KR100361884B1 - Total nucleotide sequence of korean type hepatitis c virus khcv-l2 and the amino acid sequence deduced therefrom - Google Patents

Abstract

PURPOSE: Total nucleotide sequence of Korean type hepatitis C virus KHCV-L2 and the amino acid sequence deduced therefrom are provided, thereby effectively preparing hepatitis C diagnosing reagent and vaccine. CONSTITUTION: The hepatitis C virus KHCV-L2 cDNA has the nucleotide sequence of figure 17. A polynucleotide has one nucleotide sequence selected from figures 1 to 16. The hepatitis C virus KHCV-L2 polypeptide has the amino acid sequence of figure 17. A polypeptide has one amino acid sequence selected from figures 1 to 15. A vector contains the KHCV-L2 cDNA of figure 17 or the polynucleotides of figures 1 to 16. A microorganism transformed with the vector is provided. The polypeptides of figures 1 to 15 are prepared by culturing the polynucleotides of figures 1 to 16 transformed microorganism.

Description

The present invention relates to a type of NANBH virus or hepatitis C virus designated as KHCV-L2 obtained from a Korean non-A, non-B hepatitis (NANBH) patient. It relates to a nucleotide sequence of a partial or whole cDNA gene of and the amino acid sequence inferred therefrom.

Generally, viral infections include Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), Hepatitis Delta Virus (HDV), and Hepatitis E Virus (HEV) cytomegalo. It has been known to be caused by virus (CMV) and Epsin bar virus (EBV), and its genes were identified in the 1980s, so that diagnostic reagents and vaccines were developed using them, and much progress has been made in the diagnosis and prevention of infection.

However, other forms of hepatitis, called non-A non-B infections, which differ from those described above, account for 80-90% of hepatitis after transfusion (Lancet 2,838-841 (1975)), and about 50% of patients have cirrhosis. (Cirrhosis) and Hepatocellular Carcinoma are known to be a terrible epidemic, and only the fact that human non-A non-B-type infections can infect chimpanzees is known. Hollinger, FB et al, Intervirology 10, 60-68 (1978); Bradley, D.W. et al., J. Med. Viro1. 9, 253-269 (1979)), the nature and mechanisms of antigen-antibodies associated with non-A non-B hepatitis have not been discovered until recently. The reason for this is that a small number of viruses are present in the blood of non-A non-B type infected patients and there is no information related to the antigens and antibodies of the viruses.

Bradley et al. Infected the plasma of NANBH patients with chimpanzees, secured a large amount of hepatitis serum, and established methods for the isolation and recovery of viruses (Bradley, DW et a1, Gastroenterology 88, 773-779 (1985). The physicochemical properties of the virus have been elucidated, opening the way for analysis and application at the molecular level through genetic engineering studies.

Cho et al. Obtained the NANBH virus using a large amount of chimpanzee serum obtained by the above method, and then extracted the whole nucleic acid of NANBH to clone (cPTNA) the partial cDNA, and the gene consists of about 10,000 base pairs. (Science 244, 359-362 (1989)), and proteins (5-1-1, C100) produced by expressing cloned partial gene fragments in E. coli and yeast were obtained from serum of patients with type C infection. Reacted with (Science 244, 362-364 (1989)). Meanwhile, the Japanese team cloned partial cDNA from the Japanese sera diagnosed with NANBH by the method described above (Kubo, Y. et a1., Nucleic Acids Res. 17, 10367-10372 (1989); Kato, N. et a1., Proc. Japan Acid. 65 (SerB), 219-223 (1990); Kaneko, S. et a1., Lancet 335, 976 (1990); Takeuch1, K. et a1., Gene 91, 287-291 (1990) Takeuchi, K. et a1., Nucleic Acids Res. 18 (15), 4626 (1990); Takamixawa, A. et a1., J. Virol. 65, 1105-1113 (1991)). It was claimed that the subtype was different from the nucleotide sequence of the hepatitis C virus obtained after infection.

