KR100374309B1 - PURIFICATION METHOD OF gD GLYCOPROTEIN OF HSV-2 FROM RECOMBINANT CHO CELL - Google Patents

Abstract

PURPOSE: Provided is a method for purifying gD glycoprotein of Herpes Simplex virus-2(HSV-2) expressed from the recombinant chinese hamster ovary(CHO) cell economically and stablely. CONSTITUTION: A method for purifying gD glycoprotein of Herpes Simplex virus-2(HSV-2) expressed from the recombinant chinese hamster ovary(CHO) cell comprises the steps of: culturing a recombinant CHO cell; centrifuging the cultured medium; dialyzing the supernatant; and preforming cation exchange chromatography and glucose-affinity chromatography with the dialysate.

Description

Purification method of gD glycoprotein of herpes simplex virus type 2 expressed from recombinant Chinese hamster Ovari (CHO) cells {PURIFICATION METHOD OF gD GLYCOPROTEIN OF HSV-2 FROM RECOMBINANT CHO CELL}

The present invention relates to a method for purifying the herpes simplex virus type 2 gD glycoprotein produced in secreted form in CHO cells (Chinese Hamster Ovary Cell), which is a recombinant animal cell.

Herpes Simplex Virus (HSV) is a DNA virus belonging to the genus Herpes virus and is a widespread infector in humans. Herpes simplex infection is an infection of HSV type 1 (HSV-1) and HSV type 2 (HSV-2), which is an acute bullous disease involving the mucous membranes and skin and is a common infection in people with atopy. . HSV-1 mainly forms blisters around the mouth and eye, while HSV-2 forms around the genitals. HSV infection has been reported to be severe in patients with compromised immune function and may also act as a cause of cervical cancer [Melnick, JL, Adam, E. and Rawls, W., Cancer, 34, 1355-1385 (1974); Cabral, G. A., Fry, D., Marciono-Carbral, F., Lumplcin, C., Mercer, L. and Goplerud, D., Am. J. Obstet. Gynecol., 145, 79-86 (1983). In addition, newborns and fetuses are not only able to produce HSV antibodies themselves, but because the antibodies of the mother are not passed on to the fetus, it is known that most infections have a fatal effect. Currently, about 500,000 cases of eye disease caused by HSV-1 occur in the United States alone, and more than 1,000 patients are undergoing eye transplants [Nesburn, AB, Vision Resesrch: national plan 1983- 1987, vol. II, part III, The C. V. Mosby Co., St. Louis. (1983); Smith, R. E. et al., Arch. Ophthalmol 98, 1226 (1980)]. About 95% of HSV-2 infections are known to be transmitted through sexual contact with a partner with active lesions. About 20-30% of US adults are infected with HSV-2, of which 20-50% relapse. It has been reported to have genital herpes [Johnson RE et al., N. Engl. J. Med., 321, 7 (1989); Breinig M. K. et al., J. Infect. Dis., 162, 299 (1990); Langenberg A. et al., Ann. Intern. Med., 110, 882 (1989); Koutsky L. A. et al., N. Engl. J. Med., 326, 1533 (1992).

HSV begins to replicate in skin or mucosal epidermal cells after initial infection, migrates along the nerve tissue, and lurks in the vertebral ganglion throughout life, then reactivates when immune function decreases, resulting in various diseases from the affected area. Cory L., Sexually transmitted disease, 2nd ed., New York: McGraw Hill, 1990. Nucleoside derivative Acyclovir is known to be very effective for early infection as a therapeutic agent for HSV infection [Bryson, Y. J. et al., N. Engl. J. Med., 308, 916 (1993); Mertz G. J. et al., Am. Med. Assoc., 252, 1147 (1984)], in order to maintain the effectiveness of the drug, the drug must be continuously administered, and it is known that recurrent disease caused by HSV recurs when administration is stopped [Mindel A. et al. Lancet i: 926-928 (1988); Straus S. E. et al., N. Engl. J. Med., 310, 1545 (1984)]. It has also been reported that mortality rates are high in cases of relapse after the initial infection of HSV [Nahmias, A. J. and Coleman, R. M., Immnobiology of Herpes Simplex Virus Invection CRC Press, Boca Raton, 1984, PP. 92-102].

