CN114164189B - Nicotinamide phosphoribosyl transferase mutant - Google Patents

Nicotinamide phosphoribosyl transferase mutant Download PDF

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CN114164189B
CN114164189B CN202010945768.1A CN202010945768A CN114164189B CN 114164189 B CN114164189 B CN 114164189B CN 202010945768 A CN202010945768 A CN 202010945768A CN 114164189 B CN114164189 B CN 114164189B
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CN114164189A (en
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张小飞
陆丽英
竺伟
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SYNCOZYMES (SHANGHAI) CO Ltd
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Abstract

A nicotinamide phosphoribosyl transferase mutant is disclosed that is capable of converting nicotinamide and phosphoribosyl pyrophosphate to beta-nicotinamide mononucleotide (beta-NMN). The half-life of the mutant is improved by 5.3 times compared with the wild type, the storage time and the conversion efficiency of nicotinamide phosphoribosyl transferase are improved, and the mutant is more suitable for industrial production and application.

Description

Nicotinamide phosphoribosyl transferase mutant
Technical field:
the invention belongs to the technical field of protein engineering, and particularly relates to a nicotinamide phosphoribosyl transferase mutant for preparing beta-nicotinamide mononucleotide.
The background technology is as follows:
nicotinamide mononucleotide is also called beta-nicotinamide mononucleotide (beta-Nicotinamide Mononucleotide, beta-NMN), and the structure is shown as a compound I.
Figure BDA0002675267640000011
Beta-nicotinamide mononucleotide plays an important role in human cell energy generation and participates in intracellular NAD + (nicotinamide adenine dinucleotide, an important coenzyme for cellular energy conversion) is NAD + Is one of the key precursors of (a). David Scinclair research team reported in literature Science,2017,355,1312-1317, NAD + The increase in mice can delay the signs of tissue and muscle aging in older mice, which has led to a large step forward in human development to achieve a long life dream. NAD (NAD) + The molecular weight is too large, and the oral absorption and utilization rate is low. But with respect to NAD + Research on precursor micromolecular substance beta-NMN shows that taking beta-NMN can effectively promote NAD in vivo + And significantly improves human metabolism, making beta-NMN an "laoshen drug". This has also prompted scientific researchers in the world medicine, food and cosmetics industries to continuously research and develop beta-NMN.
At present, nicotinamide phosphoribosyl transferase (NAMPT) is the most critical speed-limiting enzyme in the biological enzyme synthesis of beta-NMN, and can catalyze nicotinamide and phosphoribosyl pyrophosphoric acid to react to synthesize beta-NMN, and the conversion reaction formula is shown as Scheme 1.
Figure BDA0002675267640000021
In the catalytic reaction, the catalytic efficiency and stability of the enzyme are important indexes for industrial application, and the existing wild nicotinamide riboside phosphate transferase is poor in catalytic efficiency and stability, so that the industrial application of the beta-NMN biocatalysis technology is severely restricted.
Therefore, improving the stability of nicotinamide riboside transferase is a key factor for reducing the biocatalysis synthesis cost of beta-NMN, improving the industrial application value of nicotinamide riboside transferase and promoting the application of biocatalysis technology in the industrialized production of beta-NMN.
The invention comprises the following steps:
aiming at the problems of lower stability and lower industrial application value of nicotinamide phosphoribosyl transferase in the prior art, the invention aims to provide a nicotinamide phosphoribosyl transferase mutant with higher stability than the existing wild type nicotinamide phosphoribosyl transferase mutant.
In one aspect, the invention provides a nicotinamide riboside phosphoribosyl transferase mutant, which takes the amino acid sequence of wild nicotinamide riboside phosphoribosyl transferase shown in SEQ ID NO.1 as a reference sequence, and single-point or multi-point mutation occurs at positions 314, 315, 417, 419, 450 and 452. Wherein Ser at position 314 is mutated to Cys, gly at position 315 is mutated to Cys, pro at position 417 is mutated to Cys, ala at position 419 is mutated to Cys, leu at position 450 is mutated to Pro, and Glu at position 452 is mutated to Pro.
Further, the amino acid sequences of the nicotinamide phosphoribosyl transferase mutants are shown as SEQ ID NO.3, 5, 7, 9, 11, 13 and 15.
Further, the nucleotide sequences of the nicotinamide phosphoribosyl transferase mutants are shown as SEQ ID NO.4, 6, 8, 10, 12, 14 and 16.
Further, the nicotinamide riboside transferase mutant was derived from homosapiens, and the accession number of the wild-type template NCBI was nm_005746.3.