The present inventors isolated virus particles from about 50 ml of serum of Korean C-type infected patients, extracted whole viral ribonucleic acid, and then made a hepatitis C virus cDNA library using random primers. Immunodetection of antigen clones showing specific reaction with serum (Young, RA & Davis, RW, Proc. Natl. Acad. Sci. USA 80, 1194 (1983); Huynh, TV et a1, in DNA Cloning; A Practical Approach.VO1.1 (DMGIver, ed.) Pp 49-78, IRL Press, Oxford, UK (1985)) and cloned to determine the nucleotide sequence. The unique base sequence of the hepatitis C virus (HCV-LBC1) cDNA fragment was revealed (Korean Patent Application No. 91-9510, filed by the applicant on June 10, 1991).

The base sequence of Korean HCV has about 78% homology with that of US HCV (Choo et a1., Science 244, 359-363 (1989)), and the base sequence of two Japanese HCVs (Kato et al. al., Proc. Natl. Acad. Sci. USA 87, 9524-9528 (1990); Takamizawa et al., J. Viro. 65, 1105-1113 (1991)); It has the above homogeneity. However, since the above KHCV-LBC1 is obtained from mixed serum, it is difficult to say that it represents the base sequence of one hepatitis C virus.

Based on this fact, the present inventors have tried to find the base sequence and amino acid sequence of one type of hepatitis C virus from a Korean type hepatitis C patient. As a result, the present inventors have found the base sequence of Korean HCV (June 10, 1991). In addition to Korean Patent Application No. 91-9510, which was previously filed for, the entire cDNA of another hepatitis C virus was cloned to reveal its nucleotide sequences, thereby completing the onset.

Disclosure of the Invention An object of the present invention is to identify the nucleotide sequence of a part or whole gene of one type of Korean hepatitis C virus and the amino acid sequence deduced therefrom, thereby preparing hepatitis C diagnostic reagents and vaccines using the same to diagnose and prevent hepatitis C infection. To gear up.

Hereinafter, the onset will be described in detail.

First, RNAzol. Total RNA of HCV was extracted from serum containing Korean hepatitis C virus. Extracted by the B method (Chomczynski et al., Anal. Biochem. 162, 156-159 (1987)).

CDNA is prepared using the above RNA as a template for reverse transcriptase, cDNA synthesis, and kit. At this time, the primers of the reverse transcriptase are random primers (5'-NNNNNN-3 ', N means that base G, A, T, and C are mixed in equal proportion), and the reverse transcriptase is MMLV reverse transcriptase (Molony Murine). Leukemia Virus Reverse Transcriptase (MMLV-RT), and BD's cDNA Synthesis Kit (CDNA Synthesis Kit; Cat. No. 8267SA) are used as a synthesis kit. In addition to the random primers mentioned above, cDNA may also be synthesized using antisense primers.

On the other hand, from the nucleotide sequence information of the cDNA fragments of the Korean type hepatitis C virus (see Korean Patent Application No. 92-10039, which is filed by the applicant), a relatively conserved portion of the nucleotide sequence is selected and several polymerases corresponding thereto are selected. Synthesizing a chain acceptor primer pair. The primers for the polymerase chain reaction were synthesized by DNA synthesizer (Applied Biosystems Inc. Model 380 B, USA) using automated solid phase phosphoamidite chemistry and electrophoresed on modified polyacrylamide gels. After purification, pure water is purified on a C18 column, SEP-PAK (Waters Inc., USA). Using these primers as a template the cDNA of the Korean type hepatitis C virus prepared above using 95 ° C, 1 minute using a temperature circulator (Perkin-Elmer Cetus, U.S.A.); 55 ° C., 1 minute; The cDNA fragments are amplified by a polymerase chain reaction which repeats a temperature cycling program of 72 ° C., 3 minutes, 40 to 45 times.

The amplified cDNA fragments were separated by polyacrylamide gel electrophoresis, respectively, to make smooth ends with klenow enzyme.

Smoother terminal cDNAs were cloned into vector M13mp18 (New England Biolabs, Cat. # 408) (Maniatis et a1, Molecular Cloning, pp 51-53, A Laboratory mannual, Cold spring Harbor Laboratory (1982)), followed by Sanger's dideoxy method. The base sequence is determined according to (Sanger et al., Proc. Natl. Acad. Sci. 74, 5463 (1977)) (see FIGS. 1 to 15).

In addition, in order to determine the 5 'terminal nucleotide sequence of hepatitis C virus, a single cDNA is synthesized using a primer designed using information of the nucleotide sequence of the clone at the most 5' end of the clones. The poly d (G) tail was conjugated to the 3 'end of the single-stranded cDNA chain, amplified cDNA by PCR method, cloned into M13 vector (M13mp18), and the nucleotide sequence was determined by Sanger's dideoxy method. (See FIG. 16).

