CN104830820A - Protease and detection method for rapidly detecting ribonucleic acid at constant room temperature - Google Patents

Abstract

The invention discloses protease for rapidly detecting ribonucleic acid at a constant room temperature. The protease comprises (1) reverse transcriptase (SEQ ID No.17); (2) recombinase (SEQ ID No.18); (3) single-chain binding protein (SEQ ID No.19); (4) DNA polymerase (SEQ ID No.20); and (5) auxiliary protein (SEQ ID No.21). The invention also discloses a reagent and detection method for rapidly detecting ribonucleic acid at a constant room temperature. In the provided detection method, the RNA amplification is imitated in-vitro by using the disclosed multiple gene engineering enzymes and chemical components; the RNA amplification and detection can be finished in one step rapidly at a constant room temperature through the interactions among the components; and thus the detection of viral pathogen becomes more convenient and rapid.

Description

For proteolytic enzyme and the detection method of normal temperature isothermal rapid detection Yeast Nucleic Acid

Technical field

The present invention is a new opplication of nucleic acid isothermal amplification technology, relate to and utilize several genes engineering enzyme and chemical composition to simulate in vitro, by the interaction between them, realize under normal temperature isothermal condition, fast, amplification and the testing process of Yeast Nucleic Acid (RNA) is completed to single stage method, thus make the detection of virus type pathogenic agent more fast with easier, thus can more effectively be applied to from now in the middle of MicroRNA detection or on-site rapid inspection quarantine field.

Background technology

After polymerase chain reaction (PCR) helps to realize the amplification in vitro of thymus nucleic acid (DNA), the amplification in vitro of RNA also causes the concern of scientists thereupon, and therefore deriving technology reverse transcription PCR (RT-PCR) also just arises.Initial reverse transcription PCR, with two-step approach, namely becomes cDNA by RNA reverse transcription, is that template carries out standard PCR amplification afterwards again, thus realizes amplification or the detection of RNA with cDNA.But along with the needs developed, loaded down with trivial details two-step approach can not meet the demand of inspection and quarantine task well, the single stage method RNA amplification thereupon occurred, has carried out depth optimization by required for reaction component, has achieved the augmentation detection from RNA template to product D NA in same reaction tubes.However, PCR highly relies on the shortcoming that accurate temperature cycler carries out detecting, and is confined in centralab by the detection of RNA, also limit the ability of its rapid detection virus type pathogenic agent at the scene to a great extent.

In the nucleic acid isothermal amplification technology gone from strength to strength in recent years, also advantage and ability that some technology possesses Site Detection RNA aspect is no lack of, but because of the difference of Cleaning Principle, all emphasize particularly on different fields a little and strengths and weaknesses: 1) loop-mediated isothermal amplification technique (LAMP) utilizes 4 pairs of primer specific identifications, 6 target sites and has the Bst enzyme of strand-displacement activity, nucleic acid can be realized under 60-65 degree condition, comprise the rapid detection (<60 minute) of DNA and RNA; 2) amplification (NASBA) method of nucleotide sequence is depended on, utilize the mediation of three kinds of enzymes (AMV reversed transcriptive enzyme, RNaseH and t7 rna polymerase) and two Auele Specific Primers, incubation 1.5-2 hour under a steady temperature, can complete the enzymic process that RNA template increases fast; 3) rolling circle amplification (RCA), utilize DNA ligase and archaeal dna polymerase, with the pattern of rolling-circle replication, the strand displacement synthesis of annular template is carried out from one or more primer, thus can the genomic amplification of ring-type of achievement unit partitivirus, but the reaction times of RCA, partially to grow the advantage that (>4 hour) make it at the scene in detection field just very unobvious; 4) single primer isothermal amplification technique (SPIA), under 55-65 degree, can realize the external linear isothermal amplification of DNA by the archaeal dna polymerase of the primer of a DNA-RNA heterozygosis, RNaseH and strong strand-displacement activity in 30 minutes; The reversed transcriptive enzyme added on this basis can help the isothermal duplication realizing RNA template, Ribo-SPIA by name; 5) the enzyme dna isothermal amplification technique (HDA) that untwists is relied on, then utilize helicase, DNA single chain binding protein, archaeal dna polymerase and heat-staple reversed transcriptive enzyme acting in conjunction, under 37 or 65 degree of (temperature depends on the archaeal dna polymerase of use) conditions, with the form of two-step approach or single stage method, the rapid amplifying (~ 120 minutes) of RNA template can be realized; 6) chain substitutes amplification (SDA) technology, is the external isothermal amplification technique of DNA deriving based on enzymatic reaction and obtain, and utilizes restriction endonuclease and the archaeal dna polymerase with strand-displacement activity, realizes the amplification of nucleic acid under isothermal conditions.

