阅读说明：本技术 一种人干扰素-κ突变体及其制备方法 (Human interferon-kappa mutant and preparation method thereof ) 是由 彭继先 钱文正 柴辉 闫韵秋 戴文宇 李振森 于海勤 于 2020-12-25 设计创作，主要内容包括：本发明涉及人干扰素-κ(hIFN-κ)突变体及其制备方法。所述的hIFN-κ变体是将hIFN-κ的SEQ ID NO.1所示的氨基酸序列的第166位游离的半胱氨酸(C)突变为丝氨酸(S),或者突变为与丝氨酸结构相近的甘氨酸(G)或丙氨酸(A)。与含有野生型hIFN-κ序列的菌株相比,含有本发明的突变体序列的质粒的菌株表达产生的hIFN-κ产量较高,与干扰素-κ受体结合的亲和力更高,体外活性也更好。(The invention relates to a human interferon-kappa (hIFN-kappa) mutant and a preparation method thereof. The hIFN-kappa variant is obtained by mutating the 166 th free cysteine (C) of the amino acid sequence shown in SEQ ID NO.1 of hIFN-kappa into serine (S), or mutating the free cysteine (C) into glycine (G) or alanine (A) with similar structure with serine. Compared with a strain containing a wild type hIFN-kappa sequence, the strain containing the plasmid of the mutant sequence has higher hIFN-kappa yield, higher affinity for binding with an interferon-kappa receptor and better in vitro activity.)

1. A human interferon kappa (hIFN-. kappa.) mutant characterized in that the 166 th cysteine in the amino acid sequence of naturally occurring hIFN-. kappa.is mutated to an amino acid residue incapable of forming a disulfide bond.

2. The mutant according to claim 1, wherein in the mutant,

1) the cysteine at position 166 in SEQ ID NO.1 of the naturally occurring hIFN-. kappa.is replaced with any of the 19 other amino acids than cysteine among the 20 natural amino acids, preferably serine; or

2) The cysteine corresponding to position 166 of SEQ ID NO.1 of the amino acid sequence derived from human and having 99% homology with SEQ ID NO.1 is replaced with any of the other 19 amino acids except cysteine among the 20 natural amino acids, preferably serine; or

3) The amino acid sequence of the hIFN-kappa mutant is shown as SEQ ID NO. 2.

3. A nucleic acid molecule encoding the mutant of claim 1 or 2, wherein the nucleotide sequence of the nucleic acid molecule is as set forth in SEQ ID No. 3.

4. A nucleic acid construct comprising the nucleotide sequence of claim 3; preferably, the nucleic acid construct is a vector; more preferably, the vector is a plasmid vector; most preferably, the plasmid vector is selected from any one of pET28a, pET30a, pCZN1, pET22b, pET20b and pET-11 c.

5. A recombinant genetically engineered cell comprising the nucleic acid molecule of claim 3 or comprising the nucleic acid construct of claim 4; preferably, the genetically engineered cell is a prokaryotic host cell expressing the hIFN-kappa mutant; preferably, the prokaryotic host cell is a cell from a bacterium; more preferably, the prokaryotic host cell is a bacillus subtilis cell or an escherichia coli cell; most preferably, the prokaryotic host cell is a cell of E.coli BL21(DE 3).

6. A method for preparing hIFN-kappa mutant, wherein the hIFN-kappa mutant is obtained by culturing the genetically engineered cell of claim 5.

7. The method of claim 6, comprising:

1) culturing the gene engineering cell, and adding IPTG (isopropyl thiogalactoside) for induction expression;

2) centrifugally collecting thalli, and collecting inclusion bodies after ultrasonic crushing;

3) carrying out renaturation collection on guanidine hydrochloride to obtain an hIFN-kappa mutant;

4) purifying by chromatography to obtain protein.

8. The method of claim 7, further comprising measuring the affinity of the obtained hIFN- κ mutant for binding to a receptor and the in vitro cellular activity.

9. A pharmaceutical composition comprising the hIFN- κ mutant of claim 1 or 2.

10. Use of the hIFN-kappa mutant of claim 1 or 2 in the manufacture of a medicament for the treatment of a viral infection or a tumor.