Although vaccines have been developed to prevent such infections of HSV, vaccines that use the weakened virus itself have been banned due to the fact that the genome of HSV is directly involved in oncogenesis. Cappel, R., Sprecher, S., De Cuyper, F. and De Braekeleer, J., J. Med. Virol., 26, 137-145 (1985). Thus, the vaccine for preventing infection of HSV should be free of viral nucleic acid (DNA) and should be able to prevent not only the first infection in its function but also reinfection by a latent virus.

Recently, studies have been conducted to prepare vaccines having the above functions, and in view of efficacy and stability, it has been found that gD glycoproteins, particularly gD glycoproteins, can be used as vaccines [Torseth, JW, Cohen, GH, Eisenberg, RJ, Berman, PW, Lasky, LA, Cerrni, CP et al., J. Virol., 61, 1532-1539 (1987)].

The gD glycoprotein of HSV is a glycoprotein that functions to connect the surface part of the virus and the cell surface to be infected during HSV infection [Highlander, SL, Sutherland, SL, Gage, PJ, Johnson]. , DC, Levine, U. and Gloriose, JC, J. Virol., 61, 3356-3364 (1987); Fuller, A. O. and Spear, P. G., Proc. Natl. Acad. Sci. U. S. A., 84, 5454-5458 (1987)], plays a very important role in the initial infection of the virus. It is also an important target for cross-reactive neutralizing antibodies [Cohen, GH, Katze, M., Hydrean-Stern, C. and Ersenberg, RJ, J. Virol., 27, 172-181 (1978). It is also an important target of cellular immune responses [Blacklaws B. A., Nash, A. A. and Darby, G., J. Gen. Virol., 68, 1103-1114 (1987); Martin, S., Moss, B., Berman, P. W., Lasky, L. A. and Rouse, B. T., J. Virol., 61, 726-734 (1987)].

Burke et al. Cloned the gene containing Dihydrofolate reductase (hereinafter referred to as dhfr) and the gD gene from which the hydrophobic region at the carboxyl terminus of the gD gene of HSV-1 was removed into two different expression vectors. dhfr deficient CHO cells were simultaneously transformed, followed by amplification of the gene with mesotraxate (hereinafter referred to as MTX) to select gD protein expressing cell lines, and the purified gD protein was inoculated in guinea pigs. In addition to preventing infection, it has been suggested that it can be used as a therapeutic vaccine to prevent relapse of already infected HSV [Burke RL, REV. Infect. Dis., 13, S 906 (1991); Sanchez-Pescador L. et al., J. Immunol., 141, 1720 (1988). Mishin et al. Infected HSV-1 with Vero cells, cultivated a large amount of virus, purified gD protein and injected it into mice, guinea pigs, monkeys, etc., and then infected HSV. Relapse of HSV has also been shown to be prevented (Mishikin EM et al., Viral Immunol., 5, 151 (1992)). In addition, Bernstein and others infected HSV-2 with Vero cells, and then purified gD protein, mixed with an adjuvant, imiquimod, injected into guinea pigs, and then infected with HSV-2. Has been reported [Bernstein DI et al., J. Infect. Dis., 167, 731 (1993). Michkin et al. Cloned the gD gene of HSV-2 into an insect cell expression vector to make an expression vector, and then expressed a gD having a molecular weight of 57 kDa in the insect cell. Landolfi, V. et al., Vaccine, 11, 407 (1993). In addition, Cohen et al. Reported that gD protein, from which the hydrophobic region at the carboxyl terminus was removed from the gD gene of HSV-1, was conjugated after the melittin leader sequence and expressed in insect cells. et al., J. Virology, 68, 766 (1994).