The cDNA cross sections are linked to each other to form a nucleotide sequence, which is named KHCV-L2.

The M13 phage group (M13-KHCV-L2) containing KHCV-L2 was deposited with the ATCC under accession number ATCC 75447 on April 21, 1993.

Hereinafter, the present invention will be described in more detail based on Examples. The following examples merely illustrate the invention and do not limit the scope of the invention.

Experimental methods and procedures used in the following examples were carried out according to the following Reference Examples 1) -4) unless otherwise specified.

Reference Example 1) Cleavage of DNA Using Restriction Enzyme

A restriction enzyme and a reaction buffer solution were added to a sterile 1.5 ml eppendorf leuve to a reaction volume of 50 µl to 100 µl and reacted at 37 ° C. for 1 to 2 hours. After the reaction was completed, heat treatment was performed at 65 ° C. for 15 minutes to inactivate the restriction enzyme, and phenol extraction and ethanol precipitation were used for the restriction enzyme which was heat resistant.

Restriction enzymes and reaction buffers used were purchased from New England Biolabs, MA, USA. The composition of the 10-fold reaction buffer is as follows.

10-fold NEB buffer 1: 100 mM Tris-HC1, 100 mM MgC1 ₂ , 10 mM dithiothreitol (DTT), pH 7.0

10-fold NEB buffer 2: 100 mM Tris-HC1, 100 mM MgC1 ₂ , 500 mM NaC1, 10 mM MDTT, pH 7.0

10-fold NEB buffer 3: 100 mM Tris-HC1, 100 mM MgC1 ₂ , 1000 mM NaC1, 10 mM DTT, pH 7.0

10-fold NEB buffer 4: 200 mM tris-acetate, 100 mM magnesium acetate, 500 mM potassium acetate, 10 mM DTT, pH 7.0

Reference Example-2) Phenol Extraction and Ethanol Precipitation

Phenol extraction was used to deactivate the restriction enzyme or extract the nucleic acid after the restriction enzyme reaction was completed. Phenol was used after saturation with 10 mM Tris-HC1 (pH 8.0), 1 mM EDTA solution.

The sample and phenol were mixed at a ratio of 1: 1 (v / v), shaken vigorously, mixed, and then centrifuged (15,000 × G, 5 minutes) to transfer the supernatant to a new tube. The same procedure was repeated three times, followed by extracting the supernatant with an equal volume of chloroform solution (chloroform to isobutanol ratio 24: 1 (v / v)), adding 0.1 volume of 3M sodium acetate and 2.5 volume of 100% ethanol. It was.

This was allowed to stand at -70 ° C for 30 minutes or at -20 ° C for at least 12 hours, followed by Winsim separation (15,000xG, 20 minutes, 4 ° C) to precipitate the nucleic acid.

Reference Example 3) Ligation

T4 DNA ligase (T4 DNA ligase, New England Biolabs, Inc.) and 10-fold ligation buffer (0.5M Tris-HC1, pH7.8, 0.1M MgC1 ₂ , 0.2M dithiothreitol, 10 mM ATP) , 0.5 mg / ml BSA) was used to perform the ligation reaction. Usually, 10 units of ligase is used in a volume of 20 µl, and 100 units of ligase are used for ligation of DNA having blunt ends, and the reaction is carried out at 16 ° C. for at least 2 hours. The ligase was inactivated by heating at 占 폚 for 15 minutes.