Because of virus cause susceptibility to disease and infectivity all very strong, therefore if any one fast and accurately method or technology can help through detecting of virus (containing DNA or RNA viruses) at the scene, effectively and in time can prevent diffusion and the propagation of virus disease.Given this, nucleic acid isothermal amplification technology should be current optimal technology kind, but existing mature technology or because of incubation temperature, such as 60-65 degree, still need to rely on specific incubation equipment, or because of the incubation time still factor such as relatively long, still there is a certain distance from really realizing Site Detection.Therefore, the invention is intended to provide a kind of more fast, be applicable to the technology of Site Detection RNA more accurately.

Summary of the invention

The present invention aims to provide one under normal temperature isothermal condition, fast, can complete the amplification of Yeast Nucleic Acid (RNA) and the method for detection to single stage method.

First object of the present invention is to provide the proteolytic enzyme for normal temperature isothermal rapid detection Yeast Nucleic Acid.

For the proteolytic enzyme of normal temperature isothermal rapid detection Yeast Nucleic Acid, it is characterized in that, described proteolytic enzyme comprises: (1) reversed transcriptive enzyme; (2) recombinase; (3) single strand binding protein; (4) archaeal dna polymerase; (5) accessory protein; The aminoacid sequence of described reversed transcriptive enzyme is as shown in SEQ ID No.17; The aminoacid sequence of recombinase is as shown in SEQ ID No.18; The aminoacid sequence of single strand binding protein is as shown in SEQ ID No.19; The aminoacid sequence of archaeal dna polymerase is as shown in SEQ ID No.20; The aminoacid sequence of accessory protein is as shown in SEQ ID No.21.

Normal temperature isothermal of the present invention, refers to and to hatch under a certain steady temperature, and temperature is 40-42 DEG C (being preferably 40 DEG C), close to normal temperature, and is homogeneous constant temp.

Reversed transcriptive enzyme, be a kind of proteolytic enzyme after adopting point mutation to carry out genetic modification, being characterized in: 1) have DNA polymerase activity, take RNA as template, and catalysis dNTP is polymerized generation complementary (cDNA); 2) by the active inactivation of RNaseH that point mutation will have originally, thus avoid the RNA in DNA-RNA heterozygosis chain to be hydrolyzed; 3) this enzyme sequence derives from murine leukemia reversed transcriptive enzyme (M-MLV), transforms by point mutation; 4) consumption in reaction system is about 8-12 unit/ul.

Recombinase, is a kind of engineered protein enzyme, is characterized in: 1) have the ability in conjunction with single strand dna, therefore can form initiation complex with primer tasteless nucleotide strand; 2) there is the ability combined with double chain DNA molecule, therefore also in conjunction with DNA double chain or DNA-RNA heterozygosis chain while formation initiation complex, thus the special triple strand dna intermediate of structure could be formed; 3) there is homologous recombination activity, when single stranded DNA invades double-stranded DNA or heterozygosis chain, after searching out homologous sequence area along chain, the strand of homology can be squeezed away, form Bubble Region, so-called local, and then facilitate the intervention of follow-up archaeal dna polymerase; 4) this enzyme sequence can derive from the RecA of intestinal bacteria (E.coli), or T4 phage recombinase uvsX, by genetic engineering modified further; 5) consumption in reaction system is about 100-150ng/ul.

Single strand binding protein, is a kind of DBP, is characterized in: 1) along the Bubble Region, local formed, locally combine the homology strand being replaced out, avoid it by nuclease degradation; 2) do not possess enzymic activity, the ATP that therefore gets along well combines; 3) there is synergistic effect, therefore can multiple single strand binding protein be adjacent is incorporated on DNA single chain, be convenient to the DNA synthesis of Bubble Region; 4) this combination has short-time characteristic, therefore constantly could combine along " replication fork ", again constantly assembly again; 5) this enzyme sequence can come from the gp32 albumen of E.coli or T4; 6) consumption in reaction system is about 700-1000ng/ul.

