KR19980069021A - Histidine-labeled Korean Non-Structural Protein 3 of Hepatitis C Virus and Its Preparation Method - Google Patents

Abstract

The present invention relates to non-structural protein 3 of the histidine-labeled hepatitis C virus and a method for preparing the same.

More specifically, the present invention relates to a recombinant protein labeled with histidine at the amino terminus of a nonstructural protein 3 that exhibits protease activity important for the proliferation of Korean hepatitis C virus, an expression vector capable of producing the same in E. coli, and C using the same. The present invention relates to a method for producing non-structural protein 3 of hepatitis virus, wherein the protease protein of the present invention can be easily isolated and purified including histidine label, and widely used for screening proliferation inhibitor of hepatitis C virus. Can be.

Description

Histidine-labeled Korean Non-Structural Protein 3 of Hepatitis C Virus and Its Preparation Method

The present invention relates to non-structural protein 3 of the histidine-labeled hepatitis C virus and a method for preparing the same.

More specifically, the present invention relates to a recombinant protein labeled with histidine at the amino terminus of a nonstructural protein 3 that exhibits protease activity important for the proliferation of Korean hepatitis C virus, an expression vector capable of producing the same in E. coli, and C using the same. The present invention relates to a method for producing non-structural protein 3 of hepatitis virus, wherein the protease protein of the present invention can be easily isolated and purified including histidine label, and widely used for screening proliferation inhibitor of hepatitis C virus. Can be.

Hepatitis C Virus is a major cause of hepatitis known to cause non-A or non-B hepatitis, which not only invades humans and causes acute or chronic hepatitis, but also leads to cirrhosis or liver cancer. (Alter, HJ et al., 1989, N. Eng. J. Med, 321: 1494-1500). Hepatitis C virus has been reported to infect 1% of the world's population, with an estimated one million new cases of hepatitis C virus per year (Purcell, 1994, FEMS Microbial. Rev). , 14: 181-192; van der Poel, 1994, Hepatitis C Virus: Current Studies in Hematology and Blood Transfusion, HW Reesink ed., Pp. 137-193; Alter, HJ, 1988, AJ Zuckerman ed., Viral Hepatitis and Liver Disease, pp. 537-542). In addition, about 40-50% of patients infected with hepatitis C virus become chronic hepatitis patients, and about 20% become more advanced and become cirrhosis or liver cancer patients (Dienstag, JL, 1986). , Semin.Liver.Dis, 6: 67-81).

Hepatitis C virus can be recovered by inoculating chimpanzees with plasma from non-Hepatitis A or non-B hepatitis patients (Bradley, D. W. et al., 1988, Gatroenterology, 88: 773-779). The hepatitis C virus thus obtained has a gene in the form of a positive strand ribonucleic acid consisting of 9400 bases, from which a polyprotein consisting of 3010-3033 amino acids is produced (Choo, QL et al., 1989, Science). , 244: 359-362, Choo, QL et al., 1991, PNAS, 88: 2451-2455). Considering the gene structure described above, hepatitis C virus is similar to flaviviruses or pestiviruses, so hepatitis C virus is classified as a new genus belonging to Flaviviridae. (Van Doorn, 1994, review. J. Med. Virol, 43: 345-356).

The polyprotein of hepatitis C virus consists of core-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B from the amino terminus. That is, at the amino terminus of the polyprotein, there are structural proteins (core, E1, E2), which are components of the nucleocapsid and envelope of the virus, and the hepatitis C virus proliferates at the carboxy terminus thereof. Essential nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) are configured to be located. This polyprotein is synthesized by intracellular signal peptidase and non-structural protein 2-3 and non-structural protein 3, which are proteases present in the virus, to cut 9 specific sites of the precursor polyprotein. Produce viral proteins. Specifically, nonstructural protein 3 has been shown to act on four cleavage sites present between nonstructural proteins 3 and 4A, between 4A and 4B, between 4B and 5A, and between 5A and 5B (Hijikata, et al., 1991 , PNAS, 88: 5547-5551, Bartenschlarger, et al., 1994, J. Virol, 68: 5045-5055, Grakoui, et al., 1993, J. Virol, 67: 1385-95, Tomei, et al. , 1993, J. Virol, 67: 4017-4026).

