KR100447578B1 - A cell-based assay kit and method for measuring the activity of HCV NS5B replicase and NS3 helicase - Google Patents

Abstract

The present invention is a bacterium called Escherichia coli , which is a bacterium that expresses the activity of an autologous gene replication enzyme (RNA-dependent RNA polymerase, so-called NS5B replicaase and NS3 helicase) of hepatitis C virus (hereinafter referred to as HCV). It relates to kits and methods that can be measured in cells. The present invention can be used to screen antiviral agents by screening libraries derived from natural or non-natural products.

Description

A cell-based assay kit and method for measuring the activity of HCV NS5B replicase and NS3 helicase}

The present invention relates to kits and methods for measuring the activity of autologous replication enzymes of HCV in a cell. In the case of HCV, it does not grow well in cell culture, so it is very difficult to confirm the effect of the inhibitor intracellularly. However, the kits and methods of the present invention allow for the mass screening of candidate substances for use as HCV proliferation inhibitors in a short time.

HCV is a virus that is mainly transmitted by blood transfusions, and infects more than 3% of the world's population (see Choo, QL, etc.). Science 244 : 359-362 (1989) and Choo, QL et al., Genetic organization and diversity of the hepatitis C virus, Proc. Nat. Acad. Sci. USA. 88 : 2451-2455 (1991). HCV infection is a fatal disease that can progress from chronic hepatitis to liver cirrhosis and liver cancer. However, no effective vaccines or therapeutics have been developed. The genome of HCV is about 9.8 kilobases (kb) of positive sense RNA [Hwang LH et al., Involvement of the 5 'proximal coding sequences of hepatitis C virus with internal initiation of viral translation. Biochem Biophys Res Commun. 252 : 455-60 (1998) and Zhang J et al., A single nucleotide insertion in the 5'-untranslated region of hepatitis C virus leads to enhanced cap-independent translation. Virology 261 : 263-70 (1999)]. Self-replication of this gene is caused by NS5B replicaase (RNA-dependent RNA polymerase) produced by HCV itself (see Behrens, S. et al., Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus). . EMBO J. 15 : 12-22 (1996); 6. Yuan, Z. et al. , Expression, purification and partial characterization of HCV RNA polymerase, Biochem. Biophys. Res. Comm. 232 : 231-235 (1997); Ferrari, E. et al., Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in E. coli, J. Virol. 1649-1654 (1997); And Park, C., Kee, Y., Lee, J., Oh, J., park, J, and Myung, H. 1999. Purification and characterization of recombinant hepatitis C virus replicase. J. Microbiol. Biotechnol . 9 : 881-884 (1999). Its action is to recognize the non-toxic region (3 'UTR) at the 3' end of the HCV RNA (+) strand genome, thereby producing (-) strand RNA, and using this (-) strand RNA as a template. The enzyme, which recognizes the 3 'end of the RNA, goes through the process of producing a positive sense RNA. At this time, the double strand of the (+) and (-) RNA formed briefly is NS3 helicase of HCV [Gwack Y et al., Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein, Biochem Biophys Res Commun . 225 : 654-9 (1996), which is then released back to a single strand to serve as a template for the subsequent polymerization by the NS5B enzyme.

Both of these enzymes are essential for self-replicating HCV, but not necessary for the host, making them a good target for antiviral development.

The development of antiviral drugs depends largely on two methods. One is to design an inhibitor for that target material based on its structure. The other is to screen libraries of natural / non-natural materials using kits that can adequately analyze the activity of the target material. Retrieving the activity of hundreds of thousands of substances from a library is very time consuming and expensive. Therefore, the development of antiviral drugs is a key to success whether there is a method that can be analyzed quickly and at a low cost.

Screening of the library is again accomplished by two different methods.

One is to measure the activity of the enzyme in vitro, and the other is to measure the activity of the enzyme in cells. Both methods have advantages and disadvantages. In vitro measurements can be very rapid, but often have the problem of using radioisotopes, and their biological significance can be overlooked. That is, there is a disadvantage in that the substance can be absorbed into the cell, has no adverse effect on the cell itself, or cannot be transformed into another substance through metabolic pathways within the cell.

On the other hand, in the case of an assay using cells, the analysis period is relatively long, and the effect of a substance that is not absorbed into the cells is disadvantageous.

Therefore, the two methods can be said to be complementary in nature.