Archaeal dna polymerase, is a kind ofly rely on the engineered protein enzyme that DNA profiling carries out complementary dna sequence synthesis, is characterized in: 1) have higher continuous synthesis ability; 2) there is strand-displacement activity, while the new chain of synthesis, coordinate single strand binding protein to be continued to advance along " replication fork " direction in Bubble Region, local by strand displacement capability, thus homology chain is replaced out; 3) 3 '-5 is had ' excision enzyme (calibration capability) is active, thus the fidelity of guarantee synthesis; 4) non-refractory, reacts under being therefore highly suitable for normal temperature isothermal condition, and completes the synthesis of DNA chain; 5) sequence of this enzyme can come from the DNA polymerase i Klenow large fragment of E.coli, Bst polysaccharase, Phi-29 polysaccharase or Bacillus subtillis PolI (Bsu); 6) consumption in reaction system is about 60-90ng/ul.

Accessory protein, is a kind ofly assist or strengthen the albumen of recombinase active, is characterized in: 1) promote and stablize the combination of initiation complex and DNA double interchain; 2) the uvsY albumen of the optional T4 phage of sequence; 3) consumption in reaction system is about 20-40ng/ul.

Second object of the present invention is to provide the reagent for normal temperature isothermal rapid detection Yeast Nucleic Acid.

For the reagent of normal temperature isothermal rapid detection Yeast Nucleic Acid, it is characterized in that, described reagent mainly comprises Tris, RNase inhibitor, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT), amplimer and reversed transcriptive enzyme, recombinase, single strand binding protein, archaeal dna polymerase and accessory protein.

In described reagent, 5 kinds of albumen (enzyme) and other chemical composition prepare the optimal reaction environment of rear formation by a certain percentage: 5%PEG35000, 6% trehalose, the recombinase of 115ng/ul, the accessory protein of 35ng/ul, the single strand binding protein of 830ng/ul, the Bsu polysaccharase of 78ng/ul, the reversed transcriptive enzyme of 12 units/ul, the RNase inhibitor of 0.15 unit/ul, the ATP of 3.5mM, the dNTPs of 490uM, the Tris damping fluid of 55mM, the dithiothreitol (DTT) of 12mM, the disodium creatine phosphate of 12mM, the creatine phosphokinase of 114ng/ul, the potassium acetate of 75mM and 7% N.F,USP MANNITOL, the amplimer of 350-450nM, cumulative volume is 50ul.Detection reagent, through lyophilize process, removes liquid phase, retains solid matter, can be made into white dry powder, carry out resuspended when being convenient to field quick detection.Reaction reagent after frozen dried is white dry powder, is attached to tube wall without any viscous substance, after resuspended with damping fluid, can dissolve completely, exists without any obvious particulate material; After freeze-drying, the detection vigor of reaction system can maintain half a year.

Genetically engineered enzyme, specified chemical component and the primer utilizing function different for the reagent of normal temperature isothermal rapid detection Yeast Nucleic Acid of the present invention, can realize the amplification of RNA normal temperature.There is following characteristics: 1) genetically engineered enzyme wherein or albumen comprise 5 kinds, are reversed transcriptive enzyme, recombinase, single strand binding protein, archaeal dna polymerase and accessory protein respectively; 2) chemical composition comprises RNase inhibitor; The Triphosaden (ATP) of lasting ATP energy, disodium creatine phosphate (PCr) and creatine phosphokinase (CK) is provided to combine; The deoxyribonucleoside triphosphate (dNTP) of synthesis unit is provided; Maintain the potassium acetate (KAc) of reactive ion and potential of hydrogen environment, dithiothreitol (DTT) (DTT) and polyoxyethylene glycol (PEG); And the trehalose (Trehalose) of protein-active and N.F,USP MANNITOL (Mannitol) in maintenance freezing dry process; 3) primer is the oligonucleotide strand that a pair length is about 30-35nt, respectively the upstream and downstream of specific recognition target area.

3rd object of the present invention is to provide a kind of method of normal temperature isothermal rapid detection Yeast Nucleic Acid.