The use of gD glycoproteins as a vaccine raises technical problems that the sufficient amount of gD glycoproteins must be obtained and that the genome of HSV must be completely removed through the purification of gD glycoproteins. Bacteria [Watson, RJ, Weis, JH, Salstrom, JS and Enquist, LW, ScIence, 218, 381 (1982)], yeast [Stanberzy, MG, Bernstein, DI, Burke, RL, Pachl, C. and Myers, MG, J. Infect. Dis., 155, 914-920 (1987)], Besnia (Paoletli, E., Lipsinskas, B. R., Samsonoff, C., Mercer, S. and Panicali, D., Prol. Natl. Acad. Sci. U. S. A., 81, 193-197 (1984)], mammalian cells [Stanberry, L. R., Burke, R. L. and Myers, M. G., J. Infect. Dis., 157, 156-163 (1988)] and the production of recombinant gD glycoproteins. Production of gD glycoproteins using mammalian cells among the recombinant gD glycoproteins produced in each of these systems is necessary for post-translational modification, immunogenicity, and in vivo clearance rate. And in terms of enduring production [Charles F. Goochee, Michael J. Gramer, Dana C. Andersen, Jennifer B. Babr and James R. Rasmussen, Bio / Technology, 9, 1347-1355 (1991). )].

Based on the above, the present applicant has developed a method capable of continuously expressing the gD glycoprotein of HSV in CHO cells, which are animal cells, and filed the same as the present application.

As mentioned above, in order to develop a gD glycoprotein obtained by using genetic recombination technology as a vaccine against HSV, sufficient gD glycoprotein should be secured. Purity, yield and economic efficiency of the purification process are very important. As a method of purifying a natural gD glycoprotein, an affinity chromatography method using a monoclonal antibody has been used until now [E. M. Mishkin, J. R. Fahey, Y. Kmo, R. J. Klein, A. S. Abramovilz and S. J. Mento, Vaccine, 9, 147-153 (1991). In addition, the method of purifying the recombinant gD glycoprotein has been applied to the same method as the method of purifying the native gD glycoprotein [La. R. Stanberry, D. I. Bernstein, R. L. Burke et al., J. Infectious Disease, 155, 914-920 (1987); W. P. Sisk, J. B. Bradley et al, J. Viro., 68, 766-775 (1994); V. Landolf, C. D. Zarley, A. S. Ahramovitz et al., Vaccine, 11, 22, 407-414 (1993). However, purification of a large amount of recombinant gD glycoproteins using a purification method centered on immunoaffinity chromatography using monoclonal antibodies, yields and safety of an affinity gel matrix to be used for purification, and The problem of preparing an excess amount of monoclonal antibody is not suitable in terms of economy and efficiency.

Accordingly, the present inventors have studied the purification method of gD glycoprotein expressed in CHO cells in the form of secretion, focusing on economic and efficiency aspects, and have developed a purification method using ion exchange resins and affinity resins well known for their stability and specificity. It came to the following.

That is, it is an object of the present invention to provide a method for high-purity, high-purity purification of HSV-2 gD glycoprotein that can be used as a subunit vaccine to prevent infection of HSV.

In order to achieve the above object, in the present invention, in purifying gD glycoprotein of HSV-2 expressed from recombinant CHO cells, the culture medium of recombinant CHO cells is centrifuged, the supernatant is dialyzed, and the dialysate is subjected to cation exchange chromatography. And it provides a purification method comprising the step of affinity chromatography.

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

First, a primary culture medium containing 10% FBS is placed in a container for animal cell culture, followed by culturing recombinant CHO cells expressing gD glycoprotein of HSV-2. If CHO cells grow uniformly on the surface of the container, change to secondary culture medium and continue incubation. The culture medium containing the gD glycoprotein of HSV-2 is collected and centrifuged to obtain a supernatant. This supernatant is placed in a dialysis membrane and dialyzed with distilled water for at least 10 hours.

The dialysate is recovered and adsorbed onto the cation exchange resin and then eluted with a linear concentration gradient of sodium chloride solution. At this time, the cation exchange resin is preferably CM-Sepharose or S-Sepharose, more preferably using S-Sepharose. When performing cation exchange chromatography, the pH of the buffer is preferably in the range of 6.5 to 7.5, more preferably pH 7.0.

The buffer preferably contains sodium phosphate or bis-tris as a buffer, and it is preferable to use a sodium chloride solution of a linear concentration gradient of 0.0 to 0.3 M as eluent.

The glycoprotein solution eluted by the above cation exchange chromatography is dialyzed with a buffer solution of affinity chromatography. The dialysed gD glycoprotein solution is adsorbed onto the sugar affinity resin and then eluted to obtain pure gD glycoprotein. In this case, the sugar-affinity resin is preferably Con-A Sepharose or Lectin Sepharose, and Con-A Sepharose is more preferably used. In order to elute the adsorbed gD glycoprotein, methyl-α-D-mannopyranoside of 0.2 M to 0.5 M is preferably used, with a concentration of 0.2 M being more preferred.