HCV can only grow in humans and chimpanzees. Therefore, library screening with animals is not possible due to ethical and cost issues. It is also well known that screening with human cell lines is not possible or that HCV does not grow well in cell culture. As an alternative to this, HCV replicon has been reported, but the efficiency thereof is still low, which is not suitable for high-speed screening of the library.

Thus, the present invention has the advantages of cell-based assays, but also a kit for measuring enzyme activity and a method for measuring the inhibitory effect quickly, as well as kits for screening inhibitors that inhibit the autologous enzyme activity of HCV using the same. And a screening method.

1 illustrates the principle of operation of the intracellular assays disclosed herein.

Fig. 2 shows RT-PCR of (-) sense mRNA made by T7 RNA polymerase in a cell for intracellular assay of the present invention and (+) sense RNA made by HCV NS5B replicaase using the template as a template. The photo confirmed using.

Figure 3 is a photograph confirming the production of NS3 helicase and NS5B replicaase of HCV in cells for the intracellular assay of the present invention using Western blot.

4 is a view showing the results of performing a luciferase assay using two kinds of plasmids actually manufactured in accordance with the configuration of the present invention.

One aspect of the present invention provides a method for the simple and rapid measurement of the activity of two enzymes (NS5B replicaase and NS3 helicase) required for replication of HCV in a cell. Includes:

(a) teeth that are bacteria. Expressing autologous replication enzymes of HCV, namely NS5B replicaase and NS3 helicase, in coli cells;

(b) cloning a series of DNA sequences having a reporter gene downstream of the ribosomal binding site in another plasmid or chromosome in the same cell;

(c) transcription of the construct of step (b) to produce mRNA;

(d) replicating the mRNA produced in step (c) as a template;

(e) decoding the (+) sense mRNA generated in step (d) to express the reporter; And

(f) measuring the activity of the autologous replicating enzyme of HCV using the expressed reporter.

Such a method can be explained through FIG. 1, which schematically illustrates the configuration of the present invention.

1 is this. It is a conceptual diagram which shows typically the system for analyzing the action | action of NS5B polymerase and NS3 helicase of HCV in coli cells. That is, the luciferase gene (also negative sense) inserted downstream of the 3 'UTR of the negative sense RNA of HCV is replicated by the HCV NS5B polymerase to the opposite sense ((+) sense) and NS3 helicara After decoction by the agent, luciferase is produced when detoxification using the bacterial ribosomal binding site occurs. At this time, two HCV self-replicating enzymes are generated from a structure inserted into another plasmid or chromosome which can coexist in the same cell.

Looking at the configuration of each step in detail as follows.

Expression of Autologous Replication Enzymes of HCV in Bacteria

Expression of autologous replicating enzymes of HCV in bacteria, NS5B replicaase and NS3 helicase, is achieved by the bacterial transcription / detoxification system. Briefly, the process is first cloned into an expression vector (eg, plasmid) or chromosome that is capable of expressing these two enzymes in bacterial cells. Next, transcription occurs using a bacterium or bacteriophage promoter, and the ribosome is recognized on the generated mRNA to recognize and decode a ribosome binding site (rbs) located in front of the open reading frame. This expression then occurs within the plasmid or chromosome of the bacterium.

Substantial expression of autonomous replication enzymes of HCV can be seen from FIG. 3. Figure 3 is a photograph confirming the actual expression of the polymerase and helicase of HCV in the cells of the present invention by Western blot using anti-polymerase serum and anti-helicase serum.

Construction of Reporter Expression Constructs

Plasmids or chromosomes in the same cell are constructed to have the following configuration. A series of DNA sequences having a ribosomal binding position downstream of the 5 'UTR of HCV and a reporter gene downstream thereof are cloned downstream of the bacterial or bacteriophage promoter to have a negative sense based on the reporter gene. Using only this configuration, even if the reporter gene is transcribed by a bacteriophage phage promoter, the reporter is not expressed.

In this case, as a reporter, a reporter which is generally used, such as luciferase, beta-galactosidase, alkaline phosphatase, and lux, may be used, but is not limited thereto. The reporter gene cloned in the plasmid or chromosome causes primary transcription by a bacterium or bacteriophage promoter, and the reporter is expressed with a negative sense in the primary transcription of the construct and thus exhibits no reporter activity. In this case, as a promoter, a promoter commonly used such as lac, tac, trc, T7, T3, and SP6 may be used, but is not limited thereto. A ribosomal binding site (rbs) is located between the promoter and the reporter gene where ribosomes of the bacterium can recognize and begin to decipher. Between the rbs and the promoter, the 5 'UTR of HCV is placed in the negative sense (based on the genomic strand of HCV).

