JUP gene mutant and application thereof
阅读说明：本技术 Jup基因突变体及其应用 (JUP gene mutant and application thereof ) 是由 程翔 查灵凤 杨芬 郭爽 于 2021-08-08 设计创作，主要内容包括：本发明属于基因研究技术领域,公开了JUP基因突变体及其应用。具体公开了核酸,包含下列目标片段,目标片段与野生型JUP基因相比,核酸具有c.958C>T突变；优选的,核酸为DNA,在一些情况下含有该目标片段的RNA也在本发明范围内；多肽,与野生型JUP相比,多肽具有p.R320C突变；基因突变,与野生型JUP基因相比,基因突变具有c.958C>T突变；含有前序突变的生物模型在制备筛查、防治心肌病试剂中的应用；防治心肌病药物,包括基因载体,基因载体含有能将JUP基因的c.958位点单核苷酸T替换为单核苷酸C并表达的基因片段。本发明提供了心肌病尤其是致心律失常性右室心肌病的新的突变点,弥补了筛查、治疗上的一些空白。(The invention belongs to the technical field of gene research and discloses a JUP gene mutant and application thereof. Specifically disclosed are nucleic acids comprising a target fragment having a c.958C > T mutation in the nucleic acid, as compared to a wild-type JUP gene; preferably, the nucleic acid is DNA, and in some cases RNA containing the target fragment is also within the scope of the invention; a polypeptide having a p.r320c mutation compared to wild-type JUP; a gene mutation having a c.958C > T mutation compared to a wild-type JUP gene; the application of a biological model containing preorder mutation in the preparation of a reagent for screening and preventing and treating cardiomyopathy; the medicine for preventing and treating cardiomyopathy includes gene carrier containing gene segment capable of substituting mononucleotide T to mononucleotide C at c.958 site of JUP gene and expressing. The invention provides a new mutation point of cardiomyopathy, in particular arrhythmogenic right ventricular cardiomyopathy, and fills some gaps in screening and treatment.)
1. A nucleic acid comprising a target fragment,
said nucleic acid having a c.958C > T mutation in said target fragment as compared to a wild-type JUP gene; preferably, the nucleic acid is DNA.
2. A polypeptide characterized by having a sequence selected from the group consisting of,
the polypeptide has a p.r320c mutation compared to wild-type JUP.
3. A genetic mutation characterized in that said genetic mutation has a c.958c > T mutation as compared to the wild-type JUP gene.
4. The application of the biological model in preparing reagent for screening and preventing cardiomyopathy is characterized in that: the biological model carries at least one of the following:
a. the nucleic acid according to claim 1, wherein said nucleic acid is a nucleic acid,
b. the polypeptide of claim 2, wherein said polypeptide is,
c. a mutation of the gene of claim 3;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
5. A biological model for screening cardiomyopathy comprising a reagent that detects a JUP gene mutant, wherein the JUP gene mutant is at least one of:
a. the nucleic acid according to claim 1, wherein said nucleic acid is a nucleic acid,
b. the polypeptide of claim 2, wherein said polypeptide is,
c. a mutation of the gene of claim 3;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
6. The biological model of cardiomyopathy of claim 5 wherein the reagents capable of detecting mutants of the JUP gene comprise nucleic acid probes or primers;
preferably, the primer comprises
A forward primer having nucleotide sequence aaccagctgtcgaagaagga, and/or
The reverse primer having nucleotide sequence ggacacacggatagcacctt.
7. A construct comprising the nucleic acid of claim 1 or the genetic mutation of claim 3.
8. Recombinant cell obtained by transforming a recipient cell with the construct according to claim 7 or expressing the polypeptide according to claim 2.
9. The application of a mutation inhibitor in preparing a medicament for preventing and treating cardiomyopathy is characterized in that the mutation inhibitor is at least one of the following:
JUP gene DNA sequence c.958C > T mutation inhibitor,
JUP gene protein sequence p.R320C mutation inhibitor.
10. A pharmaceutical composition for preventing and treating myocardial infarction, which comprises at least one of the following drugs
JUP gene DNA sequence c.958C > T mutation inhibitor,
a protein sequence p.R320C mutation inhibitor of JUP gene;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy, and more preferably, the cardiomyopathy preventing and treating drug is an inhibitor of right ventricular cardiomyocyte apoptosis.