Reference Example 4) E. coli transformation

E. coli host cell JM101 (ATCC 33876) was inoculated in 100 ml of liquid LB medium (1% bactotrypton, 0.5% bacterium yeast extract, 0.5% sodium chloride) and the absorbance (OD) value at 650 nm may reach 0.25 to 0.5. Incubated at 37 ° C until Cells were then separated by centrifugation (2,500 × G, 10 minutes), washed by adding 50 ml of 0.1 M MgCl ₂ , followed by centrifugation (2,500 × G, 10 minutes), followed by 50 ml of 0.1 M CaC1 ₂ , 0.05M MgC1 ₂ solution was added and allowed to stand on ice for 30 minutes. This was again centrifuged (2,500 × G, 10 minutes) and uniformly dispersed in 5 ml of the same solution (0.1M CaC1 ₂ , 0.05M MgC1 ₂ ). All solutions and vessels used above were used after cooling at 0 ° C. 10 μl of the coupling reaction solution was added to 0.2 ml of the solution obtained above, and the mixture was left at 0 ° C. for 40 minutes and then heat-treated at 42 ° C. for 1 minute. This was added to 2.0 ml of liquid LB medium and incubated at 37 ° C. for 1 hour, and the culture solution was plated on solid LB medium containing 50 μl / ml ampicillin and then incubated at 37 ° C. overnight. After heat treatment, M13 vector DNA was subjected to 100 μl E. coli JM101 cells, 15 μl of 100 mM IPTG (isopropyl-β-D-thiogalactoside), 25 μl of 4% X-Ga1 (5-bromo-4-chloro-indo1y1-β- D-galactoside) and 3 ml of double soft solid YT medium (1% NaC1, 1% yeast extract, 1.6% bacto tryptone, 0.7% bacto agar) preheated to 48 ° C. Plated on YT medium (1% NaCl, 1% yeast extract, 1.6% bactotryptone, 1.5% bactoagar).

Example 1. Preparation of cDNA

1-1) Preparation of RNA

Cells were lysed by adding 300 μl of RNAzo1 B (Cinna / Biotecx; P.O.Box 1421, Friendswood, Texas, U.S.A.) to 100 μl of serum of C-type infected patients. After the cells were dissolved, 150 μl of chloroform was added thereto, shaken vigorously for 15 seconds, left on ice for 5 minutes, and centrifuged (12,000 × G, 4 ° C., 15 minutes). When the sample was divided into two layers, the supernatant was collected, the same volume of isopropanol was added, the mixture was allowed to stand at 4 ° C for 15 minutes, and centrifuged again (12,000xG, 4 ° C, 15 minutes) to precipitate RNA. The precipitated RNA was dissolved in distilled water treated with 10 μl of DEPC (diethy1pyrocarbonate) and immediately used to synthesize cDNA.

1-2) Preparation of cDNA

Using 3-4 μl of RNA obtained in Example 1-1 as a template for reverse transcriptase, cDNA was prepared using a cDNA synthesis kit (Cat. No. 8267SA) from BRL (PO Box 6009, Gathersburg, MD 20877 USA). Was prepared. At this time, as a primer of the reverse transcriptase, random primers (5'-NNNNNN-3 ', N means base G, A, T, and C are mixed in the same ratio) were used. To synthesize the first cDNA disconnection, 0.5 µl of 0.1 M CH ₂ HgOH was added to 3-4 µl of RNA solution obtained in Example 1-1), and the mixture was left at room temperature for 10 minutes to release the secondary structure of RNA. 1 μl of 1M beta mercaptoethanol was added and allowed to react for 5 minutes. 5 μl reverse transcriptase reaction solution (250 mM Tris-HC1, pH 8.3, 375 mM KC1 ₂ , 15 mM MgC1 ₂ , 5 mM dithiothreitol), 1.25 μl of 10 mM dNTP (dATP, dGTP, dTTP, dCTP), 1 μl DEPC-treated distilled water was added to the random primers (1.0 g / L) and 1.0 μl of RNase inhibitor (1U / μL, Promega, USA) so that the total volume was 25 μL, followed by stirring well and allowed to stand at room temperature for 10 minutes. Make sure that the mold and primer are attached well. 1.25 μl of Superscript H-reverse transcriptase (18 U / μl; BRL) was added thereto, followed by reaction at 37 ° C. for 1 hour to synthesize the first cDNA helix.

Example 2 cDNA Amplification of HCV by Polymerase Unchaining

2-1) Design of primer

Referring to the base sequence of the Korean hepatitis C virus (patent application No. 92-10039, which was previously filed by the present application), a portion of the base sequence was relatively conserved, and a primer pair for PCR was devised as follows. Above each primer pair is the sense primer and the one below is the antisense primer. The base sequence and position of the prepared primers are based on the base sequence of KHCV-LBC1 described in the patent application No. 92-10039.