A universal treatment for the effective treatment of chronic infection of hepatitis C virus has not been developed yet, and almost the only administration of interferon-alpha as a treatment for hepatitis C virus is carried out. However, when the interferon-alpha is administered to the hepatitis patients, only 15-20% of patients show the therapeutic effect (Hoofnagal, J. H., 1994, And. Intern. Med., 39: 241-275). In addition, interferon-alpha is the most widely found in the world, especially in Asia, the Americas, and Europe. Hepatitis C virus-1b is a major viral type that causes hepatitis C and has a high rate of progression to cirrhosis or liver cancer. There is a problem that the effectiveness is not widely used to treat hepatitis caused by hepatitis C virus (van Doorn, LJ, 1994, review, J. Med. Virol. 43: 345-356). Therefore, it is very important to develop new treatments for the infection of hepatitis C virus.

Nonstructural protein 3 of hepatitis C virus plays a very important role in cleaving precursor polyproteins to produce mature nonstructural proteins 3, 4A, 4B, 5A and 5B (Kim, JL et al., 1996, Cell, 87: 343-355). This nonstructural protein 3 is composed of a serine protease, nucleotide dephosphatase (NTPase), and an amino acid motif that exhibits RNA helicase activity in a single protein, and the helicase function is present at the carboxy terminus. Degradase function is known to be present at the amino terminus (Kim et al., 1995, BBRC 215: 160-165, Han et al., 1995, J. Gen. Virol 76: 985-993). During the proliferation of hepatitis C virus, the protease active site of the nonstructural protein 3 plays an important role in the production of viral particles by cleaving polyprotein, thus obtaining a nonstructural protein 3 containing the enzyme active site. If an inhibitor or the like can be selected, an effective treatment for hepatitis C can be easily developed.

In order to produce a large amount of non-structural protein 3 of hepatitis C virus, which can be used to develop an effective hepatitis C therapeutic agent, the present inventors performed a polymerase chain reaction with a viral gene as a template to perform the non-structural protein 3 gene. The resultant was cloned into an expression vector capable of producing a recombinant protein having a histidine-labeled amino acid at its amino terminus, and then the non-structural protein 3 was expressed in Escherichia coli using the expression vector and separated and purified by a histidine-labeled affinity column. By this, the present invention was completed.

It is an object of the present invention to provide a nonstructural protein 3 of hepatitis C virus labeled with histidine. Specifically, the present invention provides non-structural protein 3 of the Korean hepatitis C virus in the form of a recombinant protein comprising a histidine label and a protease active site at its amino terminus.

It is also an object of the present invention to provide an expression vector for producing non-structural protein 3 of hepatitis C virus labeled with histidine. Specifically, the present invention provides an expression vector pRSET-NS3 comprising a nucleotide sequence encoding a histidine label at the 5'-end of the nonstructural protein 3 gene.

In another aspect, the present invention provides an E. coli transformant (Accession Number: KCTC 8778P) transformed with the expression vector pRSET-NS3.

Another object of the present invention is to provide a method for preparing non-structural protein 3 of hepatitis C virus labeled with histidine using the transformant.

Specifically, the present invention cultures a transformant to induce the expression of the nonstructural protein 3, and then obtains a crude cell eluate to perform histidine-labeled affinity chromatography to perform the nonstructural protein 3 including the protease active site. Isolate and purify. At this time, it is preferable to use an imidazole concentration gradient as the elution solvent used for histidine labeled affinity chromatography.

In addition, the protein prepared above by the method of the present invention can be used to select protease inhibitors involved in the growth of the virus. In addition, the inhibitor may be used as a therapeutic agent for hepatitis, liver cirrhosis and liver cancer caused by hepatitis C virus.

Figure 1 shows the manufacturing process of the expression vector pRSET-NS3 producing non-structural protein 3 of the Korean type hepatitis C virus labeled histidine of the present invention.

Figure 2a shows the results of polyacrylamide gel electrophoresis by isolated and purified non-structural protein 3 of the Korean hepatitis C virus produced by E. coli BL21 (DE3) transformed with the expression vector pRSET-NS3 of the present invention.

Lane 1: total cell lysate (TCL) before purification

Lanes 2-6: eluted fractions

Figure 2b shows the results of Western blot separation and purification of the non-structural protein 3 of the Korean hepatitis C virus produced by E. coli BL21 (DE3) transformed with the expression vector pRSET-NS3 of the present invention.

The present invention provides a nonstructural protein 3 of hepatitis C virus labeled with histidine. Specifically, the present invention provides non-structural protein 3 in the form of histidine-labeled recombinant protein comprising a protease active site involved in its multiprotein maturation in Korean hepatitis C virus.