The application of the gene segment in preparing the medicine for preventing and treating the cardiomyopathy is characterized in that the gene segment is a gene segment of which the mononucleotide at the c.958 site of a JUP gene is C, and preferably the arrhythmogenic right ventricular cardiomyopathy is prepared.
The medicine for preventing and treating myocardial diseases is characterized by comprising a gene carrier containing
A gene fragment capable of substituting mononucleotide T at position c.958 of JUP gene into mononucleotide C and expressing, preferably, said cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
Technical Field
The invention belongs to the technical field of gene research, and particularly relates to a JUP gene mutant and application thereof.
Background
Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a genetically related cardiomyopathy with the pathological characteristics of apoptosis or necrosis of cardiomyocytes dominated by the right ventricle and replacement by adipose and fibrous connective tissue, and can also affect the left ventricle simultaneously or separately. Malignant events such as heart failure, malignant arrhythmia and SCD may occur clinically. The estimated prevalence rate in western population is 0.02% -0.05%, and the male-female ratio is 3:1, which is an important reason for SCD in population under 35 years old. The research of molecular genetics finds that desmosome gene mutation is the main pathogenesis of ARVC, the related genes comprise 13 mutant genes comprising 5 desmosome genes and 8 non-desmosome genes, but the desmosome gene mutation is considered to be a main factor causing the myocardial apoptosis of patients and the change of the myocardial cell mutation into adipose tissue. More than 50% of patients can find the 5 desmosome gene heterozygous mutation, and a few of the 5 desmosome gene heterozygous mutation or homozygous mutation are mostly autosomal dominant inheritance and a few of the 5 desmosome gene homozygous mutation are autosomal recessive inheritance. In 2000, the 1 st pathogenic gene JUP is identified in the Naxos disease, codes for desmosomal globin (PG) and is a main component of intercellular adhesion, and at present, the number of related pathogenic sites of ARVC is less than 20, so that related gene mutation is still to be further researched. Thus, the discovery and proposal of any one or a group of genes associated with arrhythmogenic right ventricular cardiomyopathy would be an important technical contribution to the art.
Disclosure of Invention
Aiming at the problems, the invention provides the JUP gene mutant and the application thereof, mainly makes up the mechanism research of the current cardiomyopathy, particularly the arrhythmogenic right ventricular cardiomyopathy, and increases the means for treating and screening the disease.
In order to solve the problems, the invention adopts the following technical scheme:
a nucleic acid comprising a target fragment,
said nucleic acid having a c.958C > T mutation in said target fragment as compared to a wild-type JUP gene; preferably, the nucleic acid is DNA, and in some cases RNA containing the target fragment is also within the scope of the invention.
A polypeptide having a p.r320c mutation compared to wild-type JUP.
A gene mutation having a c.958C > T mutation compared to a wild-type JUP gene.
Use of a biological model for the manufacture of a reagent for screening and/or preventing cardiomyopathy, said biological model carrying at least one of the following:
a. a nucleic acid having a c.958C > T mutation,
b. a polypeptide having a p.R320C mutation,
c. a mutation in a gene having a c.958C > T mutation;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
A biological model for screening cardiomyopathy, which comprises a reagent capable of detecting a JUP gene mutant, wherein the JUP gene mutant is at least one of the following:
a. a nucleic acid having a c.958C > T mutation,
b. a polypeptide having a p.R320C mutation,
c. a mutation in a gene having a c.958C > T mutation;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
In some embodiments, the reagent capable of detecting a mutant of the JUP gene comprises a primer or a nucleic acid probe;
preferably, the primer comprises
A forward primer having nucleotide sequence aaccagctgtcgaagaagga, and/or
The reverse primer having nucleotide sequence ggacacacggatagcacctt.
A construct comprising the nucleic acid or the genetic mutation described above.
Recombinant cells obtained by transforming a recipient cell with the aforementioned construct or expressing the aforementioned polypeptide.
The recombinant protein for preparing the medicine for preventing and treating the cardiomyopathy is expressed by the recombinant cell.
