Highly pathogenic feline calicivirus and vaccine and application thereof

Abstract

This invention provides a highly pathogenic feline calicivirus strain, its vaccine, and its applications. The highly pathogenic feline calicivirus was isolated from pathogenic material and identified as a highly pathogenic strain, named FCV CC475, with its cDNA sequence shown in SEQ ID NO.1. This strain exhibits strong pathogenicity, causing infected animals to develop symptoms such as fever, paw pad ulcers, and dehydration by day 3 post-infection; the viral load after 5 generations is 10. 9.80 TCID 50 / ml. This strain produces high antibody titers after immunization of cats and rabbits, demonstrating good immunogenicity. Therefore, this highly pathogenic feline calicivirus has broad application prospects in the preparation of feline calicivirus infection vaccines, diagnostic reagents, or therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of veterinary biopharmaceuticals, and more specifically, to a highly pathogenic feline calicivirus, its vaccine, and its applications. Background Technology

[0002] Feline calicivirus (FCV) is a common pathogen in cats, highly infectious, resistant to many disinfectants, and exhibiting high genetic variability. FCV belongs to the Caliciviridae family, Varicellavirus genus, and is a single-stranded, positive-sense, non-enveloped RNA virus. Due to the instability of RNA polymerase during viral replication, FCV is highly susceptible to mutation under immune stress. In recent years, highly fatal systemic diseases caused by infection with virulent systemic felinecalicivirus (VS-FCV), a variant of this virus, have been reported in many parts of the world.

[0003] The highly virulent strain of feline calicivirus (VS-FCV) has different biological characteristics and pathogenicity than traditional FCV. In addition to causing upper respiratory tract infections, it can also cause systemic clinical symptoms such as subcutaneous edema, oral ulcers, lameness, and varying degrees of ulceration on the skin of the ears and paw pads, with a high mortality rate. Cases of VS-FCV infection in pet cats have been reported both domestically and internationally.

[0004] Currently, feline triple inactivated vaccine is mainly used to prevent feline calicivirus infection. Because FCV has rich antigenic diversity and strong genetic variability, one vaccine cannot prevent infection of all wild-type strains. Studies have shown that VS-FCV infection can still occur after vaccination. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a highly pathogenic feline calicivirus strain, its vaccine, and its applications. This invention provides...

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a highly pathogenic feline calicivirus strain, wherein the highly pathogenic feline calicivirus is strain FCVCC475, and its cDNA sequence is shown in SEQ ID NO.1.

[0008] This invention isolates a feline calicivirus from pathogenic material and identifies it as a highly pathogenic strain. Immunization of cats and rabbits produces high antibody titers, indicating that the strain has good immunogenicity and can be used to prepare a vaccine to prevent infection with a virulent strain of feline calicivirus (VS-FCV).

[0009] Unlike other feline caliciviruses, FCV strain CC475 causes typical symptoms in cats, including high fever, oral ulcers, respiratory symptoms, paw pad edema, and ulceration. It belongs to the VS-FCV strain, causing fatal systemic disease in cats. Sequence alignment analysis showed that FCV strain CC475 shared 78.1% sequence similarity with the classic feline calicivirus CC3 strain and 82.9% with the classic feline calicivirus CC246 strain. This low viral homology may explain the high pathogenicity of this strain, as mutations at key sites make it prone to triggering systemic viral infections.

[0010] The FCV CC475 strain described in this invention belongs to the Caliciviridae family, Calicivirus genus, and is a non-enveloped, single-stranded positive-sense RNA virus, providing more comprehensive protection against existing FCV strains. In addition to being isolated from pathogenic material, the systemic feline calicivirus CC475 strain described in this invention can also be obtained through virus rescue, as known in SEQ ID NO.1. This involves cloning and transcribing a negative-sense RNA from full-length cDNA, packaging infectious viral particles intracellularly, and then artificially manipulating the viral cDNA in vitro, such as through mutation, deletion, insertion, and substitution. This allows for research on the gene expression and regulation mechanisms of RNA viruses, virus-host interactions, antiviral and gene therapy, exogenous gene expression, and the development of new vaccines.

