, and α-MEM was used as the medium. After 1-2 days, the cells were replaced with medium without serum when the cells were completely attached to the culture vessel. In this case, CHO-S-SFM (Gibco) was used as a medium without serum. Medium was changed every two days to separate the expressed protein. The medium of each vessel was collected and centrifuged at 5000 rpm for 10 minutes (Beckman J2-M1 centrifuge, Rotor: JA-10) to obtain a supernatant.

(Step 2) Dialysis of Culture Medium

The supernatant recovered by centrifugation in step 1 was put in a dialysis membrane (Spectrum; Mwco; 12000) and dialyzed with 20 L of distilled water. At this time, the temperature was performed for 10 hours or more while maintaining the 4 ℃.

(Step 3) Anion Exchange Chromatography

The dialysate obtained in step 2 was passed through a 10 ml Q-Sepharose packed column (Pharmacia, XK 26/20) equilibrated with buffer (25 mM phosphate, 1 mM EDTA) at a rate of 2.5 ml per minute. Subsequently, 80 ml of a buffer solution containing 0.15 M sodium chloride was flowed through the column to remove contaminating proteins adsorbed on the ion exchange resin. Then, 400 ml of buffer solution containing sodium chloride in a linear concentration gradient of 0.15 M to 0.6 M was added to elute the protein adsorbed on the resin at a rate of 2.5 ml per minute to collect each 10 ml fractions. The collected fractions were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to collect fractions containing gpI glycoproteins. The results of electrophoresis are shown in FIG. 1 and FIG.

Figure 1 shows the results of silver staining after 10% SDS-PAGE, where column 1 is the protein of standard molecular weight, column 2 is the protein that is removed from the resin during salting, and column 3 is the salt concentration gradient. Elution fractions are shown. FIG. 2 shows 10% SDS-PAGE followed by Western blotting according to Reference Example 2. The first column is a protein having a standard molecular weight, the second column is a protein which is separated from the resin when the salt is washed, and the third column is a salt concentration gradient. Fractions eluted by

As shown in FIG. 1 and FIG. 2, the purity of the target protein purified by the purification method of the present invention was 90% or more, and the yield was 80% or more as silver dyeing calculated by a known dilution method.

Example 2 Comparison of Washing Effect According to Salt Concentration During Salt Washing

Anion exchange chromatography was performed in the same manner as in step 3 of Example 1, except that a buffer solution containing 50 mM sodium chloride was used for the saline wash. The results are shown in FIGS. 3 and 4.

3 is a result of silver staining after electrophoresis, the first column is a protein of standard molecular weight, the second column is a culture medium dialysate that is added to the column, the third column is a protein that is not adsorbed to the resin, and the fourth column is eluted. Each fraction, column 5, represents the contaminating protein that is removed upon washing.

4 is a result of Western blotting according to Reference Example 2 after electrophoresis, where the first column is a protein having a standard molecular weight, the second column is a culture medium dialysate introduced into the column, and the third column is a protein not adsorbed to the resin. The fourth row represents the fractions eluted and the fifth column represents the contaminating protein that is removed upon washing.

As can be seen from the results shown in FIGS. 3 and 4, when the salt concentration is 50 mM or more, the contaminating protein can be effectively washed.

As described above, according to the purification method of the present invention, gpI glycoprotein of VZV expressed from recombinant CHO cells can be stably and economically separated and purified.

1 is a result of silver staining after electrophoresis of an ion exchange chromatographic eluate during the purification process of the present invention;

2 is a result of Western blotting after electrophoresis of an ion exchange chromatographic eluate during the purification of the present invention;

3 is a result of silver staining after electrophoresis of the eluate when low concentration of salt was washed in the ion exchange chromatography step of the purification process of the present invention;

4 is a result of Western blotting after electrophoresis of the eluent when low concentration washing was performed in the ion exchange chromatography step of the purification process of the present invention.

Claims (3)

In purifying the gpI glycoprotein of chickenpox virus (VZV) expressed from recombinant Hamster Ovary (CHO) cells, culture of recombinant CHO cells, centrifugation of the medium, dialysis of the supernatant, and dialysate anion exchange chromatography Purification method comprising the step of. The method of claim 1, In the anion exchange chromatography, a linear gradient of 0.15 M to 0.6 M in a buffer of pH 5.9 to 9.5 containing Q- or DEAE-Sepharose and containing ethanol amine, tris, bis-tris or phosphate Eluting with sodium chloride. The method of claim 2, In the anion exchange chromatography, the method characterized by washing with a salt solution of 50 mM or more before elution.