The application of a mutation inhibitor in preparing a medicament for preventing and treating cardiomyopathy, wherein the mutation inhibitor is at least one of the following:
JUP gene DNA sequence c.958C > T mutation inhibitor,
JUP gene protein sequence p.R320C mutation inhibitor.
A medicine for preventing and treating myocardial diseases contains at least one of the following medicines
JUP gene DNA sequence c.958C > T mutation inhibitor,
JUP gene protein sequence p.R320C mutation inhibitor.
In some cases, the drug is an inhibitor of right ventricular cardiomyocyte apoptosis.
The application of the gene segment in preparing the medicine for preventing and treating the cardiomyopathy is that the mononucleotide at the c.958 site of the JUP gene is C, preferably, the arrhythmogenic right ventricular cardiomyopathy is prepared.
The medicine for preventing and treating cardiomyopathy comprises a gene carrier containing
A gene fragment capable of substituting and expressing mononucleotide T at the c.958 site of JUP gene into mononucleotide C, preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy;
some expression forms of the gene vector are plasmid and adenovirus vectors.
The invention has the beneficial effects that:
provides a new mutation point of cardiomyopathy, especially arrhythmogenic right ventricular cardiomyopathy, makes up for some blank in screening and treatment, and provides some new drugs for screening and preventing cardiomyopathy.
Drawings
FIG. 1 is a family diagram of an item object;
FIG. 2 is a sequence chart of probands and healthy family members;
FIG. 3 is a screening chart for mutations in healthy people;
FIG. 4 is a study example flow chart;
FIG. 5 is a diagram of the domain of the JUP protein in which the mutation is located;
FIG. 6 shows the function prediction of mutation sites.
Detailed Description
The invention is further illustrated below:
a nucleic acid comprising a target fragment,
the target fragment has a c.958C > T mutation compared to the wild type JUP gene (one as SEQ ID NO. 1).
A polypeptide having a p.r320c mutation compared to wild type JUP (one as SEQ ID No. 2).
A gene mutation having a c.958C > T mutation compared to a wild-type JUP gene.
When the wild type JUP is subjected to mutation at a single nucleotide or amino acid sequence site of other sites of the JUP gene, the protection of the detection site related to the invention is not influenced, and as long as the site related to the invention is involved, other sites are changed relative to SEQ ID NO.1 or SEQ ID NO.2, and the protection is also within the scope of the invention.
Use of a biological model for the manufacture of a reagent for screening and/or preventing cardiomyopathy, said biological model carrying at least one of the following:
a. a nucleic acid having a c.958C > T mutation,
b. a polypeptide having a p.R320C mutation,
c. a mutation in a gene having a c.958C > T mutation;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
A biological model for screening cardiomyopathy, which comprises a reagent capable of detecting a JUP gene mutant, wherein the JUP gene mutant is at least one of the following:
a. a nucleic acid having a c.958C > T mutation,
b. a polypeptide having a p.R320C mutation,
c. a mutation in a gene having a c.958C > T mutation;
preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy.
When used in the preparation of screening reagents, reference is made to the subsequent specific use; when the biological inhibitor is used in preparation of a prevention and treatment agent, some modes are used as action targets of the medicine, or other biological medicines are used in steps of testing, extracting and the like in the preparation process, and the biological inhibitor is mainly used as auxiliary agents in the pharmaceutical process, for example, in the test of the action effect of the inhibitor in the pharmaceutical process.
In some embodiments, the reagent capable of detecting a mutant of the JUP gene comprises a primer or a nucleic acid probe;
preferably, the primer comprises
A forward primer having nucleotide sequence aaccagctgtcgaagaagga, and/or
A reverse primer having nucleotide sequence ggacacacggatagcacctt;
the nucleic acid probe may be a proximal gene detection probe, and is not particularly limited as long as it is possible.
A construct comprising the nucleic acid or the genetic mutation described above.
Recombinant cells obtained by transforming a recipient cell with the aforementioned construct or expressing the aforementioned polypeptide.
The recombinant protein for preparing the medicine for preventing and treating the cardiomyopathy is expressed by the recombinant cell.