Reporter Expression

Upon initiation of transcription by the bacterial transcription system, the reporter expression construct is transcribed by the promoter of the bacterial or bacteriophage and the RNA polymerase to produce a negative sense, wherein the sense is based on the reporter gene.

The resulting negative sense mRNA acts as a template and is replicated by NS5B replicaase and NS3 helicase of HCV expressed in the same cell. At this time, the NS5B replicator of HCV recognizes the secondary structure of 5'UTR, which is the current (-) sense, so that replication is possible. The reason is that in the mRNA state the 5 'UTR is actually located at 3', which provides the same situation as when HCV replicates its RNA genome.

When bacterial autologous replication enzymes and primary transcribed mRNA are produced in bacterial cells, the NS5B replicators of HCV produce a secondary structure (-) sense RNA of the HCV at the 3 'end of the primary transcribed mRNA. Coincides with the 3 ′ end), and this mRNA is used as a template to replicate the RNA of the opposite strand. At this time, the newly produced secondary RNA product has a structure of the rbs-reporter gene with a positive sense so that the reporter is expressed. At this time, the double-helix structure between the mRNA, which is the primary transcriptional product, and the secondary RNA, is released by NS3 helicase of HCV to help the reporter expression. In other words, RNA, a secondary product, is produced only when NS5B replicaase of HCV is active. In addition, the translation of the reporter requires unwinding of the strand by NS3 helicase of HCV.

Whether the first RNA, the first transcript ((-) sense) and the second RNA ((+) sense) replicated therefrom, may be substantially generated through FIG. 2. FIG. 2 is a diagram confirming that RNAs of (-) sense and (+) sense are respectively generated from the reporter plasmid using RT-PCR. Lane 2 detects the second RNA in the absence of autologous replication enzyme of HCV, and lane 3 detects the first RNA in the same state. The first RNA was produced in large quantities, but the second RNA produced only a small amount. Lane 4 confirms that the second RNA is produced in the presence of HCV autologous replication enzyme, and lane 5 confirms that the first RNA is produced in the same state. Comparing lanes 2 and 4, the density of lane 4 is about 8 times the density of lane 2. This is consistent with the luciferase assay results of FIG. 4.

Determination of Enzyme Activity

The RNA of the new strand cloned by the HCV enzyme becomes a (+) sense based on the reporter, and when the ribosome recognizes rbs on the RNA and the translation occurs, the reporter is expressed to measure the action of the HCV enzyme.

For example, luciferase can be used as a reporter, which is involved in oxidation reactions with oxygen and compounds known as luciferin, in which the energy produced is emitted in the form of light. As a result, it is possible to confirm the presence of luciferase by the energy emitted in the form of light, thereby confirming the presence of a self-replicating enzyme of HCV which enables the expression of luciferase.

According to another aspect of the present invention,

(a) a gene capable of expressing an autonomous replication enzyme of HCV cloned into a plasmid; And

(b) Reporter genes cloned into plasmids of (a) and other plasmids or bacterial chromosomes

Kits for measuring autologous replication enzyme activity of HCV, which include bacterial cells containing the same, are provided.

The inventors of the present invention used a gene recombination technique commonly used in the art to express reporters constructed based on pACYC184 purchased from New England Biolabs, Inc. (NEB), Beverly, Mass., USA, and the United States. A plasmid (pSKN35) expressing HCV's autonomous replication enzymes (ie, NS5B replicaase and NS3 helicase) constructed based on pBlueScriptSK purchased from Stratazine, La Jolla, Calif. It was introduced into E. coli cells using the usual transformation techniques used.

Transformed E. Coli cells were deposited on 22 October 2001 with the Biotechnology Research Institute Genetic Bank (KTCT), an international depository, under the Budapest Treaty on Microbial Deposits, and were assigned KCTC 10094BP as an accession number.