At this time, the Korean hepatitis C virus is obtained from the serum of a Korean hepatitis C patient, whose gene is different from the existing US and Japanese hepatitis C virus and is called the Korean hepatitis C virus.

The present invention produces a nonstructural protein 3 comprising a protease active site of hepatitis C virus by inserting the nonstructural protein 3 gene of hepatitis C virus into an expression vector and expressing it in E. coli.

Accordingly, the present invention provides an expression vector capable of producing non-structural protein 3 of hepatitis C virus in E. coli.

Specifically, the present invention is obtained by carrying out a polymerase chain reaction in which the nonstructural protein 3 gene of hepatitis C virus is composed of the Korean hepatitis C virus gene. At this time, oligonucleotides having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are synthesized and used as primers (see SEQ ID NO: 1 and SEQ ID NO: 2). Specifically, the primer of SEQ ID NO: 1 has a restriction enzyme Nhe I recognition site (GCTAGC) and comprises a base sequence encoding amino acids 1027 to 1032 on the polyprotein of the Korean hepatitis C virus, The primer has a restriction enzyme Eco RI recognition site (GAATTC) and one end codon and comprises amino acids 1653-1657 on the Korean hepatitis C virus polyprotein.

The expression vector of the present invention was prepared by inserting the non-structural protein 3 gene of the hepatitis C virus obtained by the above procedure into an E. coli expression vector pRSET-A comprising a nucleotide sequence corresponding to a T7 promoter and six histidines (see FIG. 1). ).

Specifically, the non-structural protein 3 gene includes a restriction enzyme recognition site to facilitate cloning into the E. coli expression vector at the 5'-end, and the corresponding 3 'end includes a termination codon and a restriction enzyme recognition site, The expression vector of is configured such that the synthesis of the protein at the start codon is started, the non-structural protein 3 including 6 histidines is expressed, and then the synthesis of the protein is completed at the artificially inserted end codon.

The expression vector of the present invention was named pRSET-NS3 and transformed into Escherichia coli, and the transformant was deposited on January 29, 1997 to the Gene Bank of Korea Institute of Science and Technology, Biotechnology Research Institute (Accession Number: KCTC). 8778P).

The present invention also provides a method for producing non-structural protein 3 of E. coli hepatitis C virus in E. coli using the above-described expression vector and E. coli transformants.

Specifically, in order to express non-structural protein 3 in the E. coli transformant transformed with the E. coli expression vector pRSET-NS3, cultivation was carried out using a liquid medium containing ampicillin and the like, followed by induction of protein expression in large quantities. Incubate. Next, E. coli cells are expressed by crushing E. coli cells expressing the recombinant protein.

Using the expression vector pRSET-NS3 of the present invention, since the nonstructural protein 3 of the Korean hepatitis C virus is produced in the form of a recombinant protein containing six histidine labels at its amino terminus, it is easily separated by histidine-labeled affinity column chromatography. It is purified. At this time, it is preferable to use an imidazole concentration gradient as the elution solvent used for histidine labeled affinity chromatography.

The non-structural protein 3 containing the histidine label prepared above is not only easily separated and purified by affinity column chromatography, etc., but also stable, so that the enzymatic activity of the protein can be maintained in the course of purification.

The molecular weight and purity of the non-structural protein 3 of the Korean-type hepatitis C virus isolated and purified above are confirmed by performing polyacrylamide gel electrophoresis and western blot (see FIGS. 2A and 2B).

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1 Preparation of Expression Vector of Nonstructural Protein 3 of Korean Hepatitis C Virus

(Step 1) Synthesis of primer for polymerase chain reaction

In order to obtain the genes of the non-structural protein 3 region of the Korean hepatitis C virus, a primer for polymerase chain reaction was synthesized using a nucleic acid synthesizer (DNA synthesizer, Applied Biosystems Inc. USA). By using the DNA sequence of the virus (Korean Patent Application No. 93-9913) is designed to include the base sequence corresponding to the 5'-end and 3'-end of the gene encoding the non-structural protein 3 site.

Specifically, the primer of SEQ ID NO: 1 has a restriction enzyme Nhe I recognition site (GCTAGC) and is designed to have a nucleotide sequence encoding amino acids 1027 to 1032 of the Korean hepatitis C virus, and the primer of SEQ ID NO: 2 is restricted. It was designed to have the enzyme Eco RI recognition site (GAATTC) and one end codon, and to have a base sequence coordinating amino acids 1653 to 1657 on the Korean hepatitis C virus polyprotein.