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to a human interferon-kappa mutant and a preparation method thereof.

Background

Interferons (IFNs) are a class of cytokines with broad-spectrum antiviral, antitumor, immunomodulatory effects. It can exert antiviral activity by binding to cognate receptor complexes on target cells.

Interferon kappa (IFN-. kappa.) is a new member of IFN discovered in recent years, and it is a type I Interferon that uses a receptor protein in combination with IFN-. alpha.and IFN-. beta.s. IFN-. kappa.consists of 207 amino acid residues, including a signal peptide of 27 amino acid residues at the N-terminus, with about 30% homology to other members of type I IFN, and IFN-. kappa.is slightly larger than other type I IFN, with a 12 amino acid residue insertion between the C and D helices. The IFN-kappa gene is located in the short arm of the 9 th pair of chromosomes, adjacent to the genes of other type I IFNs, which have no intron, but the 3' non-coding region of the IFN-kappa gene contains 1 intron. At present, researches find that the gene coding IFN-kappa is selectively expressed in epithelial keratin cells, and the recombinant hIFN-kappa is similar to interferons of other homotypes and can protect the cells from virus infection; and the expression has ethnic specificity.

The natural interferon-kappa sequence contains two pairs of disulfide bonds and a single unpaired cysteine. The in vitro expression of interferon-kappa is always a difficult problem, and prokaryotic expression methods are adopted at present, but the expression is usually carried out in the form of inclusion bodies, so that the correct folding of proteins and the correct pairing of disulfide bonds become key in the process of protein renaturation. In addition, the presence of free cysteines during renaturation tends to interfere with this correct folding and pairing, and also renders the protein inactive by forming aggregates during renaturation.

Therefore, the method improves the correct folding of the protein and the correct pairing of disulfide bonds in the protein renaturation process, reduces the protein aggregation of the interferon-kappa in the renaturation process, and is particularly important for improving the purity of the interferon-kappa protein.

Disclosure of Invention

It is an object of the present invention to provide an interferon-kappa mutant having a significant improvement in both binding affinity to a receptor and activity, as compared to a natural interferon-kappa protein which has not been mutated.

Another objective of the invention is to provide a method for preparing interferon-kappa mutants, which realizes higher renaturation efficiency through operations such as culture, induced expression, inclusion body collection, renaturation, chromatography and the like, and the obtained protein has higher activity.

The purpose of the invention is realized by the technical scheme of the application, which comprises the following steps:

according to one aspect of the present invention, there is provided a human interferon kappa (hIFN-. kappa.) mutant.

In the mutant, the 166 th cysteine in the amino acid sequence of the natural hIFN-kappa is mutated into an amino acid residue which can not form a disulfide bond, wherein the mutant can eliminate the influence on the correct folding in the renaturation process of inclusion bodies in the protein expression and purification process.

In the hIFN-kappa mutants, 1) the cysteine at position 166 in SEQ ID NO.1 of the naturally occurring hIFN-kappa is replaced with any of the 19 other amino acids than cysteine among the 20 natural amino acids, preferably serine; or, 2) the cysteine at position 166 of SEQ ID No.1 of the amino acid sequence derived from human and having 99% homology to SEQ ID No.1 is replaced with any one of 19 amino acids other than cysteine among the 20 natural amino acids, preferably serine; or, 3) the amino acid sequence of the hIFN-kappa mutant is shown as SEQ ID NO. 2.

In the present invention, the 20 natural amino acids are 20 natural amino acids known in the art, such as glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

According to the second aspect of the present invention, there is provided a nucleic acid molecule encoding the hIFN-kappa mutant, wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 3.