Use of the System-Screening of HCV Inhibitors

In addition, the present invention

(a) a gene capable of expressing an autonomous replication enzyme of HCV cloned into a plasmid; And

(b) a reporter gene cloned into the plasmid of (a) and another plasmid or said bacterial chromosome

There is provided a screening kit for screening a natural or non-naturally occurring library comprising bacterial cells containing a screening kit for screening antiviral agents, wherein the reporter is present if the kit contains a substance that inhibits HCV's autonomous replication activity. It is not expressed, and the inhibitory effect of the substance can be measured.

In addition, the kit can be used to screen for substances that inhibit the activity of NS5B replicaase and NS3 helicase of HCV.

The screening method of the present invention includes the following steps:

(a) culturing the bacterial cells of the present invention to prepare a culture;

(b) propagating the culture;

(c) adding the material to be tested to the culture and further propagating;

(d) collecting cells, removing supernatant and washing;

(e) resuspending the cells in cell lysis buffer and leaving at room temperature; And

(f) taking an appropriate amount of sample to measure the activity of the reporter.

The screening method can be used to quickly search for a material capable of inhibiting autologous replication enzymes of HCV from a library of natural or non-natural products.

Example

Hereinafter, the present invention will be further described by way of examples. However, the invention is not limited to the described embodiments.

The kit for measuring autologous enzyme activity of HCV of the present invention was tested for the effect of inhibiting autologous enzyme activity of HCV in nature.

E. coli for measuring autologous enzyme activity of HCV. Manufacturing of collie

In this experiment, a binary plasmid system containing two plasmids of HCV, one plasmid expressing NS5B replicaase and NS3 helicase (pSKNS35) and another plasmid expressing reporter (pACluc) was used.

First, plasmid pSKNS35, which expresses two autologous replication enzymes of HCV, was constructed based on pBluescriptSK, purchased from Stratazine, La Jolla, California. The helicase and NS5B polymerase domains of HCV, each of which has a ribosomal binding site upstream of the open reading frame at multiple cloning sites of pBluescriptSK, were amplified and cloned by PCR. The following primers were used to perform PCR: helicase forward-AATTGGTACCAAAGAGGAGAAATTAACTATGGCGCCCATCACGGCCTAC, where KpnI site, rbs, initiation codon, helicase N-terminus are located in turn; Helicase reverse-AATTAAGCTTTCAAGTGACGACCTCCAGGTC, followed by the HindIII site, the stop codon, and the C-terminal end of the helicase; Replicaase Forward-AATTGAATTCAAAGAGGAGAAATTAACTATGTCAATGTCCTACACATGG, with EcoRI site, rbs, initiation codon, replicant N-terminus; Replicaase reverse—AATTTCTAGATCAGCGGGGTCGGGCACGAGA, followed by the XbaI site, the stop codon, and the C-terminal end of the replicator. They were amplified using a commonly used PCR procedure with cDNA of HCV as a template and cloned into plasmids using ligase. They are transcriptionally regulated by the T7 promoter of pBluescriptSK and are translated by their respective ribosomal binding sites.

Next, the plasmid pACLuc expressing the reporter was constructed based on pACYC184 purchased from New England Biolabs, Inc., Beverly, Massachusetts. Using a restriction enzyme, the tetracycline resistance gene of pACYC184 was cut out, and the part of the bacteriophage T7 promoter, the reporter gene in the antisense direction (in this experiment, the luciferase was used as the reporter), and the ribosome binding position in the antisense direction (shine Dalgano) PACLuc was constructed by inserting the 5 'UTR (IRES) of HCV of the sequence) and (-) sense through PCR and ligase linkage, respectively. The detailed process is as follows. First, the luciferase, rbs, and 5 'UTR of HCV were cloned into the above-described pBluescripSk plasmid to have a negative sense in the above order; Luciferase forward-ATGCGACGTCATGGAAGACGCCAAAAC, which is preceded by the AatII site; Luciferase reverse-ATGCCTCGAGTTACAATTTGGACTTTC, where the XhoI site and the stop codon are located; 5 'UTR forward-ATGCGGATCCACCCAGGCCGCCAGCCC, where the BamHI site and the 5' sequence of 5 'UTR are located; 5 'UTR reverse-ATGCGACGTCCTGTGGGCGGCGGTTG, where the AatII site and the 3' sequence of the 5 'UTR are located. These two PCR products were attached with ligase using the AatII site and cloned into pBluescripSK using BamHI and XhoI at both ends. In this state, oligomers having rbs of GAAGGAG were prepared between the AatII sites, and were cloned so as to be located in front of luciferase using the AatII sites. In this state of plasmid, PCR products were cloned between the BamHI and HindIII sites of pACYC using the following two primers; Forward-AATTGGATCCTTGTAAAACGACGGCC, which in turn enters the BamHI site and the T7 promoter; Reverse-AATTAAGCTTGGCCGCCAGCCCCCG, followed by the HindIII site and the beginning of the 5 'UTR.