[0011] In a second aspect, the present invention provides the use of a highly pathogenic feline calicivirus as described in the first aspect in the preparation of a feline calicivirus infection vaccine, a detection reagent, or a therapeutic drug.

[0012] Thirdly, the present invention provides a feline calicivirus vaccine, the feline calicivirus vaccine comprising the inactivated or attenuated FCV CC475 strain described in the first aspect.

[0013] As a preferred embodiment of the present invention, the content of FCV CC475 strain in the feline calicivirus vaccine is not less than 10%. 7.00 TCID 50 / ml, for example, could be 10 7.00 TCID 50 / ml, 10 7.50 TCID 50 / ml, 10 8.00 TCID 50 / ml, 10 8.50 TCID 50 / ml, 10 9.00 TCID 50 / ml, 10 9.50 TCID 50 / ml, 10 10.00 TCID50 / ml; preferably not less than 10 9.00 TCID 50 / ml.

[0014] As a preferred embodiment of the present invention, the feline calicivirus vaccine further includes a vaccine adjuvant.

[0015] As a preferred embodiment of the present invention, the immunized animal for the feline calicivirus vaccine is a cat or a rabbit.

[0016] As a preferred embodiment of the present invention, the adjuvant of the feline calicivirus vaccine is aluminum hydroxide, and the immunized animal is a cat.

[0017] Preferably, the adjuvant for the feline calicivirus vaccine is Freund's adjuvant, and the immunized animal is a rabbit.

[0018] Since the feline calicivirus vaccine uses the feline calicivirus strain, a mild form of aluminum hydroxide is sufficient as the vaccine adjuvant. If Freund's adjuvant or other adjuvants are used, careful attention must be paid to the dosage and administration to prevent significant side effects. When preparing rabbit vaccines, multiple immunizations and the use of a more potent adjuvant like Freund's adjuvant can be considered to achieve better immunization outcomes.

[0019] Fourthly, the present invention also provides a detection kit for detecting feline calicivirus infection, the detection kit comprising a detection reagent for detecting the FCV CC475 strain as described in the first aspect.

[0020] As a preferred embodiment of the present invention, the detection reagent includes amplification primers.

[0021] As a preferred embodiment of the present invention, the amplification primers include an upper primer and a lower primer.

[0022] Preferably, the upper primer contains a base sequence as shown in SEQ ID NO.2.

[0023] Preferably, the lower primer contains a base sequence as shown in SEQ ID NO.3.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This invention isolates a viral strain from feline pathogenic material, which is identified as feline calicivirus (FCV) by PCR and sequencing, and is designated as FCV CC475 strain. This strain is highly pathogenic, causing infected animals to develop symptoms such as fever, paw pad ulcers, and dehydration as early as the 3rd day after infection; the viral load of the 5th generation is 10. 9.80 TCID 50 / ml. The inactivated vaccines prepared using this method have good immunizing effects on both cats and rabbits; for example, a vaccine prepared by uniformly mixing virus solution and aluminum hydroxide in a 7:1 volume ratio can achieve a neutralizing antibody titer of 1:512 to 1:1290 after two immunizations in cats; a vaccine prepared by uniformly mixing virus solution and Freund's adjuvant in a 1:1 volume ratio also has an immunizing effect on rabbits, with a neutralizing antibody titer of 1:2048 to 1:8192 after three immunizations. Attached Figure Description

[0026] Figure 1 For FCV identification results; bands 1-4: 4 samples to be tested; band 5: negative control; band 6: positive control; M: Marker 2000.

[0027] Figure 2 The results of FCV isolation and culture (100×) are shown; Figure A shows F81 cells infected with FCV in specimen No. 3; Figure B shows normal F81 cells.

[0028] Figure 3 For FCV PCR identification results; Band 1: P5 generation virus solution; Band 2: Positive control; Band 3: Negative control; M: Marker 2000.

[0029] Figure 4 The results are for PCR identification of exogenous virus FHV; Band 1: P5 generation virus solution; Band 2: positive control; Band 3: negative control; M: Marker 2000.