ATGCTGGACTGCAATCTGCTGAACGTTCATCTGCGTCGCGTTACCTGGCAAAATCTGCGTCATCTGAGCAGCATGAGCAATAGCTTTCCGGTTGAGTGCCTGCGCGAAAATATCGCGTTTGAACTGCCGCAGGAATTTCTGCAGTATACCCAGCCGATGAAACGCGACATCAAAAAAGCCTTCTACGAGATGAGCCTGCAGGCGTTTAACATCTTCAGCCAGCACACCTTCAAATACTGGAAAGAGCGCCACCTGAAACAGATTCAGATTGGTCTGGACCAGCAGGCAGAATATCTGAATCAGTGCCTGGAAGAAGATAAAAACGAGAACGAGGACATGAAAGAGATGAAAGAGAACGAGATGAAACCGTCTGAAGCACGCGTTCCGCAACTGAGCAGCCTGGAACTGCGTCGTTATTTTCACCGCATCGACAACTTCCTGAAAGAGAAAAAATACAGCGATTGCGCTTGGGAAATTGTACGCGTCGAAATTCGCCGCAGCCTGTACTACTTTTACAAATTCACCGCCCTGTTCCGCCGTAAATAA(SEQ ID NO:3)

According to a third aspect of the present invention, there is provided a nucleic acid construct comprising said nucleotide sequence; preferably, the nucleic acid construct is a vector; more preferably, the vector is a plasmid vector; most preferably, the plasmid vector is any one of pET28a, pET30a, pCZN1, pET22b, pET20b, and pET-11 c.

According to a fourth aspect of the present invention there is provided a recombinant genetically engineered cell comprising a nucleic acid molecule or nucleic acid construct as described above.

Wherein, the genetic engineering cell is a prokaryotic host cell for expressing and producing the hIFN-kappa mutant; preferably, the prokaryotic host cell is a cell from a bacterium; more preferably, the prokaryotic host cell is a Bacillus subtilis cell or an Escherichia coli cell; most preferably, the prokaryotic host cell is an E.coli BL21(DE3) cell.

According to a fifth aspect of the present invention, there is provided a method for preparing an hIFN- κ mutant.

Wherein, the hIFN-kappa mutant is obtained by culturing the genetic engineering cell.

Specifically, the method comprises the following steps:

1) culturing the gene engineering cell, and adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression;

2) centrifugally collecting thalli, and collecting inclusion bodies after ultrasonic crushing;

3) carrying out renaturation collection on guanidine hydrochloride to obtain an hIFN-kappa mutant;

4) purifying by chromatography to obtain protein.

Wherein in step 1) IPTG is added when the cells are cultured to an OD600 value of 0.4-1.2, preferably 0.5-1.0, most preferably 0.6-0.8; and the concentration of IPTG is 0.4-1.4mM, preferably 0.6-1.2mM, more preferably 0.8-1 mM.

In step 3), the inclusion bodies are solubilized using 4-8M guanidine hydrochloride or urea.

In step 4), firstly removing the foreign protein by anion chromatography in a flow-through manner, and then purifying the target protein by cation chromatography in a combined manner; preferably, the chromatographic column is equilibrated with 10-30mM PB binding buffer, then renaturation liquid is used for loading, after all loading, the chromatographic column is washed with the equilibration buffer until the UV value is below 20mAu, and then the target protein is eluted by using the elution buffer gradient of 10-30mM PB +300-800mM NaCl.

Wherein, the method also comprises detecting the binding affinity of the hIFN-kappa mutant with the receptor and the in vitro cell activity.

According to a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising the hIFN- κ mutant described above.

According to a seventh aspect of the present invention, there is provided the use of the hIFN- κ mutant of the invention for the preparation of a medicament for the treatment of a viral infection or a tumor.

In the present invention, the natural hIFN-kappa has the same meaning as the wild type hIFN-kappa, and refers to the hIFN-kappa protein with unchanged amino acid sequence, and can be used alternatively.

The invention has the beneficial effects that: the 166 th cysteine in the amino acid sequence of the hIFN-kappa mutant is mutated into other amino acids except cysteine, such as serine, so that the interference of free sulfydryl is removed. In addition, serine is structurally close to cysteine and is a relatively simple amino acid, so that cysteine mutation does not affect the overall structure of the hIFN-kappa protein. Under the condition that the whole structure of the hIFN-kappa protein is not influenced, the mutation obviously improves the renaturation efficiency of the protein, the binding capacity with a receptor is obviously improved under the same concentration, and the protein activity is also obviously improved, so that the production efficiency of the hIFN-kappa protein is greatly improved, and the cost is reduced.