(Step 2) Amplification of the nonstructural protein 3 gene of Korean hepatitis C virus

In order to amplify the gene of the nonstructural protein 3 of the Korean hepatitis C virus, a polymerase chain reaction was performed as follows. Put the primer of SEQ ID NO: 1 of 100 pmole and the primer of SEQ ID NO: 2 of 100 pmole obtained in (Step 1) into the reaction tube, and as a template, plasmid vector M13-KHCV-L2 (Korean Patent Application No. 93-9913, entrusted) No. ATCC 75447) then 10 μl of 10-fold polymerization buffer (500 mM potassium chloride, 100 mM Tris-hydrochloric acid pH 9.0, 1% Triton X-100, 2.5 mM magnesium chloride), 10 μl of 2 mM dNTP, 2.5 units of Taq After adding the polymerase and distilled water to make the total volume 100 mu l, the polymerase chain was subjected to the conditions of 1 minute (denatured step) at 95 ° C, 1 minute (immersion step) at 55 ° C, and 2 minutes (polymerization step) at 72 ° C. The reaction was carried out 25 times. The resulting nucleic acid fragments were isolated on 7% polyacrylamide gels to confirm that 1900 base pairs of nucleic acids were amplified. Gene clean II kits (Gene clean II kit, Bio 101, USA) were obtained from 2% agarose gels. The nucleic acid fragment of about 1.9 kb containing the nonstructural protein 3 gene was obtained by isolate | separating and purification.

(Step 3) Preparation of an E. coli expression vector containing a nonstructural protein 3 gene of the Korean hepatitis C virus

In order to prepare the expression vector of the present invention, 3 µg of plasmid vector pRSET-A (Invitrogen, USA) was added to BM (Beringermannheim, Germany) buffer solution (10 mM Tris-HCl pH 7.5, 10 mM magnesium chloride, 50 mM sodium chloride, 1 mM). Dithiothreitol) with restriction enzyme Nhe I, complete digestion with Eco RI in Gibco buffer (50 mM Tris-HCl pH 8.0, 10 mM magnesium chloride, 100 mM sodium chloride) and then in 1% agarose gel Separation by electrophoresis yielded a 2.9 kb plasmid vector nucleic acid fragment.

Meanwhile, 5 μg of the nucleic acid fragment containing the non-structural protein 3 gene obtained in step (step 2) was digested with restriction enzyme Nhe I in BM buffer, and completely digested with restriction enzyme Eco RI in Gibco buffer solution, and then 1.9. A kb nucleic acid fragment was obtained.

Into the conjugation reaction vessel, 100 ng of 2.9 kb plasmid vector nucleic acid fragment and 100 ng 1.9 kb non-structural protein 3 were placed, and 2 μl of 10-fold conjugation reaction solution (50 mM Tris-HCl pH 7.8, 10 mM magnesium chloride, 10 mM Dithio Trentol, 1 mM ATP, 25 μg / ml bovine serum albumin) and 10 units of T4 DNA ligase were added, and distilled water was added to a total volume of 20 μl and reacted at 16 ° C. for 12 hours. After the conjugation reaction, E. coli HB101 cells were transformed, and then transformants were selected on a Luria agar plate containing 50 μg / ml of ampicillin and extracted expression vectors therefrom. It was confirmed that the E. coli expression vector pRSET-NS3 was obtained. Figure 1 illustrates the process for producing the expression vector pRSET-NS3.

E. coli transformant (BL21 (DE3) / pRSET-NS3) obtained by transforming E. coli BL21 (DE3) strain with the expression vector pRSET-NS3 on January 29, 1997 Was deposited (Accession Number: KCTC 8778P).

(Step 4) Expression of Nonstructural Protein 3 Gene of Korean Hepatitis C Virus in Escherichia Coli

E. coli cells transformed in (step 3) were shaken for 12 hours in liquid Luria medium (6% bactotrypton, 0.5% yeast extract, 1% sodium chloride) containing 50 μg / ml of ampicillin. Transfer 3 ml of the culture medium to 300 ml of liquid Luria medium, shake incubate at 37 ° C for about 6 hours, change the temperature to 23 ° C when the absorbance of the culture medium is about 0.5 at 600nm, and add IPTG so that the final concentration is 1mM. It was. After 3 hours of adding IPTG, E. coli cell precipitates were collected by centrifugation (Beckman J2-21 rotor; JA10) for 10 minutes at 6,000 rpm.

