CN107868115A - A kind of non-natural ginsenoside Rd 12 and its preparation method and application - Google Patents

A kind of non-natural ginsenoside Rd 12 and its preparation method and application Download PDF

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CN107868115A
CN107868115A CN201711146324.6A CN201711146324A CN107868115A CN 107868115 A CN107868115 A CN 107868115A CN 201711146324 A CN201711146324 A CN 201711146324A CN 107868115 A CN107868115 A CN 107868115A
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CN107868115B (en
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孙媛霞
戴隆海
李娇
杨建刚
门燕
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Tianjin Institute of Industrial Biotechnology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1062—Sucrose synthase (2.4.1.13)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/20—Preparation of steroids containing heterocyclic rings
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    • C12Y204/00—Glycosyltransferases (2.4)
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01013—Sucrose synthase (2.4.1.13)

Abstract

The present invention relates to a kind of new non-natural ginsenoside Rd 12 (the O β D glucopyranosyl protopanaxadiol of 3 O β D glucopyranosyl (1 2) β D glucopyranosyl 12) and its preparation method and application, the present invention is using cheap sucrose as glucose glycosyl donor, coupling catalyst is used as by the use of glycosyl transferase and sucrose synthase, using ginseng sapoglycoside Rg 3 as glycosyl acceptor, using cheap sucrose as glycosyl donor, the C12 positions hydroxyl glycosylation production non-natural ginsenoside Rd 12 of efficient catalytic ginseng sapoglycoside Rg 3, it is good with selectivity, high catalytic efficiency, the features such as product purification is simple.In addition, non-natural ginsenoside Rd 12 prepared by the present invention can be used as antineoplastic, non-natural ginsenoside Rd 12 has significant inhibitory action to colon cancer cell, liver cancer cells, lung carcinoma cell and stomach cancer cell.

Description

A kind of non-natural ginsenoside Rd 12 and its preparation method and application
Technical field
The invention belongs to biotechnology and botany technical field, is related to a kind of non-natural ginsenoside Rd 12 and its prepares Methods and applications, and in particular to one kind utilizes glycosyl transferase and the catalysis ginseng sapoglycoside Rg 3 generation of sucrose synthase coupling reaction The method of non-natural ginsenoside Rd 12 and application.
Background technology
Ginseng (Panax ginseng C.A.Mayer) is medicinal plant important in Araliaceae Panax, main distribution In the East Asian countries such as the Changbaishan area of China northeast and Japan, South Korea, Korea；As traditional Chinese medicine, its in China Using there is thousands of years,《Sheng Nong's herbal classic》Record ginseng, which has, " main tonifying five zang organs, calms the nerves, determines soul, stop palpitate with fear, remove pathogenic factor, name Mesh, " etc. multiple efficacies.Therefore, good reputation of the ginseng labeled with " kings of hundred grass ".
Ginsenoside is the main active of ginseng, is the glycoside chemical combination connected and composed by aglycon from different glycosyls Thing.According to the difference of aglycon, ginsenoside can be divided into protopanaxadiol-type's ginsenoside, protopanaxatriol type ginsenoside and Oleanolic acid type ginsenoside three major types.Mainly including Rh2, CK, F2, Rg3, Rd etc., it is tied protopanaxadiol-type's ginsenoside Structure feature is C3-OH the and C20-OH positions that glycosyl is connected to protopanoxadiol skeleton separately or together, and protopanoxadiol C12-OH then without glycosyl connect；The difference of glycosyl number, species and glycosyl connected mode that C3-OH connects with C20-OH Formation multifarious to protopanaxadiol-type's ginsenoside structure and pharmacological activity is most important.
The glycosylation of glycosyl transferase (UDP-glycosyltransferase, UGT, EC2.4.1.X) catalysis is people Join final step modification in saponin(e biosynthesis pathway, many different structures and medicine can be produced by glycosylation Manage the ginsenoside of activity.Compared with plant origin glycosyl transferase, microbe-derived glycosyl transferase has catalytic activity The features such as height, substrate spectrum are wide, regioselectivity difference.Therefore, being excavated from microorganism new has unique catalytic activity and region The glycosyl transferase of selectivity and for ginsenoside it is glycosylation modified will be helpful to synthesis it is new and there is unique pharmacology The ginsenoside of activity.
External glycosylation has the characteristics that high catalytic efficiency, substrate tolerance are good, product purification is simple, still, body Outer glycosylation needs to use expensive uridine diphosphoglucose (UDPG, UDPG) to be used as glycosyl donor. Sucrose synthase (Sucrose synthase, SuSy, EC 2.4.1.13) can be with the cheap sucrose of efficient catalytic and the phosphorus of uridine two Sour (UDP) reaction generation uridine diphosphoglucose and fructose；In addition, sucrose synthase and glycosyl transferase can be additionally used in double enzymes Coupling reaction, the uridine diphosphoglucose of Sucrose synthesis enzymatic synthesis can be catalyzed the glycosyl of glycosylation as glycosyl transferase Donor；Meanwhile the uridine 5'-diphosphate of glycosyl transferase catalysis glycosylation generation can be used as Sucrose synthesis enzymatic synthesis uridine again The raw material of diphosphate glucose.Therefore, in glycosyl transferase and sucrose synthase dual-enzyme coupling reaction system, need to only add few The uridine 5'-diphosphate of amount and the cheap sucrose of higher concentration can realize the circular regeneration of the uridine diphosphoglucose of costliness, So as to using cheap sucrose as glucosyl group donor, efficient catalytic substrate it is glycosylation modified.
The content of the invention
The present invention provides a kind of non-natural ginsenoside Rd 12 and its preparation method and application, by methods described, by sugar Based transferase Bs-YjiC is coupled with sucrose synthase, so as to realize using cheap sucrose as glucose glycosyl donor, with ginseng soap Glycosides Rg3 is glycosyl acceptor, and the C12 positions hydroxyl glycosylation of efficient catalytic ginseng sapoglycoside Rg 3 prepares non-natural ginsenoside Rd's 12 Method.
In a first aspect, the present invention provides a kind of (3-O- β-D-glucopyranosyl (1- of non-natural ginsenoside Rd 12 2)-β-D-glucopyranosyl-12-O- β-D-glucopyranosyl-protopanaxadiol), the non-natural ginseng Saponin(e Rd12 has structure shown in Formulas I：
In the present invention, non-natural ginsenoside Rd 12 is the C12 positions hydroxyl glycosylation generation by ginseng sapoglycoside Rg 3 substrate, It is compared to other ginsenosides, and ginsenoside Rd 12 is water-soluble more preferable, and stability is higher, bioresorbable is more preferable.
Second aspect, the present invention provide a kind of catalytic body of the non-natural ginsenoside Rd 12 prepared as described in relation to the first aspect System, the catalyst system and catalyzing include ginseng sapoglycoside Rg 3, sucrose, sucrose synthase, glycosyl transferase and uridine 5'-diphosphate.
In the present invention, sucrose synthase and glycosyl transferase react available for dual-enzyme coupling, Sucrose synthesis enzymatic synthesis UDPG can be catalyzed the glycosyl donor of glycosylation as glycosyl transferase；Meanwhile glycosyl transferase catalysis substrate sugar The accessory substance UDP of glycosylation reaction generation can be used as the raw material of Sucrose synthesis enzymatic synthesis UDPG again.Therefore, shifted in glycosyl In enzyme/sucrose synthase dual-enzyme coupling reaction, only need to add a small amount of UDP and the cheap sucrose of higher concentration can realize The circular regeneration of UDPG.
Preferably, the glycosyl transferase available for the present invention is glycosyl transferase Bs-YjiC, the glycosyl transferase Bs- YjiC encoding gene derives from bacillus subtilis and/or its mutant, the amino acid sequence of the glycosyl transferase Bs-YjiC Row have the amino acid sequence of 85% homology, preferably 95% homology such as SEQ ID NO.1 or with it.
The amino acid sequence of the SEQ ID NO.1 is as follows：
In the present invention, there are at least 85% homology, preferably 95% homology with the amino acid sequence shown in SEQ ID NO.1 And the polypeptide as derived from SEQ ID NO.1 with glycosyl transferase enzymatic activity, wherein described derivative polypeptide is also included institute The amino acid sequence stated with the addition of the fusion protein formed after sequence label, signal sequence or secretory signal sequence.
Sucrose synthase (sucrose synthase, SuSy, EC2.4.1.13) is the key enzyme of carbohydate metabolism in plant, is planted Sugar stores generally in the form of starch caused by thing photosynthesis, and SuSy plays key effect in this process.SuSy is can Back reaction enzyme, can both be catalyzed a molecule UDPG and a molecule fructose synthesizes a molecule sucrose and a molecule UDP, then Synthetic starch, a molecule sucrose and UDP reactions one molecule UDPG of generation and fructose can be catalyzed again.