Drawings

FIG. 1: dissociation curves for binding of wild-type hIFN-. kappa.and hIFN-. kappa.mutants to receptors: a is the combination dissociation curve of wild type hIFN-kappa and receptor, B is the combination dissociation curve of hIFN-kappa mutant and receptor; and

FIG. 2: the activity detection results of wild type hIFN-kappa and hIFN-kappa mutant.

Detailed Description

The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments, and those skilled in the art will understand that the described embodiments are for illustrative purposes only and are not all the present invention. Based on the embodiments of the present invention, those skilled in the art will better understand and appreciate the technical solutions claimed in the present invention and the technical effects achieved thereby.

In the following examples, reagents other than the specifically prepared reagents were commercially available.

Example 1 hIFN-. kappa.mutant encoding Gene and expression vector acquisition

The amino acid sequence of hIFN-kappa is shown below:

MLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK(SEQ ID NO:1)

according to the hIFN-kappa amino acid sequence and the higher structure thereof disclosed by the prior art, the 166 th cysteine (Cys) does not participate in the formation of disulfide bonds, so that the inventor conducts site-specific mutagenesis on the hIFN-kappa amino acid sequence to construct a mutagenesis library, namely, the hIFN-kappa amino acid sequence is mutated into any one of other nineteen amino acids which do not contain sulfydryl except Cys in 20 common amino acids, and the mutated sequence is shown as follows:

MLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRXLYYFYKFTALFRRK(SEQ ID NO:4)

wherein X is any one of nineteen common amino acids except Cys.

Through bioinformatics modeling, the binding specificity of the hIFN-kappa containing the mutant sequence and the receptor thereof is analyzed, the applicant finds that the hIFN-kappa containing the mutant sequence has small influence on the higher structure of hIFN-kappa containing the mutant sequence under the condition that the hIFN-kappa is mutated into glycine, serine and alanine, the difference between the activity of hIFN-kappa containing the mutant sequence and the receptor is small, and the three mutants are selected for experimental verification. For SEQ ID NO 4 above, where X is Gly, Ala or Ser.

Considering that cysteine and serine have the most similar structures, serine substitution is finally selected for experimental verification, and the amino acid sequence is shown as follows:

MLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRSLYYFYKFTALFRRK(SEQ ID NO:2)

in order to improve the translation efficiency of the gene, the mutant hIFN-kappa amino acid sequence SEQ ID NO. 2 was subjected to E.coli codon optimization to finally obtain the nucleotide sequence of the coding gene as shown in SEQ ID NO. 3, which was synthesized by Suzhou Hongxi Biotech Limited.

The obtained coding gene shown in SEQ ID NO. 3 was transferred to pET28a (+) by Hongxn Biotechnology Co., Suzhou to obtain recombinant plasmid pET 28-hIFN-. kappa.MUT.

Example 2 protein preparation

1. The recombinant plasmid prepared in the example 1 is transferred into escherichia coli BL21(DE3) competent cells, kanamycin resistance screening is carried out, single colonies are selected for PCR identification, and positive clone cells, namely the engineering bacteria containing the recombinant plasmid pET 28-hIFN-kappa-MUT, are screened out.

2. Expression and purification of the recombinant plasmid pET 28-hIFN-. kappa.MUT in E.coli BL21(DE 3):

1) a single colony of Escherichia coli BL21(DE3) containing the recombinant plasmid pET 28-hIFN-. kappa.MUT was inoculated into 5ml of LB liquid medium containing 50. mu.g/ml kanamycin, shake-cultured at 37 ℃ at 200r/min for 16 hours, and 5ml of the bacterial solution was added to 500ml of LB liquid medium containing 50. mu.g/ml kanamycin, 1: 100 scale-up culture.

2) When the OD600 value of the escherichia coli cultured in the step 1) reaches 0.6-0.8, 1mM IPTG is added into the bacterial liquid, and shake culture is carried out for 4h at 16 ℃. Centrifugally collecting the induced thallus, re-suspending, carrying out ice bath for 30min, carrying out ultrasonic crushing for about 15min, centrifugally collecting precipitate, namely inclusion bodies, and washing the inclusion bodies for 3-5 times by using 3M urea solution.