A method for normal temperature isothermal rapid detection Yeast Nucleic Acid, is characterized in that, after detection reagent adds template, being placed in a temperature control device, to maintain incubation temperature be 40-42 DEG C, hatches 20-40 minute, hatch in process and keep homogeneous steady temperature; Visual retrieval is carried out again by agarose gel electrophoresis; Described detection reagent mainly comprises Tris, RNase inhibitor, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT), amplimer, reversed transcriptive enzyme, recombinase, single strand binding protein, archaeal dna polymerase and accessory protein.

The present invention can realize on-the-spot RNA rapid detection.After the resuspended lyophilize reaction reagent system of damping fluid, a series of detection operations carried out at normal temperatures.Damping fluid (5-6%PEG), 350-450nM primer, template, distilled water and 12-15mM magnesium acetate (MgAc) is comprised for resuspended component.Total reaction volume is 50ul; The optional 40-42 degree of incubation temperature, optimum is 40 degree; Incubation time is 20-40 minute; Can also select to be added into a fluorescent probe or fluorescence dye, realize the real-time RNA rapid detection of normal temperature.

Temperature control device of the present invention is for reacting the instrument providing appropriate incubation environment, having temperature control modules, set of time function and sample well.Such as metal bath, water-bath, incubator or other small portable incubation equipment etc.

The present invention has following technical characterstic:

1) normal temperature isothermal: by 5 kinds of engineering enzymes create together with chemical composition one at utmost simulate biological nucleic acid in vivo increase environment, and often kind of engineering enzyme all Each performs its own functions, the temperature of reaction that it is the suitableeest works, and therefore working efficiency is the highest.

2) quick: because often kind of enzyme carries out work on optimal reactive temperature, therefore activity ensure that the quick of reaction efficiently.In addition, the cDNA that enzyme which catalyzes is complementary with it based on RNA template generation, and form with it the DNA-RNA heterozygosis chain be not degraded, this heterozygosis chain is as the template of following amplification subsequently.This RNase H activity digestion by reversed transcriptive enzyme more first than RT-PCR method generates strand cDNA, and it is rapider to carry out following amplification again with strand, more saves time, therefore reacts also quicker.

3) single stage method: all amplifications from RNA to DNA or reaction are all carried out in same reaction tubes, are also only completed by disposable application of sample, therefore operate simpler.

Accompanying drawing explanation

Fig. 1 is three pairs of primers electrophorogram when being template amplification with STb gene (gDNA) or soybean RNA respectively; In figure, swimming lane 1 is gDNA amplified production, and size is 343bp, and swimming lane 2 is RNA amplification product, and size is 251bp; Swimming lane 3 is gDNA amplified production, and size is 444bp, and swimming lane 4 is RNA amplification product, and size is 352bp; Swimming lane 5 is gDNA amplified production, and size is 449bp, and swimming lane 6 is RNA amplification product, and size is 164bp; Swimming lane 7 is gDNA amplified production, and size is 472bp, and swimming lane 8 is RNA amplification product, and size is 333bp; M is DNA marker thing.

Fig. 2 is primer pair E1-F/R electrophorogram when being template amplification with STb gene (gDNA) or soybean RNA respectively; In figure, swimming lane 1,2,3 is all gDNA amplified production, and size is 343bp, is three duplicate detection; Swimming lane 4 is RNA amplification products, and size is 251bp; M is DNA marker thing.

Fig. 3 be primer pair HSP-F/R and ACT-F/R respectively with paddy rice RNA for electrophorogram during template amplification; In figure, swimming lane 1,3 is negative controls; Swimming lane 2,4 is HSP amplified production (size is 120bp) and Actin amplified production (size is 121bp) respectively; M is DNA marker thing.

Fig. 4 be primer pair CAA3-F/R and BmG-F/R respectively with silkworm RNA for electrophorogram during template amplification; In figure, swimming lane 1,4 is negative control; Swimming lane 2,3 is the amplified production of gene cytoplasmic actin A3, and size is 176bp; Swimming lane 5,6 is the amplified production of gene BmGAPDH, and size is 172bp; M is DNA marker thing.

Embodiment

Following specific embodiment is further illustrating technique and method scheme provided by the invention, but does not should be understood to limitation of the present invention.

The biological material source used in the present invention:

1.RNA template derives from Soybean Leaves, paddy rice, silkworm respectively, uses traditional Trizol method to carry out extracting and obtaining in laboratory.

2.DNA template also derives from soybean, adopts traditional Total DNA extraction method to carry out extracting acquisition.

Amplification primers all with VectorNTI7.0 design, after win polygala root biotechnology limited liability company in Beijing three and synthesize and prepare.

Embodiment 1

The present embodiment successfully can carry out normal temperature isothermal RNA rapid detection for illustration of in soybean RNA template.

1. based on the reference gene EF1b (gi353336037) of soybean and the sequence of Actin-1-like (gi353336033), according to across intron principle, devise respectively 4 to 2 pairs of primers.After experiment, 3 pairs of primers are selected in EF1b, E1-F/R (SEQ ID No.1 & 2), E2-F/R (SEQ ID No.3 & 4) and E3-F/R (SEQ ID No.5 & 6) respectively.In Actin-1-like, select 1 pair of primer, be AL1-F/R (SEQ ID No.7 & 8).

E1-F：5’-CTTCTCGATGTTAAGCCTTGGGACGATG-3’(SEQ ID No.1)

E1-R：5’-TTGAATGCAACAATGTCACAGCTCTGGA-3’(SEQ ID No.2)

E2-F：5’-ATGAGACCGACATGAAGAAGCTGGAAGA-3’(SEQ ID No.3)

E2-R：5’-CACAATTCTTGCAAGAAAACCACCATTG-3’(SEQ ID No.4)

E3-F：5’-AGGAGGATAAGAAGGCAGCAGAGGAAAG-3’(SEQ ID No.5)

E3-R：5’-TAAACCAGGCATCTCAATACTACGGACA-3’(SEQ ID No.6)

AL1-F：5’-GTGTTTCCCAGCATTGTAGGTCGTCCTC-3’(SEQ ID No.7)

AL1-R：5’-ATAGGGACAGTGTGGCTGACACCATCAC-3’(SEQ ID No.8)

2. use the freeze-drying reaction system of preparation as main reaction composition, then add the components such as primer, damping fluid, distilled water, template and magnesium acetate, complete the preparation detecting liquid.5 kinds of albumen (enzyme) and other chemical composition prepare the optimal reaction environment of rear formation by a certain percentage: 5%PEG35000,6% trehalose, the recombinase of 115ng/ul, the accessory protein of 35ng/ul, the single strand binding protein of 830ng/ul, the Bsu polysaccharase of 78ng/ul, the reversed transcriptive enzyme of 12 units/ul, the RNase inhibitor of 0.15 unit/ul, the ATP of 3.5mM, the dNTPs of 490uM, the Tris damping fluid of 55mM, the dithiothreitol (DTT) of 12mM, the disodium creatine phosphate of 12mM, the creatine phosphokinase of 114ng/ul, the potassium acetate of 75mM and 7% N.F,USP MANNITOL; The amplimer of 350-450nM, cumulative volume is 50ul.

3. amplified reaction program: 40 degree of isothermals, 40 minutes.

4. last agarose gel electrophoresis carry out detecting (Fig. 1. and Fig. 2 .).Fig. 1 be three pairs of primers respectively with STb gene (gDNA) or soybean RNA be template increase time electrophorogram.Because primer is across intron design, therefore the amplified production of obvious gDNA is greater than the product of RNA, illustrates really to achieve to obtain product from RNA sample single stage method under normal temperature isothermal condition.Fig. 2 is primer pair E1-F/R electrophorogram when being template amplification with STb gene (gDNA) or soybean RNA respectively.

Embodiment 2

The present embodiment also successfully can carry out normal temperature isothermal RNA rapid detection for illustration of in the RNA template of paddy rice and silkworm.

1., based on the sequence of reference gene HSP and Actin of paddy rice, according to across intron principle, carry out design of primers respectively.By overtesting screening, obtain primer HSP-F/R (SEQ ID No.9 & 10) and ACT-F/R (SEQ ID No.11 & 12) respectively.

HSP-F：5’-CTCAACATCTCACGTGAGATGCTCCAGCAG-3’(SEQ ID No.9)

HSP-R：5’-GTTGTAGTCTTCCTTGTTCTCAGCGATCTC-3’(SEQ ID No.10)

ACT-F：5’-GCTATGTACGTCGCCATCCAGGCCGTCCTCTC-3’(SEQ ID No.11)

ACT-R：5’-GAGCATATCCTTCATAGATGGGGACAGTGTG-3’(SEQ ID No.12)

2. based on the sequence of reference gene cytoplasmic actin A3 and BmGAPDH of silkworm, according to across intron principle of design, design and screened primer CAA3-F/R (SEQ ID No.13 & 14) and BmG-F/R (SEQ ID No.15 & 16) respectively.