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.

The composition of the buffer solution used in the examples is as follows unless otherwise specified.

Reference Example 1 Adsorption of gD Glycoprotein to Ion Exchange Resin

In order to confirm the optimal conditions of the separation and purification by examining the adsorption of the gD glycoprotein to the ion exchange resin according to the pH of the buffer and the type of buffer used, the cation exchange resins include S-, CM-sepharose and anion exchange resin. Furnace was examined for adsorption according to the following pH and buffer using Q-, DEAE-Sepharose resin.

From the above results, it was found that chromatography at pH 6.5 to 7.5 is preferred using phosphate or bis-tris as the buffer.

Reference Example 2 Western Blotting of gD Glycoprotein

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

To 20 μl of the solution to be confirmed, 20 μl of SDS-PAGE buffer was added according to the method of Lamley (Laemmli, Nature, 227, 680 (1970)), heated for 3 minutes and electrophoresed on a 10% polyacrylamide gel. Proteins isolated on gels were prepared by Tobin's method [Towbin et al., Proc. Natl. Acad. Sci., U. S.A., 72, 4750 (1979)]. The filter was placed in PBS containing 0.2% gelatin and shaken for 30 minutes to block excess protein adsorption sites. Then, rabbit anti HSV antibody (Cortex Biochem Inc., Cat.No. CR6073G) diluted 500-fold with PBS containing 0.25% gelatin, 1% Triton X-100, and 0.02% thimerosal was added for 1 hour at room temperature. Reacted by shaking gently. 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 Perosidase) 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)

(Step 1) CHO Cell Culture and Media Harvest

1 ml of recombinant CHO cell stock (KCTC 0175BP) expressing gD glycoprotein of HSV-2 stored in liquid nitrogen was thawed in a 37 ° C. water bath, followed by 10% dialysis FBS (Gibco), antibiotic (Gibco) and 1.6 μM. 10 ml of α-MEM (20% nucleoside, Gibco) medium containing MTX was added and mixed well, and centrifuged at 1,000 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 by the above method and seeded in a rotating bottle (Roller Bottle: 850 cm ² ) for mass culture.

At this time, 200 ml of the medium was added so that the cultured cell density was 2 × 10 ⁴ / cm ² , and α-MEM was used as the medium. After the medium was added, a balanced air containing 5% CO ₂ was injected for 2 minutes, and then the rotating vessel was placed in an incubator and incubated at a rotational speed of 20 RPH (revolution per hour).

After 1-2 days, when the cells were completely attached to the culture vessel, the rotational speed was increased to 60 RPH and cultured. After 5-6 days, when the cells grew to the culture vessel, the medium was completely removed and replaced with medium without serum. In this case, CHO-S-SFM (Gibco) was used as a medium without serum. 5% CO ₂ controlled air was injected even after the medium was replaced. The medium was changed every two days in order to separate the expressed protein while keeping the cells grown in the culture vessel intact. 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 placed in a dialysis membrane (Spectrum: Mwco; 12000) and dialyzed with 20 L of distilled water. At this time, the temperature is carried out for 10 hours or more while maintaining the 4 ℃.

(Step 3) Cation Exchange Chromatography

The dialysate obtained in step 2 was passed through a 50 ml S-Sepharose filled column (Pharmacia, XX 50/20) equilibrated with round solution 1 at a rate of 10 ml per minute. Subsequently, 200 ml of buffer 1 was flowed through the column to remove proteins not adsorbed on the ion exchange resin. Then, 400 ml of buffer 1 containing sodium chloride in a linear concentration gradient of 0.0 M to 0.3 M was added to elute the proteins adsorbed on the resin at a rate of 5 ml per minute to collect 10 ml fractions each. The collected fractions were 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to collect only the specific fractions containing gD glycoprotein separately. Electrophoresis results are shown in FIGS. 2 to 3.

FIG. 2 shows 10% SDS-PAGE followed by Coomassie blue staining. The first column is a protein having a standard molecular weight, the second column is a solution which is added to a cation exchange chromatography column after dialysis, and the third column is a cation exchange. Proteins not adsorbed to the resin, fourth rows, represent fractions eluted by the salt concentration gradient.