Sucrose synthase available for the present invention is not particularly limited, can be it is from various plants or microorganism, Sucrose synthase with catalysing sucrose Yu UDP fabricated in situ UDPG functions.Generally, the sucrose synthase is from plan In southern mustard, soybean, nitrous acid monad or acidophilia thermophilic thiobacillus any one or at least two combination, preferably intend In southern mustard, soybean or acidophilia thermophilic thiobacillus any one or at least two combination, the sucrose synthase is sucrose Synzyme AtSuSy；
According to the present invention, in the amino acid sequence such as SEQ ID NO.2-6 of the sucrose synthase any one or with Any one amino acid sequence with 85% homology, preferably 95% homology in SEQ ID NO.2-6；
The amino acid sequence of the SEQ ID NO.2 (arabidopsis) is as follows： MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQ EAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKY IGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEA KFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYI LDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEV WPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFS CQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEE KRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKE SKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFA TCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYG FWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDD；
The amino acid sequence of the SEQ ID NO.3 (arabidopsis) is as follows： MPTGRFETMREWVYDAISAQRNELLSLFSRYVAQGKGILQSHQLIDEFLKTVKVDGTLEDLNKSPFMKVLQSAEEAI VLPPFVALAIRPRPGVREYVRVNVYELSVDHLTVSEYLRFKEELVNGHANGDYLLELDFEPFNATLPRPTRSSSIGN GVQFLNRHLSSIMFRNKESMEPLLEFLRTHKHDGRPMMLNDRIQNIPILQGALARAEEFLSKLPLATPYSEFEFELQ GMGFERGWGDTAQKVSEMVHLLLDILQAPDPSVLETFLGRIPMVFNVVILSPHGYFGQANVLGLPDTGGQVVYILDQ VRALENEMLLRIQKQGLEVIPKILIVTRLLPEAKGTTCNQRLERVSGTEHAHILRIPFRTEKGILRKWISRFDVWPY LETFAEDASNEISAELQGVPNLIIGNYSDGNLVASLLASKLGVIQCNIAHALEKTKYPESDIYWRNHEDKYHFSSQF TADLIAMNNADFIITSTYQEIAGSKNNVGQYESHTAFTMPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSDKERR LTALHESIEELLFSAEQNDEHVGLLSDQSKPIIFSMARLDRVKNLTGLVECYAKNSKLRELANLVIVGGYIDENQSR DREEMAEIQKMHSLIEQYDLHGEFRWIAAQMNRARNGELYRYIADTKGVFVQPAFYEAFGLTVVESMTCALPTFATC HGGPAEIIENGVSGFHIDPYHPDQVAATLVSFFETCNTNPNHWVKISEGGLKRIYERYTWKKYSERLLTLAGVYAFW KHVSKLERRETRRYLEMFYSLKFRDLANSIPLATDEN；
The amino acid sequence of the SEQ ID NO.4 (arabidopsis) is as follows： MANPKLTRVLSTRDRVQDTLSAHRNELVALLSRYVDQGKGILQPHNLIDELESVIGDDETKKSLSDGPFGEILKSAM EAIVVPPFVALAVRPRPGVWEYVRVNVFELSVEQLTVSEYLRFKEELVDGPNSDPFCLELDFEPFNANVPRPSRSSS IGNGVQFLNRHLSSVMFRNKDCLEPLLDFLRVHKYKGHPLMLNDRIQSISRLQIQLSKAEDHISKLSQETPFSEFEY ALQGMGFEKGWGDTAGRVLEMMHLLSDILQAPDPSSLEKFLGMVPMVFNVVILSPHGYFGQANVLGLPDTGGQVVYI LDQVRALETEMLLRIKRQGLDISPSILIVTRLIPDAKGTTCNQRLERVSGTEHTHILRVPFRSEKGILRKWISRFDV WPYLENYAQDAASEIVGELQGVPDFIIGNYSDGNLVASLMAHRMGVTQCTIAHALEKTKYPDSDIYWKDFDNKYHFS CQFTADLIAMNNADFIITSTYQEIAGTKNTVGQYESHGAFTLPGLYRVVHGIDVFDPKFNIVSPGADMTIYFPYSEE TRRLTALHGSIEEMLYSPDQTDEHVGTLSDRSKPILFSMARLDKVKNISGLVEMYSKNTKLRELVNLVVIAGNIDVN KSKDREEIVEIEKMHNLMKNYKLDGQFRWITAQTNRARNGELYRYIADTRGAFAQPAFYEAFGLTVVEAMTCGLPTF ATCHGGPAEIIEHGLSGFHIDPYHPEQAGNIMADFFERCKEDPNHWKKVSDAGLQRIYERYTWKIYSERLMTLAGVY GFWKYVSKLERRETRRYLEMFYILKFRDLVKTVPSTADD；
The amino acid sequence of the SEQ ID NO.5 (soybean) is as follows： MATDRLTRVHSLRERLDETLTANRNEILALLSRIEAKGKGILQHHQVIAEFEEIPEENRQKLTDGAFGEVLRSTQEA IVLPPWVALAVRPRPGVWEYLRVNVHALVVEELQPAEYLHFKEELVDGSSNGNFVLELDFEPFNAAFPRPTLNKSIG NGVQFLNRHLSAKLFHDKESLHPLLEFLRLHSVKGKTLMLNDRIQNPDALQHVLRKAEEYLGTVPPETPYSEFEHKF QEIGLERGWGDNAERVLESIQLLLDLLEAPDPCTLETFLGRIPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILD QVRALENEMLHRIKQQGLDIVPRILIITRLLPDAVGTTCGQRLEKVFGTEHSHILRVPFRTEKGIVRKWISRFEVWP YLETYTEDVAHELAKELQGKPDLIVGNYSDGNIVASLLAHKLGVTQCTIAHALEKTKYPESDIYWKKLEERYHFSCQ FTADLFAMNHTDFIITSTFQEIAGSKDTVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADQTIYFPHTETSR RLTSFHPEIEELLYSSVENEEHICVLKDRSKPIIFTMARLDRVKNITGLVEWYGKNAKLRELVNLVVVAGDRRKESK DLEEKAEMKKMYGLIETYKLNGQFRWISSQMNRVRNGELYRVICDTRGAFVQPAVYEAFGLTVVEAMTCGLPTFATC NGGPAEIIVHGKSGFHIDPYHGDRAADLLVDFFEKCKLDPTHWDKISKAGLQRIEEKYTWQIYSQRLLTLTGVYGFW KHVSNLDRRESRRYLEMFYALKYRKLAESVPLAAE；
The amino acid sequence of the SEQ ID NO.6 (acidophilia thermophilic thiobacillus) is as follows： MIEALRQQLLDDPRSWYAFLRHLVASQRDSWLYTDLQRACADFREQLPEGYAEGIGPLEDFVAHTQEVIFRDPWMVF AWRPRPGRWIYVRIHREQLALEELSTDAYLQAKEGIVGLGAEGEAVLTVDFRDFRPVSRRLRDESTIGDGLTHLNRR LAGRIFSDLAAGRSQILEFLSLHRLDGQNLMLSNGNTDFDSLRQTVQYLGTLPRETPWAEIREDMRRRGFAPGWGNT AGRVRETMRLLMDLLDSPSPAALESFLDRIPMISRILIVSIHGWFAQDKVLGRPDTGGQVVYILDQARALEREMRNR LRQQGVDVEPRILIATRLIPESDGTTCDQRLEPVVGAENVQILRVPFRYPDGRIHPHWISRFKIWPWLERYAQDLER EVLAELGSRPDLIIGNYSDGNLVATLLSERLGVTQCNIAHALEKSKYLYSDLHWRDHEQDHHFACQFTADLIAMNAA DIIVTSTYQEIAGNDREIGQYEGHQDYTLPGLYRVENGIDVFDSKFNIVSPGADPRFYFSYARTEERPSFLEPEIES LLFGREPGADRRGVLEDRQKPLLLSMARMDRIKNLSGLAELYGRSSRLRGLANLVIIGGHVDVGNSRDAEEREEIRR MHEIMDHYQLDGQLRWVGALLDKTVAGELYRVVADGRGVFVQPALFEAFGLTVIEAMSSGLPVFATRFGGPLEIIED GVSGFHIDPNDHEATAERLADFLEAARERPKYWLEISDAALARVAERYTWERYAERLMTIARIFGFWRFVLDRESQV MERYLQMFRHLQWRPLAHAVPME.
In the present invention, the sucrose synthase can be one or more；
In the present invention, the glycosyl transferase can be one or more；
In the catalyst system and catalyzing of the present invention, the use ratio of glycosyl transferase and sucrose synthase is not particularly limited, can root Determined according to the species and amount of the product of required generation, generally, non-natural ginsenoside is prepared available for the present invention The concentration of glycosyl transferase and sucrose synthase is as follows：
According to the present invention, the concentration of the glycosyl transferase is 10-1000mU/mL, such as can be 10mU/mL, 20mU/ mL、30mU/mL、40mU/mL、50mU/mL、60mU/mL、80mU/mL、100mU/mL、120mU/mL、150mU/mL、200mU/ mL、250mU/mL、300mU/mL、350mU/mL、400mU/mL、450mU/mL、500mU/mL、550mU/mL、600mU/mL、 650mU/mL, 700mU/mL, 750mU/mL, 800mU/mL, 850mU/mL, 900mU/mL, 950mU/mL or 1000mU/mL, it is excellent Choosing for 160mU/mL.
According to the present invention, the concentration of the sucrose synthase is 10-1000mU/mL, such as can be 10mU/mL, 20mU/ mL、30mU/mL、40mU/mL、50mU/mL、60mU/mL、80mU/mL、100mU/mL、120mU/mL、150mU/mL、200mU/ mL、250mU/mL、300mU/mL、350mU/mL、400mU/mL、450mU/mL、500mU/mL、550mU/mL、600mU/mL、 650mU/mL, 700mU/mL, 750mU/mL, 800mU/mL, 850mU/mL, 900mU/mL, 950mU/mL or 1000mU/mL, it is excellent Elect 200mU/mL as.
The third aspect, the present invention provide a kind of carrier, and the carrier is used to express the glycosyl transfer as described in second aspect Enzyme and/or sucrose synthase.
According to the present invention, the glycosyl transferase is glycosyl transferase Bs-YjiC.
According to the present invention, the sucrose synthase is sucrose synthase AtSuSy.
In the present invention, the glycosyl transferase and sucrose synthase can be expressed on same carrier, can also be expressed It is different on carrier, be not particularly limited herein, those skilled in the art can be selected as needed.
Fourth aspect, the present invention provide a kind of host cell, and the host cell includes the carrier as described in the third aspect.
According to the present invention, the host cell is Escherichia coli, saccharomyces cerevisiae, Pichia pastoris, corynebacterium glutamicum, withered In careless bacillus or aspergillus niger any one or at least two combination.
In the present invention, the glycosyl transferase and sucrose synthase can express in different host cells respectively, Can simultaneously in same host cell heterogenous expression, be not particularly limited herein, those skilled in the art can be according to need Selected.
In the present invention, active glycosyl transferase, sucrose synthase or its combination can be recombinant polypeptide, natural more Peptide, synthesis polypeptide, polypeptide of the invention can be native purified products, or the product of chemical synthesis, or use restructuring skill Art produces from protokaryon or eucaryon host.According to the host used in recombinant production scheme, polypeptide of the invention can be glycosylation , or can be nonglycosylated.The polypeptide of the present invention may also include or do not include the methionine residues of starting.