The application of a mutation inhibitor in preparing a medicament for preventing and treating cardiomyopathy, wherein the mutation inhibitor is at least one of the following:
JUP gene DNA sequence c.958C > T mutation inhibitor,
JUP gene protein sequence p.R320C mutation inhibitor.
A medicine for preventing and treating myocardial diseases contains at least one of the following medicines
JUP gene DNA sequence c.958C > T mutation inhibitor,
JUP gene protein sequence p.R320C mutation inhibitor.
In some cases, the drug is an inhibitor of right ventricular cardiomyocyte apoptosis.
Where the aforementioned inhibitors comprise existing objectively feasible pharmaceutical agents, they should also comprise some pharmaceutical agents that are newly developed later.
The application of a gene segment in preparing a medicine for treating cardiomyopathy is disclosed, wherein the gene segment is a gene segment with C.958 site mononucleotide of JUP gene as C, and preferably, the arrhythmogenic right ventricular cardiomyopathy is disclosed.
The medicine for preventing and treating cardiomyopathy comprises a gene carrier containing
A gene fragment capable of substituting and expressing mononucleotide T at the c.958 site of JUP gene into mononucleotide C, preferably, the cardiomyopathy is arrhythmogenic right ventricular cardiomyopathy;
some expression forms of the gene vector are plasmid and adenovirus vectors.
The mutant gene is replaced to restore the normal gene sequence to realize treatment, thereby achieving the purposes of prevention and treatment. At present, the gene vector is preferably considered to be known and mature, but future emerging can gain practical recognition should also be within the scope of the present invention.
A specific study procedure is illustrated below in connection with a specific study, as shown in figure 4.
Family collection
After obtaining informed consent of the family members of the patients, clinical data of the proband and the family members are collected, a family system diagram (figure 1) is drawn, the proband is clinically diagnosed as arrhythmogenic right ventricular cardiomyopathy, and other family members are healthy. The black box refers to the diseased male, the white box to the healthy male, the white round box to the healthy female, and the arrow to the probative. 2-5ml of peripheral blood from each subject was collected using EDTA anticoagulated blood collection tubes and stored at-80 ℃.
Second, DNA extraction
Peripheral Blood whole genome DNA was extracted using a TIANAmp Blood DNA Kit Blood genome DNA extraction Kit. The purity and quality of the extracted DNA were examined by Nanodrop 2000 ultramicro nucleic acid protein assay and agarose gel electrophoresis, respectively.
Sequencing of three, all exons
DNA of proband was subjected to full exon detection. The exon detection results are shown in the following sample detection result classification statistical table:
fourth, data screening and analysis
The gene mutation information, mutation frequency and the like are inquired according to a gene mutation database (ExAC Browser), a thousand-people genome database (1000genome), a dbSNP database, an Ensembl database, an NCBI database and a UCSC database.
According to SIFT, Polyphen2, MutationTaster and other websites, the harmfulness of the mutation is predicted, the conservation of the mutation site is analyzed, and the analysis result is pathogenicity.
Sequencing by Sanger
Primer design
Designing a mutation site amplification primer by using Genetool software:
a forward primer: 5'-aaccagctgtcgaagaagga-3'
Reverse primer: 5'-ggacacacggatagcacctt-3' are provided.
DNA amplification (PCR)
The PCR reaction system amounted to 25 ul: 1ul of sample DNA, forward and reverse primers 0.5ul each, 12.5ul of 2 XTSINGKE Master Mix and 10.5ul of ddH 2O.
PCR amplification procedure: the sample is loaded on a machine, preheated at 94 ℃ for 3 minutes, circularly denatured at 94 ℃ for 30s, annealed at 59 ℃ for 30s with gradient temperature, extended at 72 ℃ for 35 times, kept at 72 ℃ for 10 minutes, and then the end temperature is 4 ℃.
PCR product detection
And detecting the PCR product by agarose gel electrophoresis. Under the indication of marker observed under a gel imager, a brighter band is arranged in the middle of the band size of 500-1000, and the position is about 800, which indicates that the PCR amplification is successful.
Sequencing
After successful PCR amplification with DNA of proband its family members, Sanger sequencing was performed.
Analysis of sequencing results
Finch TV Version 1.4.0 was used to read Sanger sequencing results as in FIG. 2.