Finally, the two constructed plasmids were transformed into E. coli using transformation techniques commonly used in the art, such as calcium chloride, electroporation, high-speed dropping, and the like. Introduced into coli cells.

Screening of Inhibitors That Inhibit the Autonomous Enzyme of HCV

E. transformed as described above. Coli was used to measure the autonomous replication inhibitory activity of HCV of unknown substances.

Above. Collie was incubated overnight under shaking conditions at 37 ^° C. in LB medium and prepared 3 ml of fresh culture the next morning. The culture was grown under shaking conditions of 37 ^° C. until the OD ⁶⁰⁰ of the culture was 0.6 while maintaining the plasmid by adding an appropriate amount of antibiotic to the culture. At this point, IPTG required for gene expression was added to a concentration of 1 mM and expression was induced for 2 hours. At this point, the test compound extracted from the algae was added to the culture and further grown for 2 hours under shaking conditions of 37 ^° C. Cells were collected by centrifugation, the supernatant was removed and washed once with distilled water. The cells were resuspended in cell lysis buffer and left at room temperature for 10 minutes. A luciferase reaction solution purchased from Promega, Madison, Wisconsin, was added, and luminescence was measured using a luminometer. The same screening method was applied in the same manner to test the inhibitory effect for several fractions separated.

result

Inhibitory Effect of HCV on Autonomous Replication Enzyme Activity Inhibitor Luciferase Relative Activity (%) Control 100 T0 25 88 T0 50 41 T2 25 21 T4 25 36 T6 25 27

As can be seen from the data in Table 1, the inhibitors of each fraction showed luciferase activity of 21% to 88% compared to the control without the inhibitor. This value is the result of normalization by measuring the cytotoxic effect of each fraction. This confirms the inhibitory effect on the autologous replication enzyme activity of HCV.

According to the present invention, a kit for measuring the activity of intracellular HCV replication enzymes (NS5B replicaase and NS3 helicase) and the same were able to quickly and largely search for HCV antiviral agents from natural / non-natural libraries.

Claims (9)

(a) a gene capable of expressing an autologous replication enzyme of HCV cloned into a plasmid; And (b) Reporter genes cloned into plasmids of (a) and other plasmids or bacterial chromosomes A kit for measuring autologous enzyme activity of HCV, comprising bacterial cells containing The gene capable of expressing the autologous replication enzyme of HCV of (a) is a gene capable of expressing NS5B replicaase and NS3 helicase, and another separate plasmid of (b) is plasmid pACLuc, and (b) The reporter gene of) is a kit for measuring autologous enzyme activity of HCV, which is a gene expressing luciferase. The kit of claim 1, wherein the gene capable of expressing the autologous replication enzyme of HCV is cloned into plasmid pBluescriptSK. delete The kit of claim 1, wherein said bacterial cell is KCTC 10094BP. delete (a) expressing an autologous replication enzyme of HCV in the bacterial cell of claim 1; (b) cloning a sequence of DNA sequences carrying the reporter gene downstream of the ribosomal binding site in another plasmid or chromosome in the same cell; (c) transcription of the construct of step (b) to produce mRNA; (d) replicating the mRNA produced in step (c) as a template; (e) decoding the (+) sense mRNA generated in step (d) to express the reporter; And (f) measuring the activity of the autologous replication enzyme of HCV using the expressed reporter Method for measuring autologous replication enzyme activity of HCV comprising a. (a) culturing the bacterial cells of claim 1 to prepare a new culture; (b) propagating the culture; (c) adding the material to be tested to the culture and further propagating; (d) collecting cells, removing supernatant and washing; (e) resuspending the cells in cell lysis buffer and leaving at room temperature; And (f) taking an appropriate amount of sample to measure the activity of the reporter A method for screening inhibitors of autologous replication enzymes of HCV, comprising. 8. The method of claim 6 or 7, wherein said bacterial cell is KCTC 10094BP. delete