5th aspect, the present invention provide a kind of catalyst system and catalyzing as described in second aspect or the host as described in fourth aspect The purposes of cell, it is characterised in that the catalyst system and catalyzing is converted into non-natural ginsenoside for being catalyzed ginseng sapoglycoside Rg 3 Rd12。
In the present invention, the ginseng sapoglycoside Rg 3 can be by carrying from ginseng or other plants containing these compositions Acquisition, general ginsenoside hydrolysis is taken to obtain or incorporate in the microbial engineering bacteria synthesis acquisition of ginsenoside route of synthesis Any one mode or various ways combination obtain.
In the present invention, glycosyl transferase and sucrose synthase in the catalyst system and catalyzing can be the places described in fourth aspect The broken crude enzyme liquid obtained of chief cell or the pure enzyme for further obtaining crude enzyme liquid through different method for purifying proteins.
6th aspect, the present invention provides a kind of method for preparing non-natural ginsenoside Rd 12, described in second aspect In the presence of catalyst system and catalyzing or host cell as described in fourth aspect, ginseng sapoglycoside Rg 3 is converted into non-natural ginsenoside Rd12；
According to the present invention, described method includes following reaction：
The reaction is included in the presence of sucrose synthase AtSuSy, catalysing sucrose and UDP reaction generation UDP- grapes Sugar and fructose；Meanwhile glycosyl transferase Bs-YjiC is catalyzed ginseng sapoglycoside Rg 3 using the UDPG generated as glycosyl donor C12 positions hydroxyl glycosylation generation non-natural ginsenoside Rd 12 and UDP.
7th aspect, the present invention provide a kind of antineoplastic, and the medicine includes non-natural as claimed in claim 1 Ginsenoside Rd 12；
In the present invention, the cancer therapy drug can only include non-natural ginsenoside Rd 12, can be with right and wrong natural ginseng soap Glycosides Rd12 and ginseng sapoglycoside Rg 3 combination, it can be gone back with right and wrong natural ginseng saponin(e Rd12 and natural ginseng saponin(e Rh2 combination Can be with right and wrong natural ginseng saponin(e Rd12 and natural ginseng saponin(e CK combination, wherein the mass ratio of each composition is not made herein Particular determination, those skilled in the art can be selected as needed.
According to the present invention, the antineoplastic also includes pharmaceutically acceptable carrier.
According to the present invention, the tumour includes any one or at least two in liver cancer, lung cancer, colon cancer or stomach cancer Combination.
Compared with prior art, the present invention has the advantages that：
(1) present invention is using cheap sucrose as glucose glycosyl donor, by the use of glycosyl transferase and sucrose synthase as Coupling catalyst, using ginseng sapoglycoside Rg 3 as glycosyl acceptor, the C12 positions hydroxyl glycosylation production of efficient catalytic ginseng sapoglycoside Rg 3 is non- Natural ginseng saponin(e Rd12, selectivity is good, transformation efficiency is high, product purification is simple.
(2) inhibitory action of non-natural ginsenoside Rd 12 prepared by the present invention to intestinal cancer, liver cancer, lung cancer and stomach cancer cell It is superior to substrate ginseng sapoglycoside Rg 3.
(3) medicine containing non-natural ginsenoside Rd 12 prepared by the present invention has huge in terms of tumour cell is treated Potentiality.
Brief description of the drawings
Fig. 1 is sucrose synthase AtSuSy and glycosyl transferase Bs-YjiC SDS-PAGE protein electrophoresis result figures；
Fig. 2 is the Reaction Mechanisms figure of non-natural ginsenoside Rd 12；
The High performance liquid chromatography-electrospray ionization mass spectrometry method combined instrument that Fig. 3 is non-natural ginsenoside Rd 12 of the present invention reflects Determine result, wherein, (A) is efficient liquid phase result figure, and (B) is mass spectral results figure.
Fig. 4 is that batch feeding is catalyzed the result figure that ginseng sapoglycoside Rg 3 synthesizes non-natural ginsenoside Rd 12.
Embodiment
For the present invention technological means and its effect taken is expanded on further, the present invention is made with reference to embodiments into Illustrate to one step.It is understood that embodiment described herein is used only for explaining the present invention, rather than to this hair Bright restriction.
In the examples where no specific technique or condition is specified, according to the technology or condition described by document in the art, Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, be can be by regular channel commercially available from The conventional products of acquisition.
Activity determination method and unit of enzyme activity are defined as follows in the present invention：
Sucrose synthase enzyme assay：Contain sucrose synthase (10 μ g), UDP in 200 μ L reaction systems (0.5mM), sucrose (300mM), Tris-HCl buffer solutions (50mM, pH 7.5), 30 DEG C of water-bath 0.5h, quickly boil termination Reaction, carry out the generation of HPLC detection fructose.Sucrose synthase enzymatic activity is defined as, per the catalysis per minute of microgram sucrose synthase The micromole's content for the fructose that sucrose hydrolysis is generated.
Glycosyl transferase enzyme assay：Contain ginseng sapoglycoside Rg 3 (1mM), uridine 5'-diphosphate in 200 μ L reaction systems Glucose (5mM), Tris-HCl buffer solutions (50mM, pH 7.5), 10 μ g glycosyl transferases Bs-YjiC, 35 DEG C of water-baths 0.5h, 200 μ L methanol terminating reactions are added, carry out the consumption of high performance liquid chromatography detection ginseng sapoglycoside Rg 3.Glycosyl transferase Enzymatic activity is defined as, per micromole's content of microgram glycosyl transferase consumed ginseng sapoglycoside Rg 3 per minute.
The clone of the glycosyl transferase of embodiment 1 and sucrose synthase, expression and purification
Glycosyl transferase Bs-YjiC in patent of the present invention is screened from bacillus subtilis (Bacillus subtilis 168), for its amino acid sequence as shown in SEQ ID NO.1, its nucleotide sequence (SEQ ID NO.7) is as follows： ATGAAAAAGTACCATATTTCGATGATCAATATCCCGGCGTACGGACATGTCAATCCTACGCTTGCTTTAGTAGAGAA GCTTTGTGAGAAAGGGCACCGTGTCACGTACGCGACGACTGAGGAGTTTGCGCCCGCTGTTCAGCAAGCCGGTGGAG AAGCATTGATCTATCATACATCCTTGAATATTGATCCTAAGCAAATCAGGGAGATGATGGAAAAGAATGACGCGCCC CTCAGCCTTTTGAAAGAATCACTCAGCATTCTGCCGCAGCTTGAGGAGTTATATAAGGATGATCAGCCTGATCTGAT CATCTATGACTTTGTTGCGCTGGCTGGTAAATTGTTTGCTGAAAAGCTTAATGTTCCGGTCATTAAGCTCTGTTCGT CATATGCCCAAAATGAATCCTTTCAGTTAGGAAATGAAGACATGCTGAAAAAAATAAGAGAAGCAGAGGCTGAATTT AAAGCCTACTTGGAGCAAGAGAAGTTGCCGGCTGTTTCATTTGAACAGTTAGCTGTGCCGGAAGCATTAAATATTGT CTTTATGCCGAAGTCTTTTCAGATTCAGCATGAGACGTTCGATGACCGTTTCTGTTTTGTCGGCCCCTCTCTCGGAG AACGGAAGGAAAAAGAAAGCCTGTTGATTGACAAGGATGATCGCCCGCTTATGCTGATTTCTTTGGGTACGGCGTTT AACGCATGGCCGGAATTTTACAAGATGTGCATCAAGGCATTTCGGGATTCTTCATGGCAAGTGATCATGTCGGTTGG GAAAACGATTGATCCAGAAAGCTTGGAGGATATTCCTGCTAACTTTACCATTCGCCAAAGTGTGCCGCAGCTTGAGG TGTTAGAGAAAGCTGATTTGTTCATCTCTCATGGCGGGATGAACAGTACGATGGAAGCGATGAACGCAGGTGTGCCG CTTGTCGTCATTCCGCAAATGTATGAGCAGGAGCTCACTGCAAATCGGGTTGATGAATTAGGCCTTGGCGTTTATTT GCCGAAAGAGGAAGTGACTGTTTCCAGCCTGCAGGAAGCGGTTCAGGCTGTATCCAGTGATCAAGAGCTGCTCAGCC GCGTCAAGAATATGCAAAAGGATGTAAAAGAAGCTGGCGGAGCGGAGCGTGCGGCAGCTGAGATTGAAGCGTTTATG AAAAAATCCGCTGTCCCGCAGTAA.
