Synthesis method, purification method and application of calprotectin fusion protein

Abstract

The invention provides a synthesis method, a purification method and application of calprotectin fusion protein, and belongs to the technical field of molecular biology. The calprotectin fusion protein disclosed by the invention is a heterodimer or a heterotetramer formed by polymerizing an S100A8 subunit and an S100A9 subunit, and six histidine affinity tags are introduced into the amino terminal of the S100A8 subunit. According to the invention, the S100A8 subunit and the S100A9 subunit of the calprotectin can be simultaneously synthesized through one strain of recombinant escherichia coli, the two subunits can complete self-assembly of the calprotectin in thallus cells, and the step of artificially mixing subunit solutions in the existing microbial fermentation synthesis method is omitted. Besides, six histidine affinity tags are introduced to the amino terminal of the S100A8 subunit, so that purification of the calprotectin fusion protein is facilitated, and the production efficiency and the product purity can be improved. The calprotectin fusion protein provided by the invention retains the transcriptional regulation activity of natural calprotectin on escherichia coli.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a synthesis method, a purification method and an application of a calprotectin fusion protein. Background Art

[0002] Calprotectin, as a member of the S100 protein family, is a multifunctional hybrid calcium-binding protein. It is self-assembled from S100A8 and S100A9 subunits and exists mainly in the form of dimers or tetramers in neutrophils.

[0003] The synthesis methods of calprotectin include in vivo separation, cell synthesis and microbial fermentation synthesis. Among them, the method of microbial fermentation synthesis requires two strains of Escherichia coli to synthesize two subunits of calprotectin respectively, and then mix the two subunits in a solution system to self-assemble into a complete calprotectin. The calprotectin obtained by this method has biological activity and relatively low production cost, which is a potential calprotectin synthesis strategy. However, the above scheme requires the construction of two recombinant bacteria to synthesize S100A8 subunits and S100A9 subunits respectively, and then mix the synthesized S100A8 subunits and S100A9 subunits for assembly, which is a relatively cumbersome process. Summary of the invention

[0004] The purpose of the present invention is to provide a synthesis method, purification method and application of a calprotectin fusion protein, to achieve the synthesis and assembly of two subunits of calprotectin through a single strain, and to optimize the downstream purification process.

[0005] The present invention provides a calprotectin fusion protein. The calprotectin fusion protein is a heterodimer or heterotetramer formed by polymerization of a S100A8 subunit and a S100A9 subunit. An affinity tag with 6 histidines is introduced into the amino terminal of the S100A8 subunit.

[0006] The present invention also provides a recombinant vector for expressing the calprotectin fusion protein described in the above scheme, with pRSFDuet-1 as the backbone plasmid, and the coding gene of the S100A8 subunit and the coding gene of the S100A9 subunit are inserted; the coding gene of the S100A8 subunit and the coding gene of the S100A9 subunit are optimized according to the codon preference of Escherichia coli.

[0007] Preferably, the nucleotide sequence of the gene encoding the S100A8 subunit is shown in SEQ ID NO.1; the gene encoding the S100A8 subunit is constructed between the BamHI and SalI restriction sites of pRSFDuet-1.

[0008] Preferably, the nucleotide sequence of the gene encoding the S100A9 subunit is shown in SEQ ID NO.2; the gene encoding the S100A9 subunit is constructed between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector.

[0009] Preferably, the nucleotide sequence of the recombinant vector is as described in SEQ ID NO.4.

[0010] The present invention also provides a recombinant Escherichia coli comprising the recombinant vector described in the above scheme.

[0011] The present invention also provides a method for synthesizing the calprotectin fusion protein described in the above scheme, comprising the following steps:

[0012] The recombinant Escherichia coli described in the above scheme is fermented and cultured, and then induced with isopropyl-β-D-thiogalactopyranoside to obtain a bacterial cell containing the calprotectin fusion protein.

[0013] The present invention also provides a method for purifying the calprotectin fusion protein described in the above scheme, comprising the following steps: the bacterial cells or crude enzyme solution of the bacterial cells containing the calprotectin fusion protein obtained by the synthesis method described in the above scheme are purified by a nickel affinity column to obtain the calprotectin fusion protein.

[0014] Preferably, the purification method comprises the following steps: the bacterial cells containing calprotectin fusion protein obtained by the synthesis method described in the above scheme are ultrasonically disrupted and then centrifuged to obtain a cell disruption supernatant; the cell disruption supernatant is successively subjected to nickel affinity column affinity adsorption and imidazole solution gradient elution to obtain a calprotectin fusion protein solution; the calprotectin fusion protein solution is successively subjected to ultrafiltration washing to remove imidazole to obtain a purified calprotectin fusion protein solution; the purified calprotectin fusion protein solution is lyophilized to obtain a purified calprotectin fusion protein; the imidazole solution gradient elution comprises sequentially using phosphate buffer containing 20, 50, 100, 200, and 500 mM imidazole for gradient elution.

[0015] The present invention also provides the calprotectin fusion protein, the recombinant vector, the recombinant Escherichia coli, the bacterial cell containing the calprotectin fusion protein obtained by the synthesis method, or the purified calprotectin fusion protein obtained by the purification method inducing Escherichia coli P ykgMO Application of promoter in downstream gene expression; ykgMO The promoter nucleotide sequence is shown in SEQ ID NO.3

[0016] The present invention provides a calprotectin fusion protein, wherein the calprotectin fusion protein is a heterodimer or heterotetramer formed by polymerization of S100A8 subunit and S100A9 subunit, and an affinity tag of 6 histidines is introduced into the amino terminus of the S100A8 subunit. The present invention can simultaneously synthesize the S100A8 subunit and the S100A9 subunit of calprotectin by a recombinant Escherichia coli, and the two subunits can complete the self-assembly of calprotectin in the bacterial cell, omitting the step of artificially mixing subunit solutions in the existing microbial fermentation synthesis method, and can simplify the preparation process. In addition, the present invention facilitates the purification of the calprotectin fusion protein by introducing an affinity tag of 6 histidines into the amino terminus of the S100A8 subunit, and can further improve the production efficiency and product purity. The calprotectin fusion protein of the present invention retains the activity of natural calprotectin on transcriptional regulation of Escherichia coli, and is of great significance for the functional research and application of calprotectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 The figure is a flow chart of fermentation synthesis and purification of calprotectin fusion protein;

[0019] Figure 2 The graph shows the results of the induction intensity gradient of the PykgMO promoter downstream fluorescent protein induced by different concentrations of calprotectin fusion protein, which is dependent on the concentration of calprotectin fusion protein. DETAILED DESCRIPTION

[0020] The present invention provides a calprotectin fusion protein. The calprotectin fusion protein is a heterodimer or heterotetramer formed by polymerization of a S100A8 subunit and a S100A9 subunit. An affinity tag with 6 histidines is introduced into the amino terminal of the S100A8 subunit.

[0021] The present invention also provides a recombinant vector pRSFDuet-His-S100A8 / A9 for expressing the calprotectin fusion protein described in the above scheme, with pRSFDuet-1 as the backbone plasmid, and the coding gene of the S100A8 subunit and the coding gene of the S100A9 subunit are inserted; the coding gene of the S100A8 subunit and the coding gene of the S100A9 subunit are optimized according to the codon preference of Escherichia coli (E. coli).

[0022] In the present invention, pRSFDuet-1 is a double gene expression vector.

[0023] In the specific implementation of the present invention, the nucleotide sequence of the gene encoding the S100A8 subunit is shown in SEQ ID NO.1, specifically:

[0024]

[0025] In the specific implementation of the present invention, the coding gene of the S100A8 subunit is constructed between the BamHI and SalI restriction sites of pRSFDuet-1, and the complete expression cassette gene sequence is shown in SEQ ID NO.1.

[0026] In the specific implementation of the present invention, the nucleotide sequence of the gene encoding the S100A9 subunit is shown in SEQ ID NO.2, specifically:

[0027]

[0028]

[0029] In the specific implementation of the present invention, the coding gene of the S100A9 subunit is constructed between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector, and the complete expression cassette gene sequence is shown in SEQ ID NO.2.

[0030] In one embodiment of the present invention, the nucleotide sequence of the recombinant vector is as described in SEQ ID NO.4, specifically:

[0031]

[0032]

[0033]

[0034]

[0035] The present invention also provides a recombinant Escherichia coli comprising the recombinant vector described in the above scheme.

[0036] In the present invention, the original strain of the recombinant Escherichia coli is preferably Escherichia coli BL21 (DE3).

[0037] In the specific implementation of the present invention, the recombinant vector pRSFDuet-His-S1100A8 / A9 is used to transform BL21 (DE3) to obtain recombinant E. coli. The present invention has no special restrictions on the transformation conditions, and the conventional transformation conditions of 42°C heat shock for 90s in the art can be used.

[0038] The present invention also provides a method for synthesizing the calprotectin fusion protein described in the above scheme, comprising the following steps:

[0039] The recombinant Escherichia coli described in the above scheme is fermented and cultured, and then induced with isopropyl-β-D-thiogalactopyranoside (IPTG) to obtain a bacterial cell containing the calprotectin fusion protein.

[0040] The invention uses Escherichia coli as a fermentation base strain to synthesize calprotectin fusion protein.

[0041] In the specific implementation process of the present invention, the recombinant Escherichia coli is cultured and activated to obtain a seed solution; the seed solution is inoculated into a fermentation medium, and after fermentation culture, IPTG is induced to express a bacterial cell containing the calprotectin fusion protein. In the present invention, the fermentation medium is preferably a TB medium; the fermentation culture is performed until the bacterial cell OD 600 Reach 0.4-0.6; the fermentation culture temperature is 37°C; the final concentration of IPTG is 500 μM; the induction time is 22h; the induction temperature is 20°C; and shaking culture is performed during the induction process.

[0042] The synthesis method of the calprotectin fusion protein provided by the present invention can obtain a high-purity calprotectin fusion protein by fermentation using Escherichia coli BL21 (DE3) and TB culture medium as a substrate. The synthesis method of the calprotectin fusion protein provided by the present invention is simple, the cost of Escherichia coli culture is low, the bacterial cell growth rate is fast, and the fermentation production intensity is high.

[0043] The present invention also provides a method for purifying the calprotectin fusion protein described in the above scheme, comprising the following steps:

[0044] The bacterial cells or crude enzyme solution of the bacterial cells containing the calprotectin fusion protein obtained by the synthesis method described in the above scheme are purified by nickel affinity column to obtain the calprotectin fusion protein.

[0045] In the present invention, the purification method comprises the following steps:

[0046] The bacterial cells containing the calprotectin fusion protein obtained by the synthesis method described in the above scheme are ultrasonically disrupted and then centrifuged to obtain a cell disruption liquid supernatant;

[0047] The cell lysis solution supernatant is subjected to affinity adsorption on a nickel affinity column and gradient elution with an imidazole solution in sequence to obtain a calprotectin fusion protein solution;

[0048] The calprotectin fusion protein solution is sequentially subjected to ultrafiltration washing to remove imidazole, thereby obtaining a purified calprotectin fusion protein solution;

[0049] The purified calprotectin fusion protein solution is freeze-dried to obtain purified calprotectin fusion protein.

[0050] First, the bacterial cells containing the calprotectin fusion protein obtained by the synthesis method described in the above scheme are ultrasonically disrupted and then centrifuged to obtain a cell disruption liquid supernatant.

[0051] In the specific implementation process of the present invention, the power of the ultrasonic crushing is 150W; the time of the ultrasonic crushing is 25-35min; the temperature of the centrifugation is 4°C; the speed of the centrifugation is 8000rpm; and the time of the centrifugation is 10min.

[0052] After obtaining the supernatant of the cell lysis solution, the supernatant of the cell lysis solution is sequentially subjected to affinity adsorption on a nickel affinity column and gradient elution with an imidazole solution to obtain a calprotectin fusion protein solution.

[0053] In the specific implementation of the present invention, the imidazole solution gradient elution includes sequentially using 20, 50, 100, 200, 500 mM imidazole in phosphate buffer for gradient elution. In the present invention, the concentration of phosphate ions in the phosphate buffer is 50 mM; the pH of the phosphate buffer is 7.4.

[0054] After obtaining the calprotectin fusion protein solution, the calprotectin fusion protein solution is subjected to ultrafiltration washing to remove imidazole, thereby obtaining a purified calprotectin fusion protein solution.

[0055] In the specific implementation process of the present invention, the molecular weight of the ultrafiltration tube used in the ultrafiltration is 10kDa.

[0056] After the calprotectin fusion protein of the present invention is purified by nickel affinity column and ultrafiltration to remove imidazole, impurities are less, and the purity and yield of the product are both high. After each 100 mL of fermentation broth is purified by nickel affinity column and ultrafiltration, 38 mg of high-purity calprotectin fusion protein can be obtained.

[0057] After obtaining the purified calprotectin fusion protein solution, the purified calprotectin fusion protein solution is lyophilized to obtain the purified calprotectin fusion protein.

[0058] The purification method provided by the present invention can be realized by using only a nickel affinity column, and the gradient elution process is simple and fast. Meanwhile, the obtained calprotectin fusion protein retains the biological activity of natural calprotectin.

[0059] The present invention also provides the calprotectin fusion protein, the recombinant vector, the recombinant Escherichia coli, the bacterial cell containing the calprotectin fusion protein obtained by the synthesis method, or the purified calprotectin fusion protein obtained by the purification method inducing Escherichia coli P ykgMO Application of promoter in downstream gene expression;ykgMO The promoter nucleotide sequence is shown in SEQ ID NO.3, specifically:

[0060]

[0061]

[0062] In the specific implementation process of the present invention, P ykgMO The promoter downstream gene uses mNeongreen fluorescent protein (GenBank EU482389) as a reporter gene to show the induced expression intensity. The nucleotide sequence of the coding gene of mNeonGreen fluorescent protein is shown in SEQ ID NO.5, specifically:

[0063]

[0064] In the present invention, the final concentration of the calprotectin fusion protein is preferably ≥100 μg / mL, more preferably 100-800 μg / mL, and most preferably 100, 200, 400 or 800 μg / mL.

[0065] In the specific implementation process of the present invention, the calprotectin fusion protein was used to induce P ykgMO The expression of mNeongreen fluorescent protein downstream of the promoter showed a concentration-dependent increase in induction intensity gradient.

[0066] The calprotectin fusion protein of the present invention retains the activity of the original natural calprotectin in regulating transcription of Escherichia coli.

[0067] To further illustrate the present invention, the synthesis method, purification method and application of a calprotectin fusion protein provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] like Figure 1 As shown, this embodiment provides a method for synthesizing and purifying a calprotectin fusion protein by Escherichia coli, comprising the following steps:

[0070] (1) Construction of pRSFDuet-His-S100A8 / A9 vector:

[0071] The nucleotide sequences of the S100A8 subunit and the S100A9 subunit were optimized according to the codon preference of Escherichia coli, and the optimized S100A8 coding sequence was shown in SEQ ID NO.1, and the optimized S100A9 coding sequence was shown in SEQ ID NO.2. The coding sequence of the S100A8 subunit was synthesized between the BamHI and SalI restriction sites of the Escherichia coli double gene expression vector pRSFDuet-1, so that it formed a fusion protein with 6 histidine tags fused to the amino terminal during the translation and synthesis of the protein. The coding sequence of the S100A8 subunit was synthesized between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector. The recombinant vector pRSFDuet-His-S100A8 / A9 was obtained, and the sequence is shown in SEQ ID NO.4.

[0072] (2) Construction of recombinant E. coli BL21 (DE3)-pRSFDuet-His-S100A8 / A9:

[0073] The recombinant vector pRSFDuet-His-S100A8 / A9 solution described in step (1) was mixed with the competent cells of E. coli BL21 (DE3), and then placed on ice for 30 minutes, followed by heat shock at 42°C for 60 seconds, and then placed on ice for 2 minutes. 1 mL of recovery medium was added to allow the competent cells to recover and culture for 1 to 2 hours, and the competent cells were coated on kanamycin-resistant plates for overnight culture and screening. Single colonies of recombinant E. coli BL21 (DE3)-pRSFDuet-His-S100A8 / A9 were obtained.

[0074] (3) Preparation of fermentation seed liquid:

[0075] The single colony obtained in step (2) was picked and inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37° C. with shaking for 10 to 12 h to obtain a recombinant E. coli seed solution.

[0076] (4) Fermentation culture and induced expression:

[0077] The recombinant E. coli seed solution obtained in step (3) was transferred to TB medium at a 1% (v / v) inoculation rate and cultured at 37°C with shaking until the bacterial OD 600 When the pH value reached 0.4 to 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 500 μM to induce the expression of calprotectin fusion protein, and the culture was shaken at 20°C for 22 h.

[0078] (5) Preparation of supernatant of cell lysis solution:

[0079] The recombinant E. coli culture fluid obtained in step (4) was centrifuged to collect the bacterial cells containing the calprotectin fusion protein and resuspended in PBS buffer. Ultrasonic disruption was performed for 25 to 35 minutes at a power of 150 W using an ultrasonic disruptor. Centrifugation was performed at 8000 rpm for 10 minutes at 4°C to separate the supernatant of the cell disruption fluid.

[0080] (6) Nickel purification column gradient elution of calprotectin fusion protein:

[0081] After the supernatant of the cell lysis solution obtained by the method described in step (5) is passed through a membrane to remove impurities, it is loaded into a nickel purification column and gradient eluted with 20, 50, 100, 200, and 500 mM imidazole phosphate buffer to obtain a gradient eluate of the calprotectin fusion protein.

[0082] (7) SDS-PAGE electrophoresis:

[0083] The gradient elution solution described in step (6) was mixed with the protein loading buffer, heated at 100°C for 10 min, and loaded onto a 12% protein gel for SDS-PAGE electrophoresis. According to the electrophoresis results, the protein samples eluted with 200 mM and 500 mM imidazole were collected to obtain a high-purity calprotectin fusion protein solution.

[0084] (8) Impurity removal of calprotectin fusion protein solution:

[0085] The high-purity calprotectin fusion protein solution described in step (7) is centrifuged through a 10 kDa molecular weight ultrafiltration tube, and then washed and centrifuged three times with PBS buffer to obtain a purified calprotectin fusion protein solution from which imidazole has been removed.

[0086] (9) Lyophilization and storage of purified calprotectin fusion protein:

[0087] The calprotectin fusion protein solution described in step (8) was refrigerated in a -80°C refrigerator overnight, and vacuum-dried for 12 h using a freeze dryer at -80°C to obtain a lyophilized powder of the calprotectin fusion protein. The protein sample was sealed and stored at 4°C.

[0088] Example 2

[0089] The induction application of the calprotectin fusion protein of Example 1 comprises the following steps:

[0090] (1) pET28a-P ykgMO - Construction of mNeongreen plasmid:

[0091] P ykgMOThe promoter (as shown in SEQ ID NO.3) and the gene encoding the mNeonGreen fluorescent protein (as shown in SEQ ID NO.5) were synthesized between the BglII and Xho I restriction sites of the pET28a vector to obtain the pET28a-PykgMO-mNeongreen plasmid.

[0092] (2) Recombinant E. coli EcN-pET28a-P ykgMO -mNeongreen build:

[0093] The recombinant vector pET28a-PykgMO-mNeongreen plasmid solution described in step (1) was mixed in the competent E. coli Nissle 1917 (EcN), and electroporated at 1.8 kV and 6 ms. The recombinant single colonies were screened on kanamycin-resistant plates.

[0094] (3) Preparation of fermentation seed liquid:

[0095] The single colony obtained in step (2) was picked and inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37° C. with shaking for 10-12 h to obtain a recombinant E. coli seed solution.

[0096] (4) Fermentation culture and induced expression:

[0097] The recombinant E. coli seed solution obtained in step (3) was transferred to LB medium at a 1% (v / v) inoculation volume. During inoculation, calprotectin fusion protein with a final concentration of 0 / 100 / 200 / 400 / 800 μg / mL was added to each group, with 3 parallels per group.

[0098] (5) Detection of induction intensity:

[0099] From the beginning of bacterial culture, samples were taken every hour, and the fluorescence signal and bacterial OD of each group of fermentation samples were monitored by an ELISA instrument at 485 / 530 nm excitation / absorption light. 600 Data from the fluorescent signal of bacterial OD 600 The fluorescence intensity change curve is drawn. The data is shown in Figure 2 As shown, the fluorescence intensity exhibited a concentration-dependent intensity difference of the calprotectin fusion protein.

[0100] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A calprotectin fusion protein, wherein the calprotectin fusion protein is a heterodimer or heterotetramer formed by polymerization of S100A8 subunit and S100A9 subunit, characterized in that: An affinity tag with six histidine residues was introduced into the amino terminus of the S100A8 subunit.

2. A recombinant vector expressing the calprotectin fusion protein according to claim 1, characterized in that: pRSFDuet-1 is used as a backbone plasmid, and a S100A8 subunit coding gene and a S100A9 subunit coding gene are inserted; the S100A8 subunit coding gene and the S100A9 subunit coding gene are optimized according to the codon preference of Escherichia coli.

3. The recombinant vector according to claim 2, characterized in that The nucleotide sequence of the gene encoding the S100A8 subunit is shown in SEQ ID NO.1; the gene encoding the S100A8 subunit is constructed between the BamHI and SalI restriction sites of pRSFDuet-1.

4. The recombinant vector according to claim 2, characterized in that The nucleotide sequence of the gene encoding the S100A9 subunit is shown in SEQ ID NO.2; the gene encoding the S100A9 subunit is constructed between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector.

5. The recombinant vector according to any one of claims 2 to 4, characterized in that: The nucleotide sequence of the recombinant vector is as shown in SEQ ID NO.

4.

6. A recombinant Escherichia coli, characterized in that A recombinant vector comprising any one of claims 2 to 5.

7. The method for synthesizing the calprotectin fusion protein according to claim 1, characterized in that: The following steps are involved: The recombinant Escherichia coli according to claim 6 is fermented and cultured, and then induced with isopropyl-β-D-thiogalactopyranoside to obtain a bacterial cell containing a calprotectin fusion protein.

8. The method for purifying the calprotectin fusion protein according to claim 1, characterized in that: The following steps are involved: The bacterial cells or crude enzyme solution of the bacterial cells containing the calprotectin fusion protein obtained by the synthesis method of claim 7 are purified by a nickel affinity column to obtain the calprotectin fusion protein.

9. The purification method according to claim 8, characterized in that The purification method The following steps are involved: The bacterial cells containing the calprotectin fusion protein obtained by the synthesis method of claim 7 are subjected to ultrasonic disruption and then centrifugation to obtain a cell disruption liquid supernatant; The cell lysis solution supernatant is subjected to affinity adsorption on a nickel affinity column and gradient elution with an imidazole solution in sequence to obtain a calprotectin fusion protein solution; The calprotectin fusion protein solution is sequentially subjected to ultrafiltration washing to remove imidazole, thereby obtaining a purified calprotectin fusion protein solution; The purified calprotectin fusion protein solution is freeze-dried to obtain a purified calprotectin fusion protein; The imidazole solution gradient elution includes sequentially using phosphate buffer containing 20, 50, 100, 200, and 500 mM imidazole for gradient elution.

10. The calprotectin fusion protein according to claim 1, the recombinant vector according to any one of claims 2 to 5, the recombinant Escherichia coli according to claim 6, the bacterial cell containing the calprotectin fusion protein obtained by the synthesis method according to claim 7, or the purified calprotectin fusion protein obtained by the purification method according to claim 8 or 9, inducing Escherichia coli P ykgMO Application of promoter in downstream gene expression; ykgMO The promoter nucleotide sequence is shown in SEQ ID NO.3.

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