[0030] Figure 5 The results are from indirect immunofluorescence (100×); Figure A shows the P5 generation of feline calicivirus strain CC475; Figure B shows normal F81 cells.

[0031] Figure 6 This is an image showing the results of FCV electron microscopy observations.

[0032] Figure 7 This image shows edema and ulceration on a cat's paw pads. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be determined by the claims.

[0034] In the following embodiments, unless otherwise specified, all reagents and consumables used were purchased from conventional reagent manufacturers in the art; unless otherwise specified, all experimental methods and techniques used were conventional methods and techniques in the art.

[0035] Example 1: Strain Screening

[0036] 1. Pathological sample collection

[0037] For swabs containing secretions from paw pad ulcers in cats suspected of having feline peduncles, use a suitable sterile swab dipped in physiological saline (containing 1000 U / ml penicillin and streptomycin), insert it into the sampling site and rotate it several times to make as much contact as possible with the secretions. After the swab is removed, immediately place it in a sample collection tube and store it in an ultra-low temperature freezer or freeze it and transport it to an ultra-low temperature freezer for storage.

[0038] 2. Treatment of diseased materials

[0039] Remove the frozen foot ulcer secretion swabs from the freezer, add 500 μl of physiological saline containing 5% FBS and 1000 U / ml penicillin and streptomycin, insert the swab head into the centrifuge tube and stir repeatedly, squeezing it against the bottom and wall of the tube multiple times (as dry as possible), freeze and thaw the treatment solution once at -70℃, incubate at 4℃ for 4-6 hours, centrifuge at 5000 r / min for 5 minutes, aseptically collect the supernatant into the centrifuge tube, filter the tissue fluid through a 0.22 μm filter membrane and aliquot, and store at -70℃.

[0040] 3. Pathological material examination, namely, sterility testing and PCR identification of isolated pathological materials.

[0041] (1) Genome preparation: RNA was extracted from the collected foot ulcer secretion swabs using a nucleic acid extraction kit, as described in the instructions. The RNA was stored at -70°C or immediately reverse transcribed into cDNA. The reverse transcription system is shown in Table 1 below, and the sample was in a water bath at 42°C for 30 minutes. The cDNA was stored at -20°C.

[0042] Table 1 Reverse Transcription System

[0043] reagents System (μl) A11Mix 4 Water 6 RNA 10 total 20

[0044] (2) PCR reaction system: The reaction system consists of 30 μl of DNA polymerase premix, 15 μl of DNA polymerase premix, 4 μl of template, 2 μl each of upstream and downstream primers (20 μmol / L), and 7 μl of ddH2O.

[0045] The primer sequences are:

[0046] FCV-681-F: 5'-CAACCTGCGCTAACGTGCTTA-3' (SEQ ID NO. 2),

[0047] FCV-681-R: 5'-TTCCCCCAAAAACYCCAGATC-3' (SEQ ID NO. 3).

[0048] (3) PCR amplification program: 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 30 seconds, 52℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 35 cycles, and finally 72℃ extension for 5 minutes.

[0049] (4) Electrophoresis detection: After PCR amplification, take 3 μl of PCR product and perform electrophoresis analysis using a 1.0% gel.

[0050] (5) Sterility test: The sterility test or pure test method in Appendix III of the 2015 edition of the Chinese Veterinary Pharmacopoeia was performed. The treated diseased material was inoculated into two tubes of thioglycolate (TG) medium and two tubes of casein agar (GA) slant, 0.2 ml each. One tube was incubated at 37℃ and the other at 25℃. Another 0.2 ml was inoculated into one tube of glucose peptone soup (GP) and incubated at 25℃. The samples were observed for 7 days.

[0051] In this embodiment, a total of four paw pad ulcer secretion samples were collected from cats suspected of having feline calicivirus, labeled as 1 to 4. All four samples passed the sterility test. PCR testing was performed on the four samples, and only sample 3 was positive for feline calicivirus. Specific results are shown in Table 2.

[0052] Table 2. Results of Pathological Examination

[0053] Pathological material number Sterility test PCR results (FCV) 1 No bacterial or mold contamination Negative 2 No bacterial or mold contamination Negative 3 No bacterial or mold contamination Positive 4 No bacterial or mold contamination Negative

[0054] The amplification product of sample No. 3 showed a band around 681bp, consistent with the expected size. (See attached image) Figure 1 .