Sixth, screening mutation of healthy population
30 healthy populations were selected for mutation screening and the results are shown in figure 3.
Results of the study
Proband carry a mutation in the JUP gene, genotype CT, as in the case of the sequencing mutant in FIG. 2, which is located in exon 6, c.958C > T, p.R320C: CGT > TGT (p.Arg320Cys), resulting in the conversion of arginine at position 320 to cysteine, as shown in FIG. 5. The other family members did not carry this mutation and were all of the CC genotypes, as the wild type of the sequencing diagrams in FIG. 2.
The mutation type is heterozygous mutation and missense mutation, also known as rs200740462, the mutation frequency is 0.0002, the mutation belongs to rare mutation, and no report is found in Chinese population, the mutation is not found in screening 30 healthy populations, and the genotypes of the 30 healthy populations are CC. As in fig. 5, the predicted harmfulness is: pathogenicity (SIFT, Polyphen2, mutationmaster), this mutated region is conserved and an important domain of the JUP protein. RegulomeDB is a database for annotating non-coding region SNPs and gene regulatory elements, and the annotation comprises Expression quantitative trait loci (eQTL), DNase I hypersensitive binding regions (DNase), transcription factor synthetic sites, known promoter regions and the like. According to the annotations, the website can comprehensively score the SNPs, and can deduce and detect the regulation mechanism possibly participated in by the interested SNPs according to the scores, thereby providing an idea for research. As shown in FIG. 6, the functional analysis of the mutation site was found to be 2b, indicating that the mutation site is likely to regulate gene expression.
It will be apparent to those skilled in the art that various modifications may be made to the above embodiments without departing from the general spirit and concept of the invention. All falling within the scope of protection of the present invention. The protection scheme of the invention is subject to the appended claims.
Sequence listing
<110> affiliated cooperation hospital of college of Tongji medical college of Huazhong university of science and technology
<120> JUP novel mutant protein, novel mutant gene and use thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2238
<212> DNA
<213> person (Huma)
<400> 1
atggaggtga tgaacctgat ggagcagcct atcaaggtga ctgagtggca gcagacatac 60
acctacgact cgggtatcca ctcgggcgcc aacacctgcg tgccctccgt cagcagcaag 120
ggcatcatgg aggaggatga ggcctgcggg cgccagtaca cgctcaagaa aaccaccact 180
tacacccagg gggtgccccc cagccaaggt gatctggagt accagatgtc cacaacagcc 240
agggccaaac gggtgcggga ggccatgtgc cctggtgtgt caggcgagga cagctcgctt 300
ctgctggcca cccaggtgga ggggcaggcc accaacctgc agcgactggc cgagccgtcc 360
cagctgctca agtcggccat tgtgcatctc atcaactacc aggacgatgc cgagctggcc 420