Further, the nicotinamide phosphoribosyl transferase mutant is expressed in E.coli BL (21) DE3.
Further, the expression vector of the nicotinamide phosphoribosyl transferase mutant is pET28a.
In another aspect, the invention provides the use of nicotinamide riboside transferase mutants that can be used in a process for preparing nicotinamide mononucleotide to catalyze the conversion of nicotinamide and phosphoribosyl pyrophosphate to nicotinamide mononucleotide.
The invention has the beneficial effects that: the invention provides a nicotinamide phosphoribosyl transferase mutant, which is finally obtained by carrying out site-directed mutagenesis on a nicotinamide phosphoribosyl transferase gene sequence, has high stability, can efficiently catalyze nicotinamide and phosphoribosyl pyrophosphate to be converted into beta-NMN, greatly reduces the cost of producing beta-NMN by industrially applying biocatalysis technology, and has higher industrial application value.
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FIG. 1 electrophoresis chart of NAMPT gene
FIG. 2 protein expression profile of wild-type NAMPT
Detailed Description
The technical content of the present invention will be further described with reference to specific embodiments, for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
EXAMPLE 1 cloning construction of wild NAMPT
NAMPT gene is amplified by designing the upstream and downstream primers: 5'-CGATCGCATATGAATCCTGCGGCAGAA-3', the underlined part is the cleavage site for the restriction enzyme NdeI; a downstream primer: 5'-ATGCTAGGAATTCCTAATGATG TG CTGCTTCCAG-3' the underlined part is the cleavage site for the restriction enzyme EcoRI.
The target gene NAMPT was amplified with the aid of Prime STAR polymerase (TaKaRa), the upstream and downstream primers. The PCR reaction conditions were: pre-denaturation at 98 ℃ for 2min, each cycle comprising denaturation at 95 ℃ for 25s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 2min for 30 cycles; and finally, derivatizing for 5min at 72 ℃. After the completion of the reaction, the PCR products were detected on a 1% agarose gel, and the results are shown in FIG. 1. The nucleic acid length is consistent with the published size of the PDB database (1473 bp), and the nucleotide sequence of the nucleic acid is shown as SEQ ID No. 2. The product was purified using a kit (Axygen Co.), the purified product was digested with restriction enzymes NdeI and EcoRI (Thermo Co.), then digested with the same restriction enzymes to obtain plasmid pET-28a, the digested gene and plasmid fragment were subjected to T40ligase ligation, the ligation was purified again, and E.coli DH 5. Alpha. Competent cells were directly transformed, after incubation at 37℃for 1 hour, directly plated on LB plates containing 25. Mu.g/ml kanamycin resistance, and the recombinant plasmid was designated pET-28a-NAMPT.
EXAMPLE 2 protein expression of wild-type NAMPT
Transferring the constructed recombinant plasmid pET28a-NAMPT into BL21 (DE 3) competent cells by a chemical conversion method, and obtaining the recombinant plasmid pET containing Kan + The resistant LB plates were incubated overnight upside down at 37 ℃. And selecting positive monoclonal cells to obtain the nicotinamide phosphoribosyl transferase gene engineering strain capable of inducing expression. BL21 (DE 3) cells containing NAMPT gene were inoculated into cells containing Kan + The primary seed culture broth was obtained by culturing overnight at 37℃in a resistant LB tube. Transferring the seed culture solution into a 2YT liquid culture medium containing resistance according to the inoculation proportion of 1%, placing the liquid culture medium into a shaking table, culturing for 3-5h at 37 ℃ and 200rpm, cooling to 20 ℃ when the OD reaches 0.6-0.8, adding IPTG, controlling the concentration to be 0.1mM, and carrying out overnight induced expression. The fermentation broth was centrifuged and the cells were collected, and the collected cells were lysed with 20mM phosphate buffer (pH 7.5) and disrupted by sonication. And then centrifuging at 12000rpm for 10min again to obtain supernatant which is transaminase protein, and detecting the protein expression condition by electrophoresis, wherein the protein expression condition is shown in figure 2.
EXAMPLE 3 construction of mutants
Site-directed mutagenesis was performed at positions 314, 315, 417, 419, 450 and 452, respectively, according to the crystal 3DHD structure of NAMPT, mutant primers were designed using Primer5 software, mutants were obtained using whole plasmid PCR, and specific Primer designs are shown in table 1.
TABLE 1 primer sequences for site-directed mutagenesis
Figure BDA0002675267640000061
The underlined sequences in Table 1 were used as mutation sites, and the whole plasmid amplification reaction was performed using the recombinant plasmid pET28a-NAMPT as a template with the participation of forward and reverse saturation primers by using Primer STAR max DNA polymerase (TaKaRa Co.), and the PCR reaction procedure was as shown in Table 2.