The base sequences of all primers are directional from the 5 'end to the 3' end, the first number in parentheses indicates the position of the first base at the 5 'end, and the last number indicates the position of the last base at the 3' end. Thus, if the preceding number is greater than the latter number, it represents an antisense primer and vice versa. Among the bases constituting the primer, the remaining bases except for 4 to 5 bases at the 3 'end can be changed little by little (2 to 3 out of 10 bases), and the 5' end is used to change the recognition site of the restriction enzyme. You can add

The primers designed as above were synthesized by a DNA synthesizer (AppliedBiosystems, Inc., model 380 B, USA) using an automated solid phase phosphoamidite chemistry, and then modified polyacrylamide gel (2M urea). , 12% acrylamide; bis (29: 1, w / w), 50 mM truss, 50 mM boric acid, 1 mM EDTA-Na). It was attached to SEP-PAK (Waters Inc., U.S.A.), a C18 group, eluted with 50%: 50% of acetonitrile: water mixture, and purified pure. The absorbance was measured at 260 nm to determine the concentration.

2-2) Polymerase Chain Reaction (PCR)

PCR reaction was carried out as follows. 5 μl of HCV cDNA obtained in 1-2) as a template, 10 μl of 10-fold concentration Taq polymerase buffer solution (10 mM Tris-C1 pH8.3, 500 mM KC1, 15 mM MgC1 ₂ , 0.1% (w / v) gelatin), 10 μl of dNTP mixture (1.25 mM of dGTP, dATP, dTTP, and dCTP), 0.2 μg each of sense and antisense primers, and 1 μl (2.5 units) of AmpliTaq DNA polymerase (Perkin Elmer Cetus, USA) The total volume was added to 100 µl. To this was added 50 μl of mineral oil to prevent evaporation of the solution. Thermer cycler (Perkin-Elmer Cetus, USA) was used and the cycle program was 95 ° C., 1 minute; 55 ° C., 1 minute; The cycle of 72 ° C., 3 minutes was repeated 40 to 45 times. In order to remove the polymerase after the reaction, the same volume of phenol / chloroform was added to the reaction mixture, the mixture was mixed well, and centrifuged. The supernatant was transferred to a new tube, mixed with 1/10 volume of 3M sodium acetate, 2.5 times volume of 100% ethanol, and centrifuged to obtain amplified double-stranded nucleic acid, which were added to 20 μl of TE buffer (10 mM Tris-HC1). , pH7.5, 1mM EDTA) was used in the next experiment.

CDNA fragments amplified by KHCVR4 and KHCVL65 were identified as B1,

CDNA fragments amplified by HCVR48 and HCVL19 were identified as B2,

CDNA fragments amplified by KR89 and HCVL16 were identified as B3,

CDNA fragments amplified by KHCVR72 and ENV1A were digested with B4,

CDNA fragments amplified by KR79D and KL61C were replaced with B5,

CDNA fragments amplified by HCVR26 and HCVL31 were identified as B6,

CDNA fragments amplified by KHCVR69 and KHCVL4 were identified as B7,

CDNA fragments amplified by JIR and HCVL18 were identified as B8,

CDNA fragments amplified by KHCVR5 and KHCVL11 were identified as B9,

CDNA fragments amplified by HCVR28 and HCVL48 were identified as B10,

CDNA fragments amplified by KHCVR8 and KHCVL19 were identified as B11,

CDNA fragments amplified by HCVR53 and KHCVL13 were identified as B12,

CDNA fragments amplified by HCVR44 and HCVL40 were identified as B13,

CDNA fragments amplified by KHCVR7 and KHCVR63 were identified as B14,

The cDNA fragments amplified by HCVR61 and GADA20 were named B15, respectively.

The cDNAs thus amplified were separated by polyacrylamide gel electrophoresis, respectively, and smooth ends were made with Klenow enzyme.

The smooth-terminal cDNAs were cloned into M13mp18 and the base sequence was determined according to Sanger's dideoxy method (see FIGS. 1 to 15).

2-3) Cloning of 5'-terminal cDNA and Identification of Nucleotide Sequences of Korean Type C Infection Virus

In Example 2-2 using JMC93 (5′-AGTCTCGCGGGGGCACGCCCAAATC-3 ′; nucleotides 249-225) designed from the terminal nucleotide sequence information of cDNA fragment B1 of hepatitis C virus prepared in Example 2-2) Dilute 1m1 TE buffer solution (10mM Tris-HC1, pH7.5, 1m EDTA) to 50µl of cDNA reactant prepared by the same method, and then transfer Centricon 100 (Centricon 100, Amicon USA, # 4200). The reaction was concentrated to 10 μl while removing residual reverse transcriptase primer and residual dNTP.