actcgcgccc tgcccgagct caccaaactg ctcaacgacg aggacccggt ggtggtgacc 480
aaggcggcca tgattgtgaa ccagctgtcg aagaaggagg cgtcgcggcg ggccctgatg 540
ggctcgcccc agctggtggc cgctgtcgtg cgtaccatgc agaataccag cgacctggac 600
acagcccgct gcaccaccag catcctgcac aacctctccc accaccggga ggggctgctc 660
gccatcttca agtcgggtgg catccctgct ctggtccgca tgctcagctc ccctgtggag 720
tcggtcctgt tctatgccat caccacgctg cacaacctgc tcctgtacca ggagggcgcc 780
aagatggccg tgcgcctggc cgacgggctg caaaagatgg tgcccctgct caacaagaac 840
aaccccaagt tcctggccat caccaccgac tgcctgcagc tcctggccta cggcaaccag 900
gagagcaagc tgatcatcct ggccaatggt gggccccagg ccctcgtgca gatcatgcgt 960
aactacagtt atgaaaagct gctctggacc accagtcgtg tgctcaaggt gctatccgtg 1020
tgtcccagca ataagcctgc cattgtggag gctggtggga tgcaggccct gggcaagcac 1080
ctgaccagca acagcccccg cctggtgcag aactgcctgt ggaccctgcg caacctctca 1140
gatgtggcca ccaagcagga gggcctggag agtgtgctga agattctggt gaatcagctg 1200
agtgtggatg acgtcaacgt cctcacctgt gccacgggca cactctccaa cctgacatgc 1260
aacaacagca agaacaagac gctggtgaca cagaacagcg gtgtggaggc tctcatccat 1320
gccatcctgc gtgctggtga caaggacgac atcacggagc ctgccgtctg cgctctgcgc 1380
cacctcacta gccgccaccc tgaggccgag atggcccaga actctgtgcg tctcaactat 1440
ggcatcccag ccatcgtgaa gctgctcaac cagcccaacc agtggccact ggtcaaggca 1500
accatcggct tgatcaggaa tctggccctg tgcccagcca accatgcccc gctgcaggag 1560
gcagcggtca tcccccgcct cgtccaactg ctggtgaagg cccaccagga tgcccagcgc 1620
cacgtagctg caggcacaca gcagccctac acggatggtg tgaggatgga ggagattgtg 1680
gagggctgca ccggagcact gcacatcctc gcccgggacc ccatgaaccg catggagatc 1740
ttccggctca acaccattcc cctgtttgtg cagctcctgt actcgtcggt ggagaacatc 1800
cagcgcgtgg ctgccggggt gctgtgtgag ctggcccagg acaaggaggc ggccgacgcc 1860
attgatgcag agggggcctc ggccccactc atggagttgc tgcactcccg caacgagggc 1920
actgccacct acgctgctgc cgtcctgttc cgcatctccg aggacaagaa cccagactac 1980
cggaagcgcg tgtccgtgga gctcaccaac tccctcttca agcatgaccc ggctgcctgg 2040
gaggctgccc agagcatgat tcccatcaat gagccctatg gagatgacat ggatgccacc 2100
taccgcccca tgtactccag cgatgtgccc cttgacccgc tggagatgca catggacatg 2160
gatggagact accccatcga cacctacagc gacggcctca ggcccccgta ccccactgca 2220
gaccacatgc tggcctag 2238
<210> 2
<211> 745
<212> PRT
<213> person (Huma)
<400> 2
Met Glu Val Met Asn Leu Met Glu Gln Pro Ile Lys Val Thr Glu Trp
1 5 10 15
Gln Gln Thr Tyr Thr Tyr Asp Ser Gly Ile His Ser Gly Ala Asn Thr
20 25 30
Cys Val Pro Ser Val Ser Ser Lys Gly Ile Met Glu Glu Asp Glu Ala
35 40 45
Cys Gly Arg Gln Tyr Thr Leu Lys Lys Thr Thr Thr Tyr Thr Gln Gly
50 55 60
Val Pro Pro Ser Gln Gly Asp Leu Glu Tyr Gln Met Ser Thr Thr Ala
65 70 75 80
Arg Ala Lys Arg Val Arg Glu Ala Met Cys Pro Gly Val Ser Gly Glu
85 90 95
Asp Ser Ser Leu Leu Leu Ala Thr Gln Val Glu Gly Gln Ala Thr Asn
100 105 110
Leu Gln Arg Leu Ala Glu Pro Ser Gln Leu Leu Lys Ser Ala Ile Val
115 120 125
His Leu Ile Asn Tyr Gln Asp Asp Ala Glu Leu Ala Thr Arg Ala Leu
130 135 140