FIG. 3 is a 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 solution introduced into a positive ion exchange chromatography column after dialysis, and a third column. Heat is a protein that is not adsorbed to the cation exchange resin, and the fourth rows represent fractions eluted by the salt concentration gradient.

FIG. 4 is a 10% SDS-PAGE followed by silver staining. The first column is a protein having a standard molecular weight, the second column is a solution introduced into a cation exchange chromatography column after dialysis, and the third column is a cation. Protein not adsorbed to the exchange resin, column 4 represents a solution in which eluted fractions are collected, and column 5 represents a solution washed with a high concentration of salt.

As shown in Figures 2 to 4, it was confirmed that the gD glycoprotein secreted in the medium was recovered without loss by the cation exchange resin.

(Step 4) Dialysis

100 ml of the protein solution containing the fractions containing the gD glycoprotein obtained in step 3 was placed in a dialysis membrane (Spectrum MWCO: 12000) and dialyzed at 2 ° C. with buffer 2 at 4 ° C. for at least 10 hours.

(Step 5) Sugar Affinity Chromatography

The protein solution obtained in step 4 was passed through a column (Bio-rad) packed with 20 ml of Con-A Sepharose equilibrated with Buffer 2 at a rate of 2 ml per minute. 40 ml of buffer 2 was then flowed to the column to remove proteins that were not adsorbed onto the sugar affinity resin. Then, buffer solution 2 containing 0.2 M of methyl-α-D-mannopyranoside was flowed to the column to elute the protein adsorbed on the resin, and collected in 3 ml fractions. The collected fractions were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by silver staining or western blotting to confirm that only gD glycoprotein was adsorbed and eluted. The results are shown in FIGS. 5 and 6.

5 is the result of silver staining after 10% SDS-PAGE, the first column is a protein of standard molecular weight, the second column is a solution added to the Con-A Sepharose column after dialysis, and the third column is Con-A 3 Proteins not adsorbed to the Paros resin, the fourth row represent fractions eluted after adsorption to Con-A.

FIG. 6 shows 10% SDS-PAGE followed by Western blotting according to Reference Example 2. The first column is a protein having a standard molecular weight, and the second column is a solution added to a Con-A Sepharose column after dialysis. Heat represents the protein that is not adsorbed to the Con-A Sepharose resin, and the fourth rows represent the fractions that are eluted after adsorption to Con-A.

The purity of the target protein purified by the purification method of the present invention was calculated by a known dilution method by silver staining, the yield was 90% or more, the yield was 50% or more.

As described above, according to the purification method of the present invention, it is possible to stably and economically separate and purified the gD glycoprotein of HSV-2 expressed from recombinant CHO cells.

1 is a flowchart of a process for isolating and purifying gD glycoprotein expressed from recombinant CHO cells,

2 is a result of staining with Coomassie blue after electrophoresis of the elution chromatography ion exchange chromatography during the purification process of the present invention,

3 is a result of Western blotting after electrophoresis of the eluate chromatographed during the purification process of the present invention,

4 is a result of silver dyeing after electrophoresis of the ion exchange chromatographic eluate during the purification process of the present invention,

5 is a result of silver dyeing after electrophoresis of the eluate chromatographed with affinity chromatography during the purification of the present invention,

Figure 6 is the result of Western blotting after electrophoresis of the eluate chromatographed affinity chromatography during the purification process of the present invention.

Claims (3)

In purifying gD glycoprotein of HSV-2 (Herpes Simplex Virus type 2) expressed from recombinant Hamster Ovary (CHO) cells, the culture medium of the recombinant CHO cells is centrifuged, the supernatant is dialyzed, and the dialysate is cationic. Purification method comprising exchange chromatography and sugar affinity chromatography. The method of claim 1, S- or CM-sepharose is used as the resin in the cation exchange chromatography, and eluted with sodium chloride in a linear concentration gradient of 0.0 to 0.3 M in a buffer solution of pH 6.5 to 7.5 containing bis-tris or phosphate. How to. The method of claim 1, In the sugar affinity chromatography using Con-A as a sugar affinity resin, characterized in that eluted with 0.2 to 0.5 M methyl-α-D- mannofranoside.