TABLE 2 Whole plasmid amplification program Table
Figure BDA0002675267640000062
Figure BDA0002675267640000071
Transferring the obtained whole plasmid mutant fragment gene into BL21 (DE 3) competent cells, and coating on Kan containing 30ug/mL + On a resistant LB plate, the strain is cultured overnight at 37 ℃, and a monoclonal is selected for sequencing to identify whether the site is mutated. After the mutants are obtained at a single point, stability changes are analyzed respectively, and the mutants with improved stability can be subjected to superposition mutation again, and the mutation method is the same as that described above.
EXAMPLE 4 comparison of mutant stability
In order to test whether the stability of the mutant is improved, firstly, shake flask expression cells of the mutant obtained by the genetic engineering means are placed in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for freezing and crushing, after the cells are placed for 2 days, 0.5g of crushed cells are respectively weighed, transferred into an EP pipe and placed in a water area pot at the temperature of 25 ℃. The sample conversion test participation vitality is respectively placed in different time periods, and the specific feeding is as follows: 50mM phosphoribosyl pyrophosphate (PRPP) is prepared by using 20mM phosphate buffer solution pH7.5, 10mL of PPRP is taken and put into a reaction kettle, 0.061g of Nicotinamide (NAM) is weighed and transferred into the reaction kettle, 0.5g of NAMPT mutant which is thermally inactivated in different time periods is put into the reaction kettle, timing reaction is started at 37 ℃, the reaction solution is heated to 90 ℃ for 5min after 4h, and the reaction is stopped. The reaction solution was centrifuged at 12000rpm for 2min and analyzed for product formation by HPLC, and the data of specific stability half-life are shown in Table 3.
TABLE 3 stability data for NAMPT mutants
Figure BDA0002675267640000081
Sequence listing
<110> Shang Ke biological medicine (Shanghai) Co., ltd
<120> Nicotinamide phosphoribosyl transferase mutant
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ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
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tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
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gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
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Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
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Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
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Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
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Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
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Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
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Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
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Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
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Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
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Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
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Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
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Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
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Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Pro Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Pro Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 6
<211> 1476
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt ctggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gagccttatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 7
<211> 491
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Cys Gly Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Cys Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 8
<211> 1476
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt gcggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggactg cgttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 9
<211> 491
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Ser Cys Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Pro Val Cys Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 10
<211> 1476
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt cttgcaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agtttgcgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 11
<211> 491
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Ser Gly Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Pro Val Ala Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Pro Glu Pro Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 12