Example 2 Purification of Korean Hepatitis C Virus Nonstructural Protein 3

(Step 1) E. coli crushing

30 g of Escherichia coli obtained in the above culture was suspended in buffer 1 (20 mM sodium phosphate pH 7.6, 300 mM sodium chloride, 1 mM magnesium chloride, 10% glycerol, 0.1% Tween-20, 1 mM β-mercaptoethanol), and then ice bath Cells were disrupted by treatment at 50% power for 2 minutes using an ultrasonic grinder (Heat Systemas-Ultrasonics, Inc. W225, USA). The solution was centrifuged at 15,000 rpm for 30 minutes using a centrifuge (Beckman J2-21 rotor; JA20, USA), and the supernatant was taken and used for the next experiment.

(Step 2) Ni-chelating Histidine Affinity Gel Chromatography

The supernatant of (step 1) was flowed into a 5 ml histidine-labeled affinity resin column (Invitrogen, USA) previously equilibrated with buffer 1 of (step 1), where 6 histidines were fused to the amino terminus. Nonstructural protein 3 was bound to the resin. Proteins weakly attached by nonspecific binding were washed out with buffer 1 containing 50 ml of 5 mM imidazole, and buffer 1 containing 50 ml of 60 mM imidazole. The histidine-labeled protein was added to 10 ml of buffer solution 1 containing 60 to 1,000 mM linear concentration of imidazole, and the fractions were collected by eluting the proteins adsorbed on the resin. The collected fractions were subjected to 15% SDS-polyacrylamide gel electrophoresis to select fractions containing nonstructural protein 3-4A and then collected. Then using a dialysis membrane (spectrum, USA) of mwco 8000 (molecular weight cut off 8000) 20 mM sodium phosphate pH 7.6, 50 mM sodium chloride, 1 mM magnesium chloride, 5 mM β-mercaptoethanol, 10% glycerol and 0.1% twin- Dialysis was performed in a buffer of 20 compositions.

Example 3 Western Blotting Using Monoclonal Antibody of Nonstructural Protein 3

Purified nonstructural protein 3 was electrophoresed on a 12% SDS-polyacrylamide gel, and then PVDF membrane was used for 1 hour at 200 mA using a semi-phor transfer kit (Semi-phor transfer kit, Hoffer Scientific Instrument, USA). Moved. The membrane was soaked in blocking solution (0.2% gelatin in phosphate buffer solution) and blocked for 1 hour. After washing the membrane with phosphate buffer solution, monochloral antibody that recognizes amino acids No. 1252 through 1335 of hepatitis C virus non-structural protein 3 was diluted with antibody (0.2% gelatin, 1% Triton × 100, 1 mM EDTA in phosphate buffer solution). After diluting 3,000 times with 0.02% Thimerosal, the membrane was soaked and reacted for 1 hour. After rinsing the membrane with phosphate buffer solution, an anti-mouse secondary antibody (Hib) connected with Horse radish peroxidase (HRP) was 3,000-fold in the antibody dilution solution. Soak in the diluted solution for 1 hour. The membrane was washed with phosphate buffer solution and developed in HRP color reaction solution (Bio-Rad. USA) to confirm the presence of nonstructural protein 3.

The present invention mass-produces the non-structural protein 3 including the protease active site of the Korean hepatitis C virus in the form of histidine-labeled recombinant protein, and allows them to be easily separated and purified by affinity column chromatography or the like.

Therefore, the histidine-labeled nonstructural protein 3 of the present invention contains a protease active site and is stable, and thus can be used in various ways. Specifically, by using the nonstructural protein 3, it is possible to easily select a proliferation inhibitor of hepatitis C virus and the like and ultimately develop a therapeutic agent such as hepatitis, cirrhosis and liver cancer caused by the virus infection.

(Sequence list)

SEQ ID NO: 1

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 1]

(Sequence list)

SEQ ID NO: 2

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 2]

Claims (4)

Nonstructural Protein of Korean Hepatitis C Virus labeled with histidine 3. An expression vector pRSET-NS3 which produces the non-structural protein 3 labeled with histidine of claim 1. E. coli transformants transformed with the expression vector pRSET-NS3 of claim 2 (Accession No .: KCTC 8778P). A method for obtaining the non-structural protein 3 of claim 1 by culturing the E. coli transformant of claim 3 to induce the expression of a protein, then obtaining a crude cell eluate and performing histidine-labeled affinity chromatography using an imidazole concentration gradient.