CAA3-F：5’-GCCATCAGGGCGTGATGGTCGGCATGGGACAGAAG-3’(SEQ ID No.13)

CAA3-R：5’-GGCGACACGCAGCTCATTGTAGAAGGTATGATGCC-3’(SEQ ID No.14)

BmG-F：5’-CATCATTCCTGCCTCTACTGGTGCTGCCAAAG-3’(SEQ ID No.15)

BmG-R：5’-GACCTTTTGCTTGATGGCTTCATAGCTTGCAG-3’(SEQ ID No.16)

3. use the freeze-drying reaction system of preparation as main reaction composition, then add the components such as primer, damping fluid, distilled water, template and magnesium acetate, complete the preparation detecting liquid.5 kinds of albumen (enzyme) and other chemical composition prepare the optimal reaction environment of rear formation by a certain percentage: 5%PEG35000,6% trehalose, the recombinase of 115ng/ul, the accessory protein of 35ng/ul, the single strand binding protein of 830ng/ul, the Bsu polysaccharase of 78ng/ul, the reversed transcriptive enzyme of 12 units/ul, the RNase inhibitor of 0.15 unit/ul, the ATP of 3.5mM, the dNTPs of 490uM, the Tris damping fluid of 55mM, the dithiothreitol (DTT) of 12mM, the disodium creatine phosphate of 12mM, the creatine phosphokinase of 114ng/ul, the potassium acetate of 75mM and 7% N.F,USP MANNITOL; The amplimer of 350-450nM, cumulative volume is 50ul.

4. amplified reaction program: 40 degree of isothermals, 40 minutes.

5. last agarose gel electrophoresis carry out detecting (Fig. 3. and Fig. 4 .).Fig. 3 be primer pair HSP-F/R and ACT-F/R respectively with paddy rice RNA for electrophorogram during template amplification.Fig. 4 be primer pair CAA3-F/R and BmG-F/R respectively with silkworm RNA for electrophoresis during template amplification.Two figure illustrate that this one step method ribonucleic acid TRAP is applicable to plant origin RNA, are also applicable to animal-origin RNA, possess methodology pardon.

SEQUENCE LISTING

 

<110> Zhejiang Taijing Biotechnology Co., Ltd.

 

<120> is used for proteolytic enzyme and the detection method of normal temperature isothermal rapid detection Yeast Nucleic Acid

 

<130>

 

<160> 21

 

<170> PatentIn version 3.3

 

<210> 1

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 1

cttctcgatg ttaagccttg ggacgatg 28

 

<210> 2

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 2

ttgaatgcaa caatgtcaca gctctgga 28

 

<210> 3

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 3

atgagaccga catgaagaag ctggaaga 28

 

<210> 4

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 4

cacaattctt gcaagaaaac caccattg 28

 

<210> 5

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 5

aggaggataa gaaggcagca gaggaaag 28

 

<210> 6

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 6

taaaccaggc atctcaatac tacggaca 28

 

<210> 7

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 7

gtgtttccca gcattgtagg tcgtcctc 28

 

<210> 8

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 8

atagggacag tgtggctgac accatcac 28

 

<210> 9

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 9

ctcaacatct cacgtgagat gctccagcag 30

 

<210> 10

<211> 30

<212> DNA

<213> artificial sequence

 

<400> 10

gttgtagtct tccttgttct cagcgatctc 30

 

<210> 11

<211> 32

<212> DNA

<213> artificial sequence

 

<400> 11

gctatgtacg tcgccatcca ggccgtcctc tc 32

 

<210> 12

<211> 31

<212> DNA

<213> artificial sequence

 

<400> 12

gagcatatcc ttcatagatg gggacagtgt g 31

 

<210> 13

<211> 35

<212> DNA

<213> artificial sequence

 

<400> 13

gccatcaggg cgtgatggtc ggcatgggac agaag 35

 

<210> 14

<211> 35

<212> DNA

<213> artificial sequence

 

<400> 14

ggcgacacgc agctcattgt agaaggtatg atgcc 35

 

<210> 15

<211> 32

<212> DNA

<213> artificial sequence

 

<400> 15

catcattcct gcctctactg gtgctgccaa ag 32

 

 

<210> 16

<211> 32

<212> DNA

<213> artificial sequence

 

<400> 16

gaccttttgc ttgatggctt catagcttgc ag 32

 

<210> 17

<211> 1737

<212> PRT

<213> artificial sequence

 

<400> 17

 

Met Gly Gln Thr Val Thr Thr Pro Leu Ser Leu Thr Leu Gly His Trp

1 5 10 15

 

Lys Asp Val Glu Arg Ile Ala His Asn Gln Ser Val Asp Val Lys Lys

20 25 30

 

Arg Arg Trp Val Thr Phe Cys Ser Ala Glu Trp Pro Thr Phe Asn Val

35 40 45

 

Gly Trp Pro Arg Asp Gly Thr Phe Asn Arg Asp Leu Ile Thr Gln Val

50 55 60

 

Lys Ile Lys Val Phe Ser Pro Gly Pro His Gly His Pro Asp Gln Val

65 70 75 80

 

Pro Tyr Ile Val Thr Trp Glu Ala Leu Ala Phe Asp Pro Pro Pro Trp

85 90 95

 

Val Lys Pro Phe Val His Pro Lys Pro Pro Pro Pro Leu Pro Pro Ser

100 105 110

 

Ala Pro Ser Leu Pro Leu Glu Pro Pro Arg Ser Thr Pro Pro Arg Ser

115 120 125

 

Ser Leu Tyr Pro Ala Leu Thr Pro Ser Leu Gly Ala Lys Pro Lys Pro

130 135 140

 

Gln Val Leu Ser Asp Ser Gly Gly Pro Leu Ile Asp Leu Leu Thr Glu

145 150 155 160

 

Asp Pro Pro Pro Tyr Arg Asp Pro Arg Pro Pro Pro Ser Asp Arg Asp

165 170 175

 

Gly Asn Gly Gly Glu Ala Thr Pro Ala Gly Glu Ala Pro Asp Pro Ser

180 185 190

 

Pro Met Ala Ser Arg Leu Arg Gly Arg Arg Glu Pro Pro Val Ala Asp

195 200 205

 

Ser Thr Thr Ser Gln Ala Phe Pro Leu Arg Ala Gly Gly Asn Gly Gln

210 215 220

 

Leu Gln Tyr Trp Pro Phe Ser Ser Ser Asp Leu Tyr Asn Trp Lys Asn

225 230 235 240

 

Asn Asn Pro Ser Phe Ser Glu Asp Pro Gly Lys Leu Thr Ala Leu Ile

245 250 255

 

Glu Ser Val Leu Ile Thr His Gln Pro Thr Trp Asp Asp Cys Gln Gln

260 265 270

 

Leu Leu Gly Thr Leu Leu Thr Gly Glu Glu Lys Gln Arg Val Leu Leu

275 280 285

 

Glu Ala Arg Lys Ala Val Arg Gly Asp Asp Gly Arg Pro Thr Gln Leu

290 295 300

 

Pro Asn Glu Val Asp Ala Ala Phe Pro Leu Glu Arg Pro Asp Trp Asp

305 310 315 320

 

Tyr Thr Thr Gln Ala Gly Arg Asn His Leu Val His Tyr Arg Gln Leu

325 330 335

 

Leu Leu Ala Gly Leu Gln Asn Ala Gly Arg Ser Pro Thr Asn Leu Ala

340 345 350

 

Lys Val Lys Gly Ile Thr Gln Gly Pro Asn Glu Ser Pro Ser Ala Phe

355 360 365

 

Leu Glu Arg Leu Lys Glu Ala Tyr Arg Arg Tyr Thr Pro Tyr Asp Pro

370 375 380

 

Glu Asp Pro Gly Gln Glu Thr Asn Val Ser Met Ser Phe Ile Trp Gln

385 390 395 400

 

Ser Ala Pro Asp Ile Gly Arg Lys Leu Glu Arg Leu Glu Asp Leu Lys

405 410 415

 

Asn Lys Thr Leu Gly Asp Leu Val Arg Glu Ala Glu Lys Ile Phe Asn

420 425 430

 

Lys Arg Glu Thr Pro Glu Glu Arg Glu Glu Arg Ile Arg Arg Glu Thr

435 