Sucrose synthase comes from arabidopsis in patent of the present invention, and amino acid sequence is as shown in SEQ ID NO.2, its nucleic acid sequence It is as follows to arrange (SEQ ID NO.8)：
ATGGCAAACGCTGAACGTATGATAACGCGCGTCCACAGCCAACGTGAGCGTTTGAACGAAACGCTTGTTTCTGAGAG AAACGAAGTCCTTGCCTTGCTTTCCAGGGTTGAAGCCAAAGGTAAAGGTATTTTACAACAAAACCAGATCATTGCTG AATTCGAAGCTTTGCCTGAACAAACCCGGAAGAAACTTGAAGGTGGTCCTTTCTTTGACCTTCTCAAATCCACTCAG GAAGCAATTGTGTTGCCACCATGGGTTGCTCTAGCTGTGAGGCCAAGGCCTGGTGTTTGGGAATACTTACGAGTCAA TCTCCATGCTCTTGTCGTTGAAGAACTCCAACCTGCTGAGTTTCTTCATTTCAAGGAAGAACTCGTTGATGGAGTTA AGAATGGTAATTTCACTCTTGAGCTTGATTTCGAGCCATTCAATGCGTCTATCCCTCGTCCAACACTCCACAAATAC ATTGGAAATGGTGTTGACTTCCTTAACCGTCATTTATCGGCTAAGCTCTTCCATGACAAGGAGAGTTTGCTTCCATT GCTTAAGTTCCTTCGTCTTCACAGCCACCAGGGCAAGAACCTGATGTTGAGCGAGAAGATTCAGAACCTCAACACTC TGCAACACACCTTGAGGAAAGCAGAAGAGTATCTAGCAGAGCTTAAGTCCGAAACACTGTATGAAGAGTTTGAGGCC AAGTTTGAGGAGATTGGTCTTGAGAGGGGATGGGGAGACAATGCAGAGCGTGTCCTTGACATGATACGTCTTCTTTT GGACCTTCTTGAGGCGCCTGATCCTTGCACTCTTGAGACTTTTCTTGGAAGAGTACCAATGGTGTTCAACGTTGTGA TCCTCTCTCCACATGGTTACTTTGCTCAGGACAATGTTCTTGGTTACCCTGACACTGGTGGACAGGTTGTTTACATT CTTGATCAAGTTCGTGCTCTGGAGATAGAGATGCTTCAACGTATTAAGCAACAAGGACTCAACATTAAACCAAGGAT TCTCATTCTAACTCGACTTCTACCTGATGCGGTAGGAACTACATGCGGTGAACGTCTCGAGAGAGTTTATGATTCTG AGTACTGTGATATTCTTCGTGTGCCCTTCAGAACAGAGAAGGGTATTGTTCGCAAATGGATCTCAAGGTTCGAAGTC TGGCCATATCTAGAGACTTACACCGAGGATGCTGCGGTTGAGCTATCGAAAGAATTGAATGGCAAGCCTGACCTTAT CATTGGTAACTACAGTGATGGAAATCTTGTTGCTTCTTTATTGGCTCACAAACTTGGTGTCACTCAGTGTACCATTG CTCATGCTCTTGAGAAAACAAAGTACCCGGATTCTGATATCTACTGGAAGAAGCTTGACGACAAGTACCATTTCTCA TGCCAGTTCACTGCGGATATTTTCGCAATGAACCACACTGATTTCATCATCACTAGTACTTTCCAAGAAATTGCTGG AAGCAAAGAAACTGTTGGGCAGTATGAAAGCCACACAGCCTTTACTCTTCCCGGATTGTATCGAGTTGTTCACGGGA TTGATGTGTTTGATCCCAAGTTCAACATTGTCTCTCCTGGTGCTGATATGAGCATCTACTTCCCTTACACAGAGGAG AAGCGTAGATTGACTAAGTTCCACTCTGAGATCGAGGAGCTCCTCTACAGCGATGTTGAGAACAAAGAGCACTTATG TGTGCTCAAGGACAAGAAGAAGCCGATTCTCTTCACAATGGCTAGGCTTGATCGTGTCAAGAACTTGTCAGGTCTTG TTGAGTGGTACGGGAAGAACACCCGCTTGCGTGAGCTAGCTAACTTGGTTGTTGTTGGAGGAGACAGGAGGAAAGAG TCAAAGGACAATGAAGAGAAAGCAGAGATGAAGAAAATGTATGATCTCATTGAGGAATACAAGCTAAACGGTCAGTT CAGGTGGATCTCCTCTCAGATGGACCGGGTAAGGAACGGTGAGCTGTACCGGTACATCTGTGACACCAAGGGTGCTT TTGTCCAACCTGCATTATATGAAGCCTTTGGGTTAACTGTTGTGGAGGCTATGACTTGTGGTTTACCGACTTTCGCC ACTTGCAAAGGTGGTCCAGCTGAGATCATTGTGCACGGTAAATCGGGTTTCCACATTGACCCTTACCATGGTGATCA GGCTGCTGATACTCTTGCTGATTTCTTCACCAAGTGTAAGGAGGATCCATCTCACTGGGATGAGATCTCAAAAGGAG GGCTTCAGAGGATTGAGGAGAAATACACTTGGCAAATCTATTCACAGAGGCTCTTGACATTGACTGGTGTGTATGGA TTCTGGAAGCATGTCTCGAACCTTGACCGTCTTGAGGCTCGCCGTTACCTTGAAATGTTCTATGCATTGAAGTATCG CCCATTGGCTCAGGCTGTTCCTCTTGCACAAGATGATTGA.
Specific preparation method is as follows：
(1) using the cDNA of the genomic DNA of bacillus subtilis and arabidopsis as masterplate, performing PCR amplification is entered.Primer sequence It is as follows：
Note：GGATCC：The restriction enzyme sites of BamH I；GTCGAC:The restriction enzyme sites of Sal I；GTCGAC：The restriction enzyme sites of Nde I；GCGGCCGC：Not I restriction enzyme sites.
(2) two genes are inserted respectively into the pET32 carriers through identical restriction enzyme ferment treatment, structure restructuring Carrier pET32-Bs-YjiC and pET32-AtSuSy, take 1 μ L recombinant plasmids pET32-Bs-YjiC, pET32-AtSuSy and sky Plasmid pET32, mix and converted with 100 μ L E. colis BL21 (DE3) competent cells respectively, convert bar Part is：Ice bath 30min, 42 DEG C of water-baths heat shock 90s, ice bath 1-2min；
(3) bacterium solution being totally converted is coated on to the LB solid plates containing 100 μ g/L ampicillins or kanamycins On, 37 DEG C are incubated overnight 14-16h；
(4) it will verify that correct recombinant plasmid is transferred to E.coli BL21 (DE3) host cell, induce the table of destination protein Reach；Inductive condition is：(10g/L NaCl, 10g/L in the LB culture mediums containing 100 μ g/L ampicillins or kanamycins Peptone and 5g/L dusty yeasts), 37 DEG C of culture 3~4h to thalline OD600For 0.6~0.8, IPTG to final concentration of 0.3mM is added To induce the expression of destination protein, the condition of culture of inducible protein is 16 DEG C, 200r/min cultivates 16-20h.Meanwhile to be transferred to PET32 E.coliBL21 cultivates as control under the same conditions；
(5) after the completion of cultivating, 6000r/min centrifugations 5min collects thalline, by thalline 25mmol/LTris-HCl, pH After 7.0 buffer solutions suspend, ultrasonication, pass through SDS-PAGE protein electrophoresis analysis purpose protein Bs s-YjiC and AtSuSy table Up to situation, as a result as shown in Figure 1.
As shown in Fig. 1 SDS-PAGE protein adhesives, compared with the recombinant bacterial strain for expressing empty plasmid, glycosyl transferase is recombinated Bs-YjiC and the sucrose synthase solubility expression in broken liquid supernatant, wherein, glycosyl transferase Bs-YjiC size is about For 62kDa, and the size of sucrose synthase is about 110kDa.Further acquisition purity can be purified by nickel post nucleophilic chromatography to exceed 95% glycosyl transferase Bs-YjiC and sucrose synthase AtSuSy.
The coupling catalysed ginseng sapoglycoside Rg 3s of the glycosyl transferase Bs-YjiC of embodiment 2 and sucrose synthase AtSuSy synthesize non-day Right ginsenoside Rd 12
With ginseng sapoglycoside Rg 3 (3-O- [β-D-glucopyranosyl (1-2)-β-D-glucopyranosyl] -20 (S) - Protopanaxadiol it is) substrate, using cheap sucrose as glycosyl donor, passes through the double enzymes of glycosyl transferase-sucrose synthase Coupling reaction, the (3-O- [β-D- of C12-OH glycosylation generation non-naturals ginsenoside Rd 12 of ginseng sapoglycoside Rg 3 can be catalyzed glucopyranosyl(1-2)-β-D-glucopyranosyl]-12-β-D-glucopyranosyl-proto Panaxadiol), specifically react as shown in Fig. 2 specific as follows：
The enzyme reaction system includes：2mM Rg3,0.5mM UDP, 300mM sucrose, 160U/mL glycosyl transferases Bs-YjiC With 200U/mL sucrose synthases AtSuSy, 35 DEG C, 0.5h is reacted.
Enzyme reaction product is reflected by high performance liquid chromatography electrospray ionization mass spectrometry combined instrument (HPLC-ESI-MS) It is fixed, as a result as shown in Figure 3.
From figure 3, it can be seen that be not added with sucrose in figure (A) goes out new product as the blank control reaction detection of glycosyl donor Generation, and with the addition of glycosyl transferase Bs-YjiC and sucrose synthase AtSuSy coupling reaction of the sucrose as glycosyl donor Ginseng sapoglycoside Rg 3 can be catalyzed and generate the single product of non-natural ginsenoside Rd 12.Meanwhile shown from figure (B) mass spectral results The molecular weight of the product is divided into [M+H]+m/z+~947.5689]+[M+H-H2O]+m/z+~929.5474, so as to further true It is ginsenoside Rd12 to have determined the product.
The batch feeding of embodiment 3 synthesis non-natural ginsenoside Rd 12
In order to avoid avoiding disposably adding suppression of the excessive ginseng sapoglycoside Rg 3 to glycosyl transferase activity, reflect simultaneously Can be by 2mM ginseng sapoglycoside Rg 3 glycosyls in 0.5h in glycosyl transferase Bs-YjiC and sucrose synthase AtSuSy coupling reactions Metaplasia is into non-natural ginsenoside Rd 12.Therefore, further mended respectively in time point 1h, 2h, 4h and 8h by batch feeding 2mM ginseng sapoglycoside Rg 3s are filled, to synthesize the non-natural ginsenoside Rd 12 of higher concentration.
From fig. 4, it can be seen that the supplement ginseng sapoglycoside Rg 3 substrate by 4 batches, reacts 18h, may finally obtain 9.8mM ginsenoside Rds 12 (9.3g/L), the conversion ratio of ginseng sapoglycoside Rg 3 reach 98%, and the synthesis speed of ginsenoside Rd 12 Rate reaches 0.5g/L/h.
The active anticancer of the ginsenoside Rd 12 of embodiment 4
Take the logarithm the tumour cell of phase growth, including colon cancer cell Lovo, hepatocellular carcinoma H22, lung carcinoma cell DMS53 And stomach cancer cell SNU719.First, cell suspending liquid is made in the cancer cell of exponential phase of growth and is inoculated with every μ L of hole 100 The preculture 48h in 96 orifice plates is until cell density is 1.0 × 104Per hole.After cell attachment growth, 100 μ L are separately added into The non-natural ginsenoside Rd 12 of various concentrations and ginseng sapoglycoside Rg 3, control experiment is used as using ginseng sapoglycoside Rg 3.It is incubated 48h Afterwards, 15 μ L MTT solution are added into every orifice plate and cause final concentration of 5mg/mL, continues to be incubated tumour cell 4h, terminates and cultivate and inhale Remove nutrient solution.Then, 150 μ L dimethyl sulfoxide (DMSO) is added in every hole, fully shaking makes dissolution of crystals.Then existed using ELIASA The OD value in each hole is surveyed at 570nm, passes through OD570Value calculates inhibiting rate of the ginsenoside to various cancers cell.