<211> 1476
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt ctggaaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggaccc agttgctgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagaccct gagccttatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 13
<211> 491
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Cys Cys Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Cys Val Cys Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Leu Glu Glu Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 14
<211> 1476
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt gctgcaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggactg cgtttgcgat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagacctt gaggaatatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476
<210> 15
<211> 491
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Met Asn Pro Ala Ala Glu Ala Glu Phe Asn Ile Leu Leu Ala Thr Asp
1 5 10 15
Ser Tyr Lys Val Thr His Tyr Lys Gln Tyr Pro Pro Asn Thr Ser Lys
20 25 30
Val Tyr Ser Tyr Phe Glu Cys Arg Glu Lys Lys Thr Glu Asn Ser Lys
35 40 45
Leu Arg Lys Val Lys Tyr Glu Glu Thr Val Phe Tyr Gly Leu Gln Tyr
50 55 60
Ile Leu Asn Lys Tyr Leu Lys Gly Lys Val Val Thr Lys Glu Lys Ile
65 70 75 80
Gln Glu Ala Lys Asp Val Tyr Lys Glu His Phe Gln Asp Asp Val Phe
85 90 95
Asn Glu Lys Gly Trp Asn Tyr Ile Leu Glu Lys Tyr Asp Gly His Leu
100 105 110
Pro Ile Glu Ile Lys Ala Val Pro Glu Gly Phe Val Ile Pro Arg Gly
115 120 125
Asn Val Leu Phe Thr Val Glu Asn Thr Asp Pro Glu Cys Tyr Trp Leu
130 135 140
Thr Asn Trp Ile Glu Thr Ile Leu Val Gln Ser Trp Tyr Pro Ile Thr
145 150 155 160
Val Ala Thr Asn Ser Arg Glu Gln Lys Lys Ile Leu Ala Lys Tyr Leu
165 170 175
Leu Glu Thr Ser Gly Asn Leu Asp Gly Leu Glu Tyr Lys Leu His Asp
180 185 190
Phe Gly Tyr Arg Gly Val Ser Ser Gln Glu Thr Ala Gly Ile Gly Ala
195 200 205
Ser Ala His Leu Val Asn Phe Lys Gly Thr Asp Thr Val Ala Gly Leu
210 215 220
Ala Leu Ile Lys Lys Tyr Tyr Gly Thr Lys Asp Pro Val Pro Gly Tyr
225 230 235 240
Ser Val Pro Ala Ala Glu His Ser Thr Ile Thr Ala Trp Gly Lys Asp
245 250 255
His Glu Lys Asp Ala Phe Glu His Ile Val Thr Gln Phe Ser Ser Val
260 265 270
Pro Val Ser Val Val Ser Asp Ser Tyr Asp Ile Tyr Asn Ala Cys Glu
275 280 285
Lys Ile Trp Gly Glu Asp Leu Arg His Leu Ile Val Ser Arg Ser Thr
290 295 300
Gln Ala Pro Leu Ile Ile Arg Pro Asp Cys Cys Asn Pro Leu Asp Thr
305 310 315 320
Val Leu Lys Val Leu Glu Ile Leu Gly Lys Lys Phe Pro Val Thr Glu
325 330 335
Asn Ser Lys Gly Tyr Lys Leu Leu Pro Pro Tyr Leu Arg Val Ile Gln
340 345 350
Gly Asp Gly Val Asp Ile Asn Thr Leu Gln Glu Ile Val Glu Gly Met
355 360 365
Lys Gln Lys Met Trp Ser Ile Glu Asn Ile Ala Phe Gly Ser Gly Gly
370 375 380
Gly Leu Leu Gln Lys Leu Thr Arg Asp Leu Leu Asn Cys Ser Phe Lys
385 390 395 400
Cys Ser Tyr Val Val Thr Asn Gly Leu Gly Ile Asn Val Phe Lys Asp
405 410 415
Cys Val Cys Asp Pro Asn Lys Arg Ser Lys Lys Gly Arg Leu Ser Leu
420 425 430
His Arg Thr Pro Ala Gly Asn Phe Val Thr Leu Glu Glu Gly Lys Gly
435 440 445
Asp Pro Glu Pro Tyr Gly Gln Asp Leu Leu His Thr Val Phe Lys Asn
450 455 460
Gly Lys Val Thr Lys Ser Tyr Ser Phe Asp Glu Ile Arg Lys Asn Ala
465 470 475 480
Gln Leu Asn Ile Glu Leu Glu Ala Ala His His
485 490
<210> 16
<211> 1476
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
atgaatcctg cggcagaagc cgagttcaac atcctcctgg ccaccgactc ctacaaggtt 60
actcactata aacaatatcc acccaacaca agcaaagttt attcctactt tgaatgccgt 120
gaaaagaaga cagaaaactc caaattaagg aaggtgaaat atgaggaaac agtattttat 180
gggttgcagt acattcttaa taagtactta aaaggtaaag tagtaaccaa agagaaaatc 240
caggaagcca aagatgtcta caaagaacat ttccaagatg atgtctttaa tgaaaaggga 300
tggaactaca ttcttgagaa gtatgatggg catcttccaa tagaaataaa agctgttcct 360
gagggctttg tcattcccag aggaaatgtt ctcttcacgg tggaaaacac agatccagag 420
tgttactggc ttacaaattg gattgagact attcttgttc agtcctggta tccaatcaca 480
gtggccacaa attctagaga gcagaagaaa atattggcca aatatttgtt agaaacttct 540
ggtaacttag atggtctgga atacaagtta catgattttg gctacagagg agtctcttcc 600
caagagactg ctggcatagg agcatctgct cacttggtta acttcaaagg aacagataca 660
gtagcaggac ttgctctaat taaaaaatat tatggaacga aagatcctgt tccaggctat 720
tctgttccag cagcagaaca cagtaccata acagcttggg ggaaagacca tgaaaaagat 780
gcttttgaac atattgtaac acagttttca tcagtgcctg tatctgtggt cagcgatagc 840
tatgacattt ataatgcgtg tgagaaaata tggggtgaag atctaagaca tttaatagta 900
tcaagaagta cacaggcacc actaataatc agacctgatt gctgcaaccc tcttgacact 960
gtgttaaagg ttttggagat tttaggtaag aagtttcctg ttactgagaa ctcaaagggt 1020
tacaagttgc tgccacctta tcttagagtt attcaagggg atggagtaga tattaatacc 1080
ttacaagaga ttgtagaagg catgaaacaa aaaatgtgga gtattgaaaa tattgccttc 1140
ggttctggtg gaggtttgct acagaagttg acaagagatc tcttgaattg ttccttcaag 1200
tgtagctatg ttgtaactaa tggccttggg attaacgtct tcaaggacct gcttgtgcat 1260
cccaacaaaa ggtccaaaaa gggccgatta tctttacata ggacgccagc agggaatttt 1320
gttacactgg aggaaggaaa aggagaccct gagccttatg gtcaggatct tctccatact 1380
gtcttcaaga atggcaaggt gacaaaaagc tattcatttg atgaaataag aaaaaatgca 1440
cagctgaata ttgaactgga agcagcacat cattag 1476