[0055] The target band was gel-cleaved, sequenced, and compared with the NCBI website. The results showed that it was highly similar to the feline calicivirus gene.

[0056] Example 2: Isolation of the virus strain

[0057] The sterile specimens from Example 1 that passed the sterility test and were identified as feline calicivirus by PCR were used for virus isolation.

[0058] Virus isolation and culture: F81 cells were passaged to 25 cm⁻¹. 2 Once the cells have grown into a monolayer, wash them three times with serum-free MEM medium. Add the tissue fluid obtained after processing the pathogenic material to the cell culture flask and incubate at 37°C with 5% CO2 for 1 hour. After incubation, add 4.5 ml of MEM medium containing 2% serum to the cell culture flask. Mix the liquid in the cell culture flask well and place it in a 37°C, 5% CO2 incubator. Observe the cytopathic effect every 12 hours. If no cytopathic effect occurs within 3-4 days after inoculation, discard the flask.

[0059] If cytopathic effects occur, the virus solution is harvested by repeated freeze-thaw cycles (P1 generation). During virus isolation, normal cells are used as a control to observe the cytopathic effects. The harvested virus solution is then inoculated into F81 cells and passaged until the P5 generation.

[0060] Combination Figure 2 It can be seen that when tissue fluid from the No. 3 pathogen was inoculated into F81 cells and passaged continuously to the third generation, and cultured at 37°C for 48–72 hours, cell detachment, shrinkage, and grape-like lesions were observed (Figure A), while normal F81 cells showed no cytopathic effects (Figure B).

[0061] Example 3: Identification of Toxic Strains

[0062] 1. Feline calicivirus detection: The isolated P5 generation virus solution was tested according to the method described in Example 1, and the results were observed.

[0063] 2. Feline Infectious Rhinotracheitis Virus Detection: The isolated P5 generation virus solution was tested, and the results were observed. The specific method is as follows:

[0064] (1) Genome preparation: DNA was extracted from the isolated P5 generation virus using a nucleic acid extraction kit and stored at -20°C.

[0065] (2) PCR reaction system: The total reaction system is 30 μl, DNA polymerase premix 15 μl, template 4 μl, upstream and downstream primers (20 μmol / L) 2 μl each, and ddH2O 7 μl.

[0066] The primer sequences are:

[0067] FHV-401-F: 5'-GCGGATTACACCACCACTCT-3' (SEQ ID NO.4),

[0068] FHV-401-R: 5'-AAAGGCTAAGGGCGTGTACC-3' (SEQ ID NO. 5).

[0069] (3) PCR amplification program: 95℃ pre-denaturation for 5 minutes, 95℃ denaturation for 30 seconds, 63℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 35 cycles, and finally 72℃ extension for 5 minutes.

[0070] (4) Electrophoresis detection: After PCR amplification, take 3 μl of PCR product and perform electrophoresis analysis using a 1.0% gel.

[0071] The harvested P5 generation virus fluid was identified by PCR using FCV identification primers, and the results are as follows: Figure 3 As shown, the PCR amplification product was 681 bp, and the band size was consistent with expectations. The detection results for the exogenous virus FHV were all negative. Figure 4 This strain was named feline calicivirus CC475.

[0072] 2. Determination of viral load: The P5 generation virus solution was serially diluted 10-fold using MEM, and 10... -6 10 -7 10 -8 10 -9 10 -10 The virus at different dilutions was inoculated into 96-well cell culture plates containing a monolayer of F81 cells, 100 μl per well, with eight replicates per dilution. Normal cell controls were also included. Cells were cultured at 37°C and 5% CO2 for 3–5 days, and cell proliferative events (CPEs) were observed under a light microscope. TCID was calculated using the Reed-Muench method. 50 The feline calicivirus CC475 strain P5 generation virus content is 10. 9.80 TCID 50 / ml.