1 different ginsenosides of table analyze (μ g/mL) to the inhibitory activity of cancer cell
503nhibiting concentration (IC50) be evaluate pharmaceutical activity important parameter, IC50Numerical value is higher, illustrates the work of the compound Property is lower, and IC50Numerical value is lower, illustrates that the activity of the compound is higher.Rare ginsenoside Rg3 is anticancer important in ginseng Material, as shown in table 1,503nhibiting concentration (IC of the ginseng sapoglycoside Rg 3 to colon-cancer cell Lovo and lung carcinoma cell DMS5350) exceed 300 μ g/mL, to hepatocellular carcinoma H22 and stomach cancer cell SNU719 503nhibiting concentration (IC50) it is respectively 286 μ g/mL and 281 μ g/mL；Rather than natural ginseng saponin(e Rd12 is to colon-cancer cell Lovo, hepatocellular carcinoma H22, lung carcinoma cell DMS53 and stomach cancer cell SNU719 503nhibiting concentration (IC50) it is respectively 198 μ g/mL, 160 μ g/mL, 26 μ g/mL and 112 μ g/mL.Illustrate non-natural people Ginseng saponin(e Rd12 is respectively provided with significant inhibitory activity to this several cancer, while also illustrates the C12 positions hydroxyl sugar of ginseng sapoglycoside Rg 3 After base, the non-natural ginsenoside Rd 12 generated relatively joins soap to the inhibitory action of stomach cancer cell, intestinal cancer, liver cancer and lung cancer Glycosides Rg3 is obviously improved.Therefore, compared with ginseng sapoglycoside Rg 3, the medicine containing non-natural ginsenoside Rd 12 is swollen in treatment There is bigger application potential in terms of oncocyte.
Pay attention to, above are only presently preferred embodiments of the present invention and institute's application technology principle.It will be appreciated by those skilled in the art that The invention is not restricted to specific embodiment described here, can carry out for a person skilled in the art various obvious changes, Readjust and substitute without departing from protection scope of the present invention.Therefore, although being carried out by above example to the present invention It is described in further detail, but the present invention is not limited only to above example, without departing from the inventive concept, also Other more equivalent embodiments can be included, and the scope of the present invention is determined by scope of the appended claims.
Sequence table
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>A kind of non-natural ginsenoside Rd 12 and its preparation method and application
<130> PYS201711773EC
<141> 2017-11-17
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Met Lys Lys Tyr His Ile Ser Met Ile Asn Ile Pro Ala Tyr Gly His
1 5 10 15
Val Asn Pro Thr Leu Ala Leu Val Glu Lys Leu Cys Glu Lys Gly His
20 25 30
Arg Val Thr Tyr Ala Thr Thr Glu Glu Phe Ala Pro Ala Val Gln Gln
35 40 45
Ala Gly Gly Glu Ala Leu Ile Tyr His Thr Ser Leu Asn Ile Asp Pro
50 55 60
Lys Gln Ile Arg Glu Met Met Glu Lys Asn Asp Ala Pro Leu Ser Leu
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Leu Lys Glu Ser Leu Ser Ile Leu Pro Gln Leu Glu Glu Leu Tyr Lys
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Asp Asp Gln Pro Asp Leu Ile Ile Tyr Asp Phe Val Ala Leu Ala Gly
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Lys Leu Phe Ala Glu Lys Leu Asn Val Pro Val Ile Lys Leu Cys Ser
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Ser Tyr Ala Gln Asn Glu Ser Phe Gln Leu Gly Asn Glu Asp Met Leu
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Lys Lys Ile Arg Glu Ala Glu Ala Glu Phe Lys Ala Tyr Leu Glu Gln
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Glu Lys Leu Pro Ala Val Ser Phe Glu Gln Leu Ala Val Pro Glu Ala
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Leu Asn Ile Val Phe Met Pro Lys Ser Phe Gln Ile Gln His Glu Thr
180 185 190
Phe Asp Asp Arg Phe Cys Phe Val Gly Pro Ser Leu Gly Glu Arg Lys
195 200 205
Glu Lys Glu Ser Leu Leu Ile Asp Lys Asp Asp Arg Pro Leu Met Leu
210 215 220
Ile Ser Leu Gly Thr Ala Phe Asn Ala Trp Pro Glu Phe Tyr Lys Met
225 230 235 240
Cys Ile Lys Ala Phe Arg Asp Ser Ser Trp Gln Val Ile Met Ser Val
245 250 255
Gly Lys Thr Ile Asp Pro Glu Ser Leu Glu Asp Ile Pro Ala Asn Phe
260 265 270
Thr Ile Arg Gln Ser Val Pro Gln Leu Glu Val Leu Glu Lys Ala Asp
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Leu Phe Ile Ser His Gly Gly Met Asn Ser Thr Met Glu Ala Met Asn
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Ala Gly Val Pro Leu Val Val Ile Pro Gln Met Tyr Glu Gln Glu Leu
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Thr Ala Asn Arg Val Asp Glu Leu Gly Leu Gly Val Tyr Leu Pro Lys
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Glu Glu Val Thr Val Ser Ser Leu Gln Glu Ala Val Gln Ala Val Ser
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Ser Asp Gln Glu Leu Leu Ser Arg Val Lys Asn Met Gln Lys Asp Val
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Lys Glu Ala Gly Gly Ala Glu Arg Ala Ala Ala Glu Ile Glu Ala Phe
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Met Lys Lys Ser Ala Val Pro Gln
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Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu
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Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu
20 25 30
Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln
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Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu
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Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile
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Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val
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Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu
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Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val
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Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala
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Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp
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Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser
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Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys
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Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His
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Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr
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Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg
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Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu
245 250 255
Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu
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Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly
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Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln
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Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu
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Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile
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Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg
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Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro
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Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu
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Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu
385 390 395 400
Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser
405 410 415
Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr
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Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser
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Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln
450 455 460
Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr
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Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr
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Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His
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Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala
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Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr
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Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn
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Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe
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Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu
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Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val
595 600 605
Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala
610 615 620
Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly
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Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu
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Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala
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Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly
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Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val
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His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala
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Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser
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His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys
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Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val
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Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg
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Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln
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Ala Val Pro Leu Ala Gln Asp Asp
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Met Pro Thr Gly Arg Phe Glu Thr Met Arg Glu Trp Val Tyr Asp Ala
1 5 10 15
Ile Ser Ala Gln Arg Asn Glu Leu Leu Ser Leu Phe Ser Arg Tyr Val
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Ala Gln Gly Lys Gly Ile Leu Gln Ser His Gln Leu Ile Asp Glu Phe
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Leu Lys Thr Val Lys Val Asp Gly Thr Leu Glu Asp Leu Asn Lys Ser
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Pro Phe Met Lys Val Leu Gln Ser Ala Glu Glu Ala Ile Val Leu Pro
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Pro Phe Val Ala Leu Ala Ile Arg Pro Arg Pro Gly Val Arg Glu Tyr
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Val Arg Val Asn Val Tyr Glu Leu Ser Val Asp His Leu Thr Val Ser
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Glu Tyr Leu Arg Phe Lys Glu Glu Leu Val Asn Gly His Ala Asn Gly
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Asp Tyr Leu Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala Thr Leu Pro
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Arg Pro Thr Arg Ser Ser Ser Ile Gly Asn Gly Val Gln Phe Leu Asn
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Arg His Leu Ser Ser Ile Met Phe Arg Asn Lys Glu Ser Met Glu Pro
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Leu Leu Glu Phe Leu Arg Thr His Lys His Asp Gly Arg Pro Met Met
180 185 190
Leu Asn Asp Arg Ile Gln Asn Ile Pro Ile Leu Gln Gly Ala Leu Ala
195 200 205
Arg Ala Glu Glu Phe Leu Ser Lys Leu Pro Leu Ala Thr Pro Tyr Ser
210 215 220
Glu Phe Glu Phe Glu Leu Gln Gly Met Gly Phe Glu Arg Gly Trp Gly
225 230 235 240
Asp Thr Ala Gln Lys Val Ser Glu Met Val His Leu Leu Leu Asp Ile
245 250 255
Leu Gln Ala Pro Asp Pro Ser Val Leu Glu Thr Phe Leu Gly Arg Ile
260 265 270
Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe Gly
275 280 285
Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln Val Val Tyr
290 295 300
Ile Leu Asp Gln Val Arg Ala Leu Glu Asn Glu Met Leu Leu Arg Ile
305 310 315 320
Gln Lys Gln Gly Leu Glu Val Ile Pro Lys Ile Leu Ile Val Thr Arg
325 330 335
Leu Leu Pro Glu Ala Lys Gly Thr Thr Cys Asn Gln Arg Leu Glu Arg
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Val Ser Gly Thr Glu His Ala His Ile Leu Arg Ile Pro Phe Arg Thr
355 360 365
Glu Lys Gly Ile Leu Arg Lys Trp Ile Ser Arg Phe Asp Val Trp Pro
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Tyr Leu Glu Thr Phe Ala Glu Asp Ala Ser Asn Glu Ile Ser Ala Glu
385 390 395 400
Leu Gln Gly Val Pro Asn Leu Ile Ile Gly Asn Tyr Ser Asp Gly Asn
405 410 415
Leu Val Ala Ser Leu Leu Ala Ser Lys Leu Gly Val Ile Gln Cys Asn
420 425 430
Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Glu Ser Asp Ile Tyr
435 440 445
Trp Arg Asn His Glu Asp Lys Tyr His Phe Ser Ser Gln Phe Thr Ala
450 455 460
Asp Leu Ile Ala Met Asn Asn Ala Asp Phe Ile Ile Thr Ser Thr Tyr
465 470 475 480
Gln Glu Ile Ala Gly Ser Lys Asn Asn Val Gly Gln Tyr Glu Ser His
485 490 495
Thr Ala Phe Thr Met Pro Gly Leu Tyr Arg Val Val His Gly Ile Asp
500 505 510
Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Met Thr
515 520 525
Ile Tyr Phe Pro Tyr Ser Asp Lys Glu Arg Arg Leu Thr Ala Leu His
530 535 540
Glu Ser Ile Glu Glu Leu Leu Phe Ser Ala Glu Gln Asn Asp Glu His
545 550 555 560
Val Gly Leu Leu Ser Asp Gln Ser Lys Pro Ile Ile Phe Ser Met Ala
565 570 575
Arg Leu Asp Arg Val Lys Asn Leu Thr Gly Leu Val Glu Cys Tyr Ala
580 585 590
Lys Asn Ser Lys Leu Arg Glu Leu Ala Asn Leu Val Ile Val Gly Gly
595 600 605
Tyr Ile Asp Glu Asn Gln Ser Arg Asp Arg Glu Glu Met Ala Glu Ile
610 615 620
Gln Lys Met His Ser Leu Ile Glu Gln Tyr Asp Leu His Gly Glu Phe
625 630 635 640
Arg Trp Ile Ala Ala Gln Met Asn Arg Ala Arg Asn Gly Glu Leu Tyr
645 650 655
Arg Tyr Ile Ala Asp Thr Lys Gly Val Phe Val Gln Pro Ala Phe Tyr
660 665 670
Glu Ala Phe Gly Leu Thr Val Val Glu Ser Met Thr Cys Ala Leu Pro
675 680 685
Thr Phe Ala Thr Cys His Gly Gly Pro Ala Glu Ile Ile Glu Asn Gly
690 695 700
Val Ser Gly Phe His Ile Asp Pro Tyr His Pro Asp Gln Val Ala Ala
705 710 715 720
Thr Leu Val Ser Phe Phe Glu Thr Cys Asn Thr Asn Pro Asn His Trp
725 730 735
Val Lys Ile Ser Glu Gly Gly Leu Lys Arg Ile Tyr Glu Arg Tyr Thr
740 745 750
Trp Lys Lys Tyr Ser Glu Arg Leu Leu Thr Leu Ala Gly Val Tyr Ala
755 760 765
Phe Trp Lys His Val Ser Lys Leu Glu Arg Arg Glu Thr Arg Arg Tyr
770 775 780
Leu Glu Met Phe Tyr Ser Leu Lys Phe Arg Asp Leu Ala Asn Ser Ile
785 790 795 800
Pro Leu Ala Thr Asp Glu Asn
805
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<211> 809
<212> PRT
<213>Artificial synthesized sequence ()
<400> 4
Met Ala Asn Pro Lys Leu Thr Arg Val Leu Ser Thr Arg Asp Arg Val
1 5 10 15
Gln Asp Thr Leu Ser Ala His Arg Asn Glu Leu Val Ala Leu Leu Ser
20 25 30
Arg Tyr Val Asp Gln Gly Lys Gly Ile Leu Gln Pro His Asn Leu Ile
35 40 45
Asp Glu Leu Glu Ser Val Ile Gly Asp Asp Glu Thr Lys Lys Ser Leu
50 55 60
Ser Asp Gly Pro Phe Gly Glu Ile Leu Lys Ser Ala Met Glu Ala Ile
65 70 75 80
Val Val Pro Pro Phe Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val
85 90 95
Trp Glu Tyr Val Arg Val Asn Val Phe Glu Leu Ser Val Glu Gln Leu
100 105 110
Thr Val Ser Glu Tyr Leu Arg Phe Lys Glu Glu Leu Val Asp Gly Pro
115 120 125
Asn Ser Asp Pro Phe Cys Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala
130 135 140
Asn Val Pro Arg Pro Ser Arg Ser Ser Ser Ile Gly Asn Gly Val Gln
145 150 155 160
Phe Leu Asn Arg His Leu Ser Ser Val Met Phe Arg Asn Lys Asp Cys
165 170 175
Leu Glu Pro Leu Leu Asp Phe Leu Arg Val His Lys Tyr Lys Gly His
180 185 190
Pro Leu Met Leu Asn Asp Arg Ile Gln Ser Ile Ser Arg Leu Gln Ile
195 200 205
Gln Leu Ser Lys Ala Glu Asp His Ile Ser Lys Leu Ser Gln Glu Thr
210 215 220
Pro Phe Ser Glu Phe Glu Tyr Ala Leu Gln Gly Met Gly Phe Glu Lys
225 230 235 240
Gly Trp Gly Asp Thr Ala Gly Arg Val Leu Glu Met Met His Leu Leu
245 250 255
Ser Asp Ile Leu Gln Ala Pro Asp Pro Ser Ser Leu Glu Lys Phe Leu
260 265 270
Gly Met Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly
275 280 285
Tyr Phe Gly Gln Ala Asn Val Leu Gly Leu Pro Asp Thr Gly Gly Gln
290 295 300
Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Thr Glu Met Leu
305 310 315 320
Leu Arg Ile Lys Arg Gln Gly Leu Asp Ile Ser Pro Ser Ile Leu Ile
325 330 335
Val Thr Arg Leu Ile Pro Asp Ala Lys Gly Thr Thr Cys Asn Gln Arg
340 345 350
Leu Glu Arg Val Ser Gly Thr Glu His Thr His Ile Leu Arg Val Pro
355 360 365
Phe Arg Ser Glu Lys Gly Ile Leu Arg Lys Trp Ile Ser Arg Phe Asp
370 375 380
Val Trp Pro Tyr Leu Glu Asn Tyr Ala Gln Asp Ala Ala Ser Glu Ile
385 390 395 400
Val Gly Glu Leu Gln Gly Val Pro Asp Phe Ile Ile Gly Asn Tyr Ser
405 410 415
Asp Gly Asn Leu Val Ala Ser Leu Met Ala His Arg Met Gly Val Thr
420 425 430
Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser
435 440 445
Asp Ile Tyr Trp Lys Asp Phe Asp Asn Lys Tyr His Phe Ser Cys Gln
450 455 460
Phe Thr Ala Asp Leu Ile Ala Met Asn Asn Ala Asp Phe Ile Ile Thr
465 470 475 480
Ser Thr Tyr Gln Glu Ile Ala Gly Thr Lys Asn Thr Val Gly Gln Tyr
485 490 495
Glu Ser His Gly Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His
500 505 510
Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala
515 520 525
Asp Met Thr Ile Tyr Phe Pro Tyr Ser Glu Glu Thr Arg Arg Leu Thr
530 535 540
Ala Leu His Gly Ser Ile Glu Glu Met Leu Tyr Ser Pro Asp Gln Thr