Claims (4)

1. The nicotinamide phosphoribosyl transferase mutant is characterized in that the amino acid sequence of the nicotinamide phosphoribosyl transferase mutant is shown as SEQ ID NO.3, 5, 7, 9, 11, 13 and 15.
2. The nicotinamide riboside phosphate transferase mutant of claim 1, wherein the ketoreductase mutant has a gene nucleotide sequence shown in SEQ ID nos. 4, 6, 8, 10, 12, 14, 16.
3. The nicotinamide riboside transferase mutant of claim 1, wherein the nicotinamide riboside transferase mutant is expressed in e.coli bl (21) DE3.
4. A nicotinamide riboside transferase mutant according to claim 1, wherein the nicotinamide riboside transferase mutant is capable of converting nicotinamide and phosphoribosyl pyrophosphate to a β -nicotinamide mononucleotide.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108026517A (en) * 2016-07-30 2018-05-11 邦泰生物工程(深圳)有限公司 A kind of Nampt mutant and its application
CN108048420A (en) * 2017-11-29 2018-05-18 天津市湖滨盘古基因科学发展有限公司 The Nampt mutain of people a kind of and its application
CN109022383A (en) * 2018-07-10 2018-12-18 天津市湖滨盘古基因科学发展有限公司 The Nampt precursor mutain of people a kind of and its application
CN109666658A (en) * 2018-12-27 2019-04-23 成都及禾生物科技有限公司 It is used to prepare Nampt, encoding gene, recombinant vector and the application of NMN

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108026517A (en) * 2016-07-30 2018-05-11 邦泰生物工程(深圳)有限公司 A kind of Nampt mutant and its application
CN108048420A (en) * 2017-11-29 2018-05-18 天津市湖滨盘古基因科学发展有限公司 The Nampt mutain of people a kind of and its application
CN109022383A (en) * 2018-07-10 2018-12-18 天津市湖滨盘古基因科学发展有限公司 The Nampt precursor mutain of people a kind of and its application
CN109666658A (en) * 2018-12-27 2019-04-23 成都及禾生物科技有限公司 It is used to prepare Nampt, encoding gene, recombinant vector and the application of NMN

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