[0073] 3. Indirect immunofluorescence detection: P5 generation virus solution was serially diluted 10-fold and inoculated into F81 cells. The cells were cultured at 37℃ and 5% CO2. After 3 days, the cells were fixed with 80% cold acetone, stained with FCV-specific monoclonal antibody as primary antibody and FITC-labeled goat anti-mouse IgG as secondary antibody, and the results were observed under a fluorescence microscope.

[0074] The P5 generation of feline calicivirus CC475 was inoculated into F81 cells and cultured for 3 days before indirect immunofluorescence detection. Results are as follows: Figure 5 As shown, virus-infected F81 cells exhibit high-intensity green fluorescence (Figure A), while control group cells show no visible fluorescence (Figure B), indicating that feline calicivirus has been isolated.

[0075] 4. Transmission electron microscopy observation: P5 generation virus solution was adsorbed onto a metal grid and stained with 1% phosphotungstic acid for 2-3 minutes. Then, the residual staining solution on the metal grid was absorbed with filter paper. The results were observed and recorded using an electron microscope (JEM 1200EXII).

[0076] The morphological characteristics of feline calicivirus CC475 strain P5 were observed under a transmission microscope after negative staining. Figure 6 As shown, under an electron microscope, calicivirus-like particles were observed. They were spherical, without an envelope, and had a diameter of about 35 nm.

[0077] 5. Specificity test: Dilute P5 generation virus solution to 2000 TCID. 50 / ml, mixed with an equal volume of feline calicivirus positive serum, incubated at 37°C for 1 hour, and then inoculated into 4 wells of a 96-well cell culture plate that has grown into a monolayer of F81 cells, 0.1ml per well. Normal cell control and virus control were also set up. The cells were incubated at 37°C in a 5% CO2 incubator and observed for 3-5 days. The virus control wells showed cytopathic effects, while the positive serum wells and normal cell wells did not show cytopathic effects. The virus solution was then determined to be positive for feline calicivirus.

[0078] The CPE induced in F81 cells by the P5 generation of feline calicivirus CC475 strain could be specifically inhibited by feline calicivirus-positive serum, indicating that the isolated virus was feline calicivirus.

[0079] 6. Aseptic testing, mycoplasma testing, and exogenous virus testing shall be conducted in accordance with the testing methods recorded in the appendices of Part III of the Chinese Veterinary Pharmacopoeia.

[0080] Sterility testing of feline calicivirus CC475 strain P5 generation showed no bacterial or fungal contamination.

[0081] Mycoplasma testing was performed on the P5 generation of feline calicivirus CC475. No color change was observed when inoculated into liquid mycoplasma culture medium, and no mycoplasma colonies were observed to grow on solid culture medium. However, in the positive control, the liquid culture medium turned yellow on the 3rd day, and "fried egg-shaped" mycoplasma colonies were observed under a low-power microscope after 5 days of culture on solid culture medium.

[0082] Exogenous virus testing was performed on the P5 generation of feline calicivirus strain CC475. Neither normal control cells nor sample-inoculated cells showed CPE or erythrocyte adsorption. Fluorescence detection showed specific fluorescence in the positive control, while neither normal control cells nor sample-inoculated cells showed fluorescence. This indicates that the P5 generation of feline calicivirus strain CC475 is free from exogenous virus contamination.

[0083] 7. Pathogenicity test: Take the P5 generation virus solution and challenge 5 healthy susceptible cats by nasal drops. Each cat is inoculated with 0.5 ml of virus and observed after challenge.

[0084] On the third day after the experimental cats were inoculated with feline calicivirus CC475 strain P5, they began to show symptoms such as fever and paw pad ulcers. Figure 7 Symptoms such as dehydration, as shown in the image, prove that the feline calicivirus CC475 strain is a highly pathogenic strain.

[0085] Example 3: Application of the virus strain

[0086] 1. Using cats as experimental animals

[0087] (1) Vaccine preparation: Take P5 generation virus solution, add BEI with a final concentration of 0.005mol / L, inactivate at 37℃ for 48 hours, add sodium thiosulfate with a final concentration of 0.005mol / L to neutralize BEI, and mix the inactivated virus solution that has passed the inactivation test with aluminum hydroxide in a volume ratio of 7:1 to prepare an inactivated vaccine.