545 550 555 560
Asp Glu His Val Gly Thr Leu Ser Asp Arg Ser Lys Pro Ile Leu Phe
565 570 575
Ser Met Ala Arg Leu Asp Lys Val Lys Asn Ile Ser Gly Leu Val Glu
580 585 590
Met Tyr Ser Lys Asn Thr Lys Leu Arg Glu Leu Val Asn Leu Val Val
595 600 605
Ile Ala Gly Asn Ile Asp Val Asn Lys Ser Lys Asp Arg Glu Glu Ile
610 615 620
Val Glu Ile Glu Lys Met His Asn Leu Met Lys Asn Tyr Lys Leu Asp
625 630 635 640
Gly Gln Phe Arg Trp Ile Thr Ala Gln Thr Asn Arg Ala Arg Asn Gly
645 650 655
Glu Leu Tyr Arg Tyr Ile Ala Asp Thr Arg Gly Ala Phe Ala Gln Pro
660 665 670
Ala Phe Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys
675 680 685
Gly Leu Pro Thr Phe Ala Thr Cys His Gly Gly Pro Ala Glu Ile Ile
690 695 700
Glu His Gly Leu Ser Gly Phe His Ile Asp Pro Tyr His Pro Glu Gln
705 710 715 720
Ala Gly Asn Ile Met Ala Asp Phe Phe Glu Arg Cys Lys Glu Asp Pro
725 730 735
Asn His Trp Lys Lys Val Ser Asp Ala Gly Leu Gln Arg Ile Tyr Glu
740 745 750
Arg Tyr Thr Trp Lys Ile Tyr Ser Glu Arg Leu Met Thr Leu Ala Gly
755 760 765
Val Tyr Gly Phe Trp Lys Tyr Val Ser Lys Leu Glu Arg Arg Glu Thr
770 775 780
Arg Arg Tyr Leu Glu Met Phe Tyr Ile Leu Lys Phe Arg Asp Leu Val
785 790 795 800
Lys Thr Val Pro Ser Thr Ala Asp Asp
805
<210> 5
<211> 805
<212> PRT
<213>Artificial synthesized sequence ()
<400> 5
Met Ala Thr Asp Arg Leu Thr Arg Val His Ser Leu Arg Glu Arg Leu
1 5 10 15
Asp Glu Thr Leu Thr Ala Asn Arg Asn Glu Ile Leu Ala Leu Leu Ser
20 25 30
Arg Ile Glu Ala Lys Gly Lys Gly Ile Leu Gln His His Gln Val Ile
35 40 45
Ala Glu Phe Glu Glu Ile Pro Glu Glu Asn Arg Gln Lys Leu Thr Asp
50 55 60
Gly Ala Phe Gly Glu Val Leu Arg Ser Thr Gln Glu Ala Ile Val Leu
65 70 75 80
Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val Trp Glu
85 90 95
Tyr Leu Arg Val Asn Val His Ala Leu Val Val Glu Glu Leu Gln Pro
100 105 110
Ala Glu Tyr Leu His Phe Lys Glu Glu Leu Val Asp Gly Ser Ser Asn
115 120 125
Gly Asn Phe Val Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala Ala Phe
130 135 140
Pro Arg Pro Thr Leu Asn Lys Ser Ile Gly Asn Gly Val Gln Phe Leu
145 150 155 160
Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser Leu His
165 170 175
Pro Leu Leu Glu Phe Leu Arg Leu His Ser Val Lys Gly Lys Thr Leu
180 185 190
Met Leu Asn Asp Arg Ile Gln Asn Pro Asp Ala Leu Gln His Val Leu
195 200 205
Arg Lys Ala Glu Glu Tyr Leu Gly Thr Val Pro Pro Glu Thr Pro Tyr
210 215 220
Ser Glu Phe Glu His Lys Phe Gln Glu Ile Gly Leu Glu Arg Gly Trp
225 230 235 240
Gly Asp Asn Ala Glu Arg Val Leu Glu Ser Ile Gln Leu Leu Leu Asp
245 250 255
Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu Gly Arg
260 265 270
Ile Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe
275 280 285
Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val
290 295 300
Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Asn Glu Met Leu His Arg
305 310 315 320
Ile Lys Gln Gln Gly Leu Asp Ile Val Pro Arg Ile Leu Ile Ile Thr
325 330 335
Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Gln Arg Leu Glu
340 345 350
Lys Val Phe Gly Thr Glu His Ser His Ile Leu Arg Val Pro Phe Arg
355 360 365
Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp
370 375 380
Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Val Ala His Glu Leu Ala Lys
385 390 395 400
Glu Leu Gln Gly Lys Pro Asp Leu Ile Val Gly Asn Tyr Ser Asp Gly
405 410 415
Asn Ile Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys
420 425 430
Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Glu Ser Asp Ile
435 440 445
Tyr Trp Lys Lys Leu Glu Glu Arg Tyr His Phe Ser Cys Gln Phe Thr
450 455 460
Ala Asp Leu Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr
465 470 475 480
Phe Gln Glu Ile Ala Gly Ser Lys Asp Thr Val Gly Gln Tyr Glu Ser
485 490 495
His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His Gly Ile
500 505 510
Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Gln
515 520 525
Thr Ile Tyr Phe Pro His Thr Glu Thr Ser Arg Arg Leu Thr Ser Phe
530 535 540
His Pro Glu Ile Glu Glu Leu Leu Tyr Ser Ser Val Glu Asn Glu Glu
545 550 555 560
His Ile Cys Val Leu Lys Asp Arg Ser Lys Pro Ile Ile Phe Thr Met
565 570 575
Ala Arg Leu Asp Arg Val Lys Asn Ile Thr Gly Leu Val Glu Trp Tyr
580 585 590
Gly Lys Asn Ala Lys Leu Arg Glu Leu Val Asn Leu Val Val Val Ala
595 600 605
Gly Asp Arg Arg Lys Glu Ser Lys Asp Leu Glu Glu Lys Ala Glu Met
610 615 620
Lys Lys Met Tyr Gly Leu Ile Glu Thr Tyr Lys Leu Asn Gly Gln Phe
625 630 635 640
Arg Trp Ile Ser Ser Gln Met Asn Arg Val Arg Asn Gly Glu Leu Tyr
645 650 655
Arg Val Ile Cys Asp Thr Arg Gly Ala Phe Val Gln Pro Ala Val Tyr
660 665 670
Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro
675 680 685
Thr Phe Ala Thr Cys Asn Gly Gly Pro Ala Glu Ile Ile Val His Gly
690 695 700
Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Arg Ala Ala Asp
705 710 715 720
Leu Leu Val Asp Phe Phe Glu Lys Cys Lys Leu Asp Pro Thr His Trp
725 730 735
Asp Lys Ile Ser Lys Ala Gly Leu Gln Arg Ile Glu Glu Lys Tyr Thr
740 745 750
Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val Tyr Gly
755 760 765
Phe Trp Lys His Val Ser Asn Leu Asp Arg Arg Glu Ser Arg Arg Tyr
770 775 780
Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Lys Leu Ala Glu Ser Val
785 790 795 800
Pro Leu Ala Ala Glu
805
<210> 6
<211> 793
<212> PRT
<213>Artificial synthesized sequence ()
<400> 6
Met Ile Glu Ala Leu Arg Gln Gln Leu Leu Asp Asp Pro Arg Ser Trp
1 5 10 15
Tyr Ala Phe Leu Arg His Leu Val Ala Ser Gln Arg Asp Ser Trp Leu
20 25 30
Tyr Thr Asp Leu Gln Arg Ala Cys Ala Asp Phe Arg Glu Gln Leu Pro
35 40 45
Glu Gly Tyr Ala Glu Gly Ile Gly Pro Leu Glu Asp Phe Val Ala His
50 55 60
Thr Gln Glu Val Ile Phe Arg Asp Pro Trp Met Val Phe Ala Trp Arg
65 70 75 80
Pro Arg Pro Gly Arg Trp Ile Tyr Val Arg Ile His Arg Glu Gln Leu
85 90 95
Ala Leu Glu Glu Leu Ser Thr Asp Ala Tyr Leu Gln Ala Lys Glu Gly
100 105 110
Ile Val Gly Leu Gly Ala Glu Gly Glu Ala Val Leu Thr Val Asp Phe
115 120 125
Arg Asp Phe Arg Pro Val Ser Arg Arg Leu Arg Asp Glu Ser Thr Ile
130 135 140
Gly Asp Gly Leu Thr His Leu Asn Arg Arg Leu Ala Gly Arg Ile Phe
145 150 155 160
Ser Asp Leu Ala Ala Gly Arg Ser Gln Ile Leu Glu Phe Leu Ser Leu
165 170 175
His Arg Leu Asp Gly Gln Asn Leu Met Leu Ser Asn Gly Asn Thr Asp
180 185 190
Phe Asp Ser Leu Arg Gln Thr Val Gln Tyr Leu Gly Thr Leu Pro Arg
195 200 205
Glu Thr Pro Trp Ala Glu Ile Arg Glu Asp Met Arg Arg Arg Gly Phe
210 215 220
Ala Pro Gly Trp Gly Asn Thr Ala Gly Arg Val Arg Glu Thr Met Arg
225 230 235 240
Leu Leu Met Asp Leu Leu Asp Ser Pro Ser Pro Ala Ala Leu Glu Ser
245 250 255
Phe Leu Asp Arg Ile Pro Met Ile Ser Arg Ile Leu Ile Val Ser Ile
260 265 270
His Gly Trp Phe Ala Gln Asp Lys Val Leu Gly Arg Pro Asp Thr Gly
275 280 285
Gly Gln Val Val Tyr Ile Leu Asp Gln Ala Arg Ala Leu Glu Arg Glu
290 295 300
Met Arg Asn Arg Leu Arg Gln Gln Gly Val Asp Val Glu Pro Arg Ile
305 310 315 320
Leu Ile Ala Thr Arg Leu Ile Pro Glu Ser Asp Gly Thr Thr Cys Asp
325 330 335
Gln Arg Leu Glu Pro Val Val Gly Ala Glu Asn Val Gln Ile Leu Arg
340 345 350
Val Pro Phe Arg Tyr Pro Asp Gly Arg Ile His Pro His Trp Ile Ser
355 360 365
Arg Phe Lys Ile Trp Pro Trp Leu Glu Arg Tyr Ala Gln Asp Leu Glu
370 375 380
Arg Glu Val Leu Ala Glu Leu Gly Ser Arg Pro Asp Leu Ile Ile Gly
385 390 395 400
Asn Tyr Ser Asp Gly Asn Leu Val Ala Thr Leu Leu Ser Glu Arg Leu
405 410 415
Gly Val Thr Gln Cys Asn Ile Ala His Ala Leu Glu Lys Ser Lys Tyr
420 425 430
Leu Tyr Ser Asp Leu His Trp Arg Asp His Glu Gln Asp His His Phe
435 440 445
Ala Cys Gln Phe Thr Ala Asp Leu Ile Ala Met Asn Ala Ala Asp Ile
450 455 460
Ile Val Thr Ser Thr Tyr Gln Glu Ile Ala Gly Asn Asp Arg Glu Ile
465 470 475 480
Gly Gln Tyr Glu Gly His Gln Asp Tyr Thr Leu Pro Gly Leu Tyr Arg
485 490 495
Val Glu Asn Gly Ile Asp Val Phe Asp Ser Lys Phe Asn Ile Val Ser
500 505 510
Pro Gly Ala Asp Pro Arg Phe Tyr Phe Ser Tyr Ala Arg Thr Glu Glu
515 520 525
Arg Pro Ser Phe Leu Glu Pro Glu Ile Glu Ser Leu Leu Phe Gly Arg
530 535 540
Glu Pro Gly Ala Asp Arg Arg Gly Val Leu Glu Asp Arg Gln Lys Pro
545 550 555 560
Leu Leu Leu Ser Met Ala Arg Met Asp Arg Ile Lys Asn Leu Ser Gly
565 570 575
Leu Ala Glu Leu Tyr Gly Arg Ser Ser Arg Leu Arg Gly Leu Ala Asn
580 585 590
Leu Val Ile Ile Gly Gly His Val Asp Val Gly Asn Ser Arg Asp Ala
595 600 605
Glu Glu Arg Glu Glu Ile Arg Arg Met His Glu Ile Met Asp His Tyr
610 615 620
Gln Leu Asp Gly Gln Leu Arg Trp Val Gly Ala Leu Leu Asp Lys Thr
625 630 635 640
Val Ala Gly Glu Leu Tyr Arg Val Val Ala Asp Gly Arg Gly Val Phe
645 650 655
Val Gln Pro Ala Leu Phe Glu Ala Phe Gly Leu Thr Val Ile Glu Ala
660 665 670
Met Ser Ser Gly Leu Pro Val Phe Ala Thr Arg Phe Gly Gly Pro Leu
675 680 685
Glu Ile Ile Glu Asp Gly Val Ser Gly Phe His Ile Asp Pro Asn Asp
690 695 700
His Glu Ala Thr Ala Glu Arg Leu Ala Asp Phe Leu Glu Ala Ala Arg
705 710 715 720
Glu Arg Pro Lys Tyr Trp Leu Glu Ile Ser Asp Ala Ala Leu Ala Arg
725 730 735