Pro Glu Leu Thr Lys Leu Leu Asn Asp Glu Asp Pro Val Val Val Thr
145 150 155 160
Lys Ala Ala Met Ile Val Asn Gln Leu Ser Lys Lys Glu Ala Ser Arg
165 170 175
Arg Ala Leu Met Gly Ser Pro Gln Leu Val Ala Ala Val Val Arg Thr
180 185 190
Met Gln Asn Thr Ser Asp Leu Asp Thr Ala Arg Cys Thr Thr Ser Ile
195 200 205
Leu His Asn Leu Ser His His Arg Glu Gly Leu Leu Ala Ile Phe Lys
210 215 220
Ser Gly Gly Ile Pro Ala Leu Val Arg Met Leu Ser Ser Pro Val Glu
225 230 235 240
Ser Val Leu Phe Tyr Ala Ile Thr Thr Leu His Asn Leu Leu Leu Tyr
245 250 255
Gln Glu Gly Ala Lys Met Ala Val Arg Leu Ala Asp Gly Leu Gln Lys
260 265 270
Met Val Pro Leu Leu Asn Lys Asn Asn Pro Lys Phe Leu Ala Ile Thr
275 280 285
Thr Asp Cys Leu Gln Leu Leu Ala Tyr Gly Asn Gln Glu Ser Lys Leu
290 295 300
Ile Ile Leu Ala Asn Gly Gly Pro Gln Ala Leu Val Gln Ile Met Arg
305 310 315 320
Asn Tyr Ser Tyr Glu Lys Leu Leu Trp Thr Thr Ser Arg Val Leu Lys
325 330 335
Val Leu Ser Val Cys Pro Ser Asn Lys Pro Ala Ile Val Glu Ala Gly
340 345 350
Gly Met Gln Ala Leu Gly Lys His Leu Thr Ser Asn Ser Pro Arg Leu
355 360 365
Val Gln Asn Cys Leu Trp Thr Leu Arg Asn Leu Ser Asp Val Ala Thr
370 375 380
Lys Gln Glu Gly Leu Glu Ser Val Leu Lys Ile Leu Val Asn Gln Leu
385 390 395 400
Ser Val Asp Asp Val Asn Val Leu Thr Cys Ala Thr Gly Thr Leu Ser
405 410 415
Asn Leu Thr Cys Asn Asn Ser Lys Asn Lys Thr Leu Val Thr Gln Asn
420 425 430
Ser Gly Val Glu Ala Leu Ile His Ala Ile Leu Arg Ala Gly Asp Lys
435 440 445
Asp Asp Ile Thr Glu Pro Ala Val Cys Ala Leu Arg His Leu Thr Ser
450 455 460
Arg His Pro Glu Ala Glu Met Ala Gln Asn Ser Val Arg Leu Asn Tyr
465 470 475 480
Gly Ile Pro Ala Ile Val Lys Leu Leu Asn Gln Pro Asn Gln Trp Pro
485 490 495
Leu Val Lys Ala Thr Ile Gly Leu Ile Arg Asn Leu Ala Leu Cys Pro
500 505 510
Ala Asn His Ala Pro Leu Gln Glu Ala Ala Val Ile Pro Arg Leu Val
515 520 525
Gln Leu Leu Val Lys Ala His Gln Asp Ala Gln Arg His Val Ala Ala
530 535 540
Gly Thr Gln Gln Pro Tyr Thr Asp Gly Val Arg Met Glu Glu Ile Val
545 550 555 560
Glu Gly Cys Thr Gly Ala Leu His Ile Leu Ala Arg Asp Pro Met Asn
565 570 575
Arg Met Glu Ile Phe Arg Leu Asn Thr Ile Pro Leu Phe Val Gln Leu
580 585 590
Leu Tyr Ser Ser Val Glu Asn Ile Gln Arg Val Ala Ala Gly Val Leu
595 600 605
Cys Glu Leu Ala Gln Asp Lys Glu Ala Ala Asp Ala Ile Asp Ala Glu
610 615 620
Gly Ala Ser Ala Pro Leu Met Glu Leu Leu His Ser Arg Asn Glu Gly
625 630 635 640
Thr Ala Thr Tyr Ala Ala Ala Val Leu Phe Arg Ile Ser Glu Asp Lys
645 650 655
Asn Pro Asp Tyr Arg Lys Arg Val Ser Val Glu Leu Thr Asn Ser Leu
660 665 670
Phe Lys His Asp Pro Ala Ala Trp Glu Ala Ala Gln Ser Met Ile Pro
675 680 685
Ile Asn Glu Pro Tyr Gly Asp Asp Met Asp Ala Thr Tyr Arg Pro Met
690 695 700
Tyr Ser Ser Asp Val Pro Leu Asp Pro Leu Glu Met His Met Asp Met
705 710 715 720
Asp Gly Asp Tyr Pro Ile Asp Thr Tyr Ser Asp Gly Leu Arg Pro Pro
725 730 735
Tyr Pro Thr Ala Asp His Met Leu Ala
740 745