[0088] (2) Experimental grouping: 10 experimental cats were divided into an immunization group and a control group, with 5 cats in each group. The immunization group was injected subcutaneously into the neck and back of the body with inactivated vaccine, while the control group was injected subcutaneously into the neck and back of the body with MEM. The immunization was performed twice, 1 ml each time, with an interval of 21 days.

[0089] (3) Antibody detection: 21 days after booster immunization, blood was collected to detect the FCV neutralizing antibody titer. The antibody titers produced by the experimental cats in each group were statistically analyzed. The specific results are shown in Table 3. The FCV neutralizing antibody titer of the experimental cats in the control group was no higher than 1:4, and the FCV neutralizing antibody titer of the immunized group was 1:512 to 1:1290.

[0090] Table 3 Neutralizing antibody titers in experimental cats

[0091]

[0092]

[0093] 2. Using rabbits as experimental animals

[0094] (1) Vaccine preparation: Take P5 generation virus solution, add BEI with a final concentration of 0.005mol / L, inactivate at 37℃ for 48 hours, add sodium thiosulfate with a final concentration of 0.005mol / L to neutralize BEI, mix the inactivated virus solution that has passed the inactivation test with Freund's adjuvant in a 1:1 ratio to prepare an inactivated vaccine.

[0095] (2) Experimental grouping: 10 healthy rabbits were divided into an immunization group and a control group, with 5 rabbits in each group. The immunization group was injected with inactivated vaccine, and the control group was injected with MEM. The immunization was performed three times, 1 ml each time, with an interval of 14 days.

[0096] (3) Antibody detection: Blood was collected from the marginal ear vein 14 days after the third immunization to detect the FCV neutralizing antibody titer. The antibody titers produced by the experimental cats in each group were statistically analyzed. The specific results are shown in Table 4. The FCV neutralizing antibody titers in the control group were all less than 1:2, while the FCV neutralizing antibody titers in the immunized group were 1:2048 to 1:8192.

[0097] Table 4. Rabbit neutralizing antibody titers

[0098]

[0099]

[0100] In summary, the feline calicivirus CC475 strain provided by this invention is a highly pathogenic strain that can cause infected animals to develop symptoms such as fever, paw pad ulcers, and dehydration by the third day after infection; the vaccine prepared using this strain has good immunity in cats and rabbits.

[0101] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A highly pathogenic feline calicivirus, characterized in that, The highly pathogenic feline calicivirus is FCV CC475 strain, and the cDNA sequence of FCV CC475 strain is shown in SEQ ID NO.

1.

2. The use of the highly pathogenic feline calicivirus as described in claim 1 in the preparation of feline calicivirus infection vaccines, diagnostic reagents, or therapeutic drugs.

3. A feline calicivirus vaccine, characterized in that, The feline calicivirus vaccine comprises the inactivated or attenuated FCV CC475 strain as described in claim 1.

4. The feline calicivirus vaccine according to claim 3, characterized in that, The feline calicivirus vaccine contains no less than 10% FCVCC475 strain. 7.00 TCID 50 / ml.

5. The feline calicivirus vaccine according to claim 3, characterized in that, The feline calicivirus vaccine also includes a vaccine adjuvant.

6. The feline calicivirus vaccine according to claim 3, characterized in that, The feline calicivirus vaccine is administered to cats or rabbits.

7. The feline calicivirus vaccine according to claim 5 or 6, characterized in that, The adjuvant for the feline calicivirus vaccine is aluminum hydroxide, and the immunized animal is a cat. The adjuvant for the feline calicivirus vaccine is Freund's adjuvant, and the immunized animal is a rabbit.

8. A detection kit for feline calicivirus infection, characterized in that, The test kit contains a test reagent for detecting the FCV CC475 strain as described in claim 1.

9. The detection kit according to claim 8, characterized in that, The detection reagent contains amplification primers.

10. The detection kit according to claim 9, characterized in that, The amplification primers include an upper primer and a lower primer; The upper primer contains the base sequence shown in SEQ ID NO.2; The lower primer contains the base sequence shown in SEQ ID NO.3.

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