4 μl of 5-fold tailing buffer (5 × tailing buffer, 0.5M potassium cacodylate, pH7.2, 10 mM CaC1 ₂ , 1 mM DTT), 4 μl of conjugated poly d (G) tail to solid cDNA. 1 mM dGTP and 10 monomeric terminal deoxynucleotide transferase (BRL, USA, # 80085B) were added to 10 µl of the cDNA solution, and diluted to 50 µl with distilled water, followed by 37 ° C. The reaction was carried out for 30 minutes at, followed by heat treatment at 65 ° C. for 5 minutes.

The primers DC17R1RO (5'-AAGGATCCGTCGACATCGATAATACGACTCATATAGGGAC17-3 ') and R0 (5'-AAGGATCCGTCGACATC-3') designed to determine the 5'-terminus were synthesized and used in conjunction with the primer JMC93 in Example 2-2) PCR was carried out in the same manner to amplify the cDNA having a poly d (G) tail. Thus amplified cDNA (hereinafter referred to as 5UTR) was isolated by polyacrylamide gel electrophoresis and a smooth end was made with Klenow enzyme.

The smooth-terminal cDNA was cloned into the vector M13mp18, and then the base sequence was determined by Sanger's dideoxy method (see FIG. 16).

Fragments 5UTR and Fragments B1 through B16 can be linked together to form a single nucleotide sequence and the sequence thus formed is named KHCV-L2 and is shown in FIG. 17.

In addition, the nucleotide sequences of KHCV-L2 and KHCV-LBC1 are compared and shown in FIG. 18.

The present invention is expected to contribute to hepatitis C diagnostic research and medical research by revealing the entire nucleotide sequence and amino acid sequence of another type of Korean hepatitis C virus.

1 shows the nucleotide sequence of fragment B1 and the amino acid sequence deduced therefrom,

2 shows the nucleotide sequence of fragment B2 and the amino acid sequence deduced therefrom,

3 shows the nucleotide sequence of fragment B3 and the amino acid sequence deduced therefrom,

4 shows the nucleotide sequence of fragment B4 and the amino acid sequence deduced therefrom,

5 shows the nucleotide sequence of fragment B5 and the amino acid sequence deduced therefrom,

6 shows the nucleotide sequence of fragment B6 and the amino acid sequence deduced therefrom,

7 shows the nucleotide sequence of fragment B7 and the amino acid sequence deduced therefrom,

8 shows the nucleotide sequence of fragment B8 and the amino acid sequence deduced therefrom,

9 shows the nucleotide sequence of fragment B9 and the amino acid sequence deduced therefrom,

10 shows the nucleotide sequence of fragment B10 and the amino acid sequence deduced therefrom,

11 shows the nucleotide sequence of fragment B11 and the amino acid sequence deduced therefrom,

12 shows the nucleotide sequence of fragment B12 and the amino acid sequence deduced therefrom,

13 shows the nucleotide sequence of fragment B13 and the amino acid sequence deduced therefrom,

14 shows the nucleotide sequence of fragment B14 and the amino acid sequence deduced therefrom,

15 shows the nucleotide sequence of fragment B15 and the amino acid sequence deduced therefrom,

16 shows the nucleotide sequence of fragment 5UTR,

Figure 17 shows the total nucleotide sequence of KHCV-L2 and the amino acid sequence deduced therefrom,

18 shows a comparison of the entire nucleotide sequences of KHCV-L2 and KHCV-LBC1.

Claims (7)

Korean type hepatitis C virus (KHCV) -L2 cDNA having the nucleotide sequence of FIG. Polynucleotide having a nucleotide sequence of any one of FIGS. 1 to 16. KHCV-L2 polypeptide having the amino acid sequence of FIG. Polypeptides having the amino acid sequence of any one of FIGS. A vector comprising the KHCV-L2 cDNA of claim 1 or the polynucleotide of claim 2. A microorganism transformed with the vector of claim 5. A method of preparing the polypeptide of claim 3 or 4 by culturing the microorganism of claim 6.