Val Ala Glu Arg Tyr Thr Trp Glu Arg Tyr Ala Glu Arg Leu Met Thr
740 745 750
Ile Ala Arg Ile Phe Gly Phe Trp Arg Phe Val Leu Asp Arg Glu Ser
755 760 765
Gln Val Met Glu Arg Tyr Leu Gln Met Phe Arg His Leu Gln Trp Arg
770 775 780
Pro Leu Ala His Ala Val Pro Met Glu
785 790
<210> 7
<211> 1179
<212> DNA
<213>Artificial synthesized sequence ()
<400> 7
atgaaaaagt accatatttc gatgatcaat atcccggcgt acggacatgt caatcctacg 60
cttgctttag tagagaagct ttgtgagaaa gggcaccgtg tcacgtacgc gacgactgag 120
gagtttgcgc ccgctgttca gcaagccggt ggagaagcat tgatctatca tacatccttg 180
aatattgatc ctaagcaaat cagggagatg atggaaaaga atgacgcgcc cctcagcctt 240
ttgaaagaat cactcagcat tctgccgcag cttgaggagt tatataagga tgatcagcct 300
gatctgatca tctatgactt tgttgcgctg gctggtaaat tgtttgctga aaagcttaat 360
gttccggtca ttaagctctg ttcgtcatat gcccaaaatg aatcctttca gttaggaaat 420
gaagacatgc tgaaaaaaat aagagaagca gaggctgaat ttaaagccta cttggagcaa 480
gagaagttgc cggctgtttc atttgaacag ttagctgtgc cggaagcatt aaatattgtc 540
tttatgccga agtcttttca gattcagcat gagacgttcg atgaccgttt ctgttttgtc 600
ggcccctctc tcggagaacg gaaggaaaaa gaaagcctgt tgattgacaa ggatgatcgc 660
ccgcttatgc tgatttcttt gggtacggcg tttaacgcat ggccggaatt ttacaagatg 720
tgcatcaagg catttcggga ttcttcatgg caagtgatca tgtcggttgg gaaaacgatt 780
gatccagaaa gcttggagga tattcctgct aactttacca ttcgccaaag tgtgccgcag 840
cttgaggtgt tagagaaagc tgatttgttc atctctcatg gcgggatgaa cagtacgatg 900
gaagcgatga acgcaggtgt gccgcttgtc gtcattccgc aaatgtatga gcaggagctc 960
actgcaaatc gggttgatga attaggcctt ggcgtttatt tgccgaaaga ggaagtgact 1020
gtttccagcc tgcaggaagc ggttcaggct gtatccagtg atcaagagct gctcagccgc 1080
gtcaagaata tgcaaaagga tgtaaaagaa gctggcggag cggagcgtgc ggcagctgag 1140
attgaagcgt ttatgaaaaa atccgctgtc ccgcagtaa 1179
<210> 8
<211> 2427
<212> DNA
<213>Artificial synthesized sequence ()
<400> 8
atggcaaacg ctgaacgtat gataacgcgc gtccacagcc aacgtgagcg tttgaacgaa 60
acgcttgttt ctgagagaaa cgaagtcctt gccttgcttt ccagggttga agccaaaggt 120
aaaggtattt tacaacaaaa ccagatcatt gctgaattcg aagctttgcc tgaacaaacc 180
cggaagaaac ttgaaggtgg tcctttcttt gaccttctca aatccactca ggaagcaatt 240
gtgttgccac catgggttgc tctagctgtg aggccaaggc ctggtgtttg ggaatactta 300
cgagtcaatc tccatgctct tgtcgttgaa gaactccaac ctgctgagtt tcttcatttc 360
aaggaagaac tcgttgatgg agttaagaat ggtaatttca ctcttgagct tgatttcgag 420
ccattcaatg cgtctatccc tcgtccaaca ctccacaaat acattggaaa tggtgttgac 480
ttccttaacc gtcatttatc ggctaagctc ttccatgaca aggagagttt gcttccattg 540
cttaagttcc ttcgtcttca cagccaccag ggcaagaacc tgatgttgag cgagaagatt 600
cagaacctca acactctgca acacaccttg aggaaagcag aagagtatct agcagagctt 660
aagtccgaaa cactgtatga agagtttgag gccaagtttg aggagattgg tcttgagagg 720
ggatggggag acaatgcaga gcgtgtcctt gacatgatac gtcttctttt ggaccttctt 780
gaggcgcctg atccttgcac tcttgagact tttcttggaa gagtaccaat ggtgttcaac 840
gttgtgatcc tctctccaca tggttacttt gctcaggaca atgttcttgg ttaccctgac 900
actggtggac aggttgttta cattcttgat caagttcgtg ctctggagat agagatgctt 960
caacgtatta agcaacaagg actcaacatt aaaccaagga ttctcattct aactcgactt 1020
ctacctgatg cggtaggaac tacatgcggt gaacgtctcg agagagttta tgattctgag 1080
tactgtgata ttcttcgtgt gcccttcaga acagagaagg gtattgttcg caaatggatc 1140
tcaaggttcg aagtctggcc atatctagag acttacaccg aggatgctgc ggttgagcta 1200
tcgaaagaat tgaatggcaa gcctgacctt atcattggta actacagtga tggaaatctt 1260
gttgcttctt tattggctca caaacttggt gtcactcagt gtaccattgc tcatgctctt 1320
gagaaaacaa agtacccgga ttctgatatc tactggaaga agcttgacga caagtaccat 1380
ttctcatgcc agttcactgc ggatattttc gcaatgaacc acactgattt catcatcact 1440
agtactttcc aagaaattgc tggaagcaaa gaaactgttg ggcagtatga aagccacaca 1500
gcctttactc ttcccggatt gtatcgagtt gttcacggga ttgatgtgtt tgatcccaag 1560
ttcaacattg tctctcctgg tgctgatatg agcatctact tcccttacac agaggagaag 1620
cgtagattga ctaagttcca ctctgagatc gaggagctcc tctacagcga tgttgagaac 1680
aaagagcact tatgtgtgct caaggacaag aagaagccga ttctcttcac aatggctagg 1740
cttgatcgtg tcaagaactt gtcaggtctt gttgagtggt acgggaagaa cacccgcttg 1800
cgtgagctag ctaacttggt tgttgttgga ggagacagga ggaaagagtc aaaggacaat 1860
gaagagaaag cagagatgaa gaaaatgtat gatctcattg aggaatacaa gctaaacggt 1920
cagttcaggt ggatctcctc tcagatggac cgggtaagga acggtgagct gtaccggtac 1980
atctgtgaca ccaagggtgc ttttgtccaa cctgcattat atgaagcctt tgggttaact 2040
gttgtggagg ctatgacttg tggtttaccg actttcgcca cttgcaaagg tggtccagct 2100
gagatcattg tgcacggtaa atcgggtttc cacattgacc cttaccatgg tgatcaggct 2160
gctgatactc ttgctgattt cttcaccaag tgtaaggagg atccatctca ctgggatgag 2220
atctcaaaag gagggcttca gaggattgag gagaaataca cttggcaaat ctattcacag 2280
aggctcttga cattgactgg tgtgtatgga ttctggaagc atgtctcgaa ccttgaccgt 2340
cttgaggctc gccgttacct tgaaatgttc tatgcattga agtatcgccc attggctcag 2400
gctgttcctc ttgcacaaga tgattga 2427
<210> 9
<211> 32
<212> DNA
<213>Artificial synthesized sequence ()
<400> 9
cgcggatcca tgaaaaagta ccatatttcg at 32
<210> 10
<211> 31
<212> DNA
<213>Artificial synthesized sequence ()
<400> 10
cgcgtcgact tactgcggga cagcggattt t 31
<210> 11
<211> 35
<212> DNA
<213>Artificial synthesized sequence ()
<400> 11
gcgtcgacaa atggcaaacg ctgaacgtat gataa 35
<210> 12
<211> 35
<212> DNA
<213>Artificial synthesized sequence ()
<400> 12
ttgcggccgc ttatcatacg ttcagcgttt gccat 35

Claims (10)

1. a kind of non-natural ginsenoside Rd 12, it is characterised in that the non-natural ginsenoside Rd 12 has to tie shown in Formulas I Structure：
2. a kind of catalystic converter system for preparing non-natural ginsenoside Rd 12 as claimed in claim 1, it is characterised in that described Catalystic converter system includes ginseng sapoglycoside Rg 3, sucrose, sucrose synthase, glycosyl transferase and uridine 5'-diphosphate.
3. catalystic converter system according to claim 2, it is characterised in that the glycosyl transferase is glycosyl transferase Bs-YjiC；
Preferably, the encoding gene of the glycosyl transferase Bs-YjiC derives from bacillus subtilis and/or its mutant；
Preferably, the amino acid sequence of the glycosyl transferase Bs-YjiC such as SEQ ID NO.1 or homologous with 85% with it Property, the preferably amino acid sequence of 95% homology.
4. catalyst system and catalyzing according to claim 2, it is characterised in that the sucrose synthase from arabidopsis, soybean, In nitrous acid monad or acidophilia thermophilic thiobacillus any one or at least two combination, preferably arabidopsis, soybean In acidophilia thermophilic thiobacillus any one or at least two combination；
Preferably, any one in the amino acid sequence of the sucrose synthase such as SEQ ID NO.2-6 or with SEQ ID Any one amino acid sequence with 85% homology, preferably 95% homology in NO.2-6；
Preferably, that the sucrose synthase is the sucrose synthase AtSuSy from arabidopsis, its amino acid sequence such as SEQ Shown in ID NO.2；
Preferably, the concentration of the glycosyl transferase is 10-1000mU/mL, preferably 160mU/mL；
Preferably, the concentration of the sucrose synthase is 10-1000mU/mL, preferably 200mU/mL.
5. a kind of carrier, it is characterised in that the carrier is used for sugar of the expression as described in claim 2-4 separately or together Based transferase and/or sucrose synthase；
Preferably, the glycosyl transferase is glycosyl transferase Bs-YjiC；
Preferably, the sucrose synthase is sucrose synthase AtSuSy.
6. a kind of host cell, it is characterised in that the host cell includes carrier as claimed in claim 5；
Preferably, the host cell is Escherichia coli, saccharomyces cerevisiae, Pichia pastoris, corynebacterium glutamicum, bacillus subtilis In bacterium or aspergillus niger any one or at least two combination.
A kind of 7. use of catalyst system and catalyzing as any one of claim 2-4 or host cell as claimed in claim 6 On the way, it is characterised in that the catalyst system and catalyzing is converted into non-natural ginsenoside Rd 12 for being catalyzed ginseng sapoglycoside Rg 3.
A kind of 8. method for preparing non-natural ginsenoside Rd 12, it is characterised in that any one of claim 2-4 In the presence of catalyst system and catalyzing or host cell as claimed in claim 6, ginseng sapoglycoside Rg 3 is converted into non-natural ginsenoside Rd12。
9. a kind of antineoplastic, it is characterised in that the medicine includes non-natural ginsenoside as claimed in claim 1 Rd12；
Preferably, the antineoplastic also includes pharmaceutically acceptable carrier.
10. antineoplastic according to claim 9, it is characterised in that the tumour includes liver cancer, lung cancer, colon cancer In stomach cancer any one or at least two combination.
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* Cited by examiner, † Cited by third party
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CN103849672A (en) * 2012-12-06 2014-06-11 中国科学院上海生命科学研究院 Group of glycosyl transferase and application thereof
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