3) Dissolving the inclusion body by 6M guanidine hydrochloride solution, diluting and renaturing, filtering and clarifying after renaturation, and collecting supernatant for later use; firstly, the chromatographic column is equilibrated by 20mM PB binding buffer, the supernatant is loaded, after all the samples are loaded, the chromatographic column is washed by the equilibration buffer until the UV value is below 20mAu, then the gradient elution is carried out by the elution buffer containing 20mM PB and 500mM NaCl, and the eluent containing the target protein is collected.

Wherein, in the step 1), the optimal OD600 value is determined by adopting a single-factor variable method: IPGT was added at OD600 values of 0.4, 0.6, 0.8, 1.0, 1.2, and then SDS-PAGE was followed by Coomassie blue staining to determine protein expression concentration, and the optimal concentration of protein was obtained when the optimal bacterial solution OD600 was determined to be 0.6-0.8.

In step 2), the optimum IPTG concentration is determined by: when the bacteria were cultured to OD600 of 0.6-0.8, IPGT was added at final concentrations of 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM, and 1.4mM to induce expression for 4 hours, and a portion of the cells was collected and subjected to SDS-PAGE gel electrophoresis, and Coomassie blue staining to verify the protein concentration, thereby finally confirming that the optimum target protein concentration was obtained at an IPGT concentration of 0.8-1 mM.

Example 3 Activity assay

1. Affinity assay for binding of interferon-kappa proteins to their receptors

The binding affinity of interferon- κ to its receptor was determined using a gator unlabeled biomolecular analyzer (probelife) from the suzhou crystal laboratories, following the instructions for its use: firstly diluting purified interferon-kappa by multiple proportion, wherein the total concentration is 5, then combining an anti-huFc probe with an interferon receptor, reacting with interferon kappa with different concentrations, combining the receptor with interferon-kappa protein to form a dynamic curve, putting the reacted probe into a dissociation buffer solution, slowly dissociating the receptor and the interferon-kappa protein to form a dynamic curve, and finally calculating the affinity according to the curve and the concentration. Specific results are shown in Table 1 and FIGS. 1A-B.

TABLE 1

Sample (I)	Koff(1/s)	Kon(1/Ms)	KD(M)	Response to
					Wild type interferon-kappa	0.00422	5.21E+04	8.10E-08	0.146
Interferon-kappa mutants	0.00734	1.13E+05	6.51E-08	0.187

As shown in Table 1 and FIG. 1, the KD value of the interferon-. kappa.mutants of the present invention is lower than that of the wild-type interferon-. kappa.which is a sufficient indication that the interferon-. kappa.mutants bind to their receptors more strongly than the wild-type interferon-. kappa.. In addition, the response value of the interferon-kappa mutant after cysteine mutation is also obviously greater than that of the wild-type non-mutated interferon-kappa protein. The result shows that the mutation of free cysteine in the amino acid sequence of interferon-kappa into serine which can not form disulfide bond for connecting two peptide chains avoids the abnormal connection between the free cysteine and other normally paired cysteine, so that the mutation greatly promotes the stability of interferon-kappa protein in solution in a certain sense, and further improves the binding affinity of the protein and its receptor.

2. In vitro Activity assay for Interferon-kappa proteins

According to the reporter gene plasmid culture method, luciferase reporter gene plasmids are transfected in HEK293 cells, positive clones are screened by antibiotics, positive monoclonals are screened by a limiting dilution method, cells are amplified and plated, interferon-kappa after gradient dilution is added for co-culture, then a fluorescence indicator is added, signals are recorded and analyzed, and the result is shown in figure 2.

As can be seen from FIG. 2, the interferon-. kappa.mutants of the present invention have significantly higher activity than the non-mutated interferon-. kappa.s. The activity of the non-mutated interferon-kappa fluctuates greatly at higher drug concentrations. This also fully indicates that the interferon-kappa mutants of the present invention have higher activity and stability even at larger drug concentrations.

Although the present invention has been described with reference to the preferred embodiments, it is not intended to limit the present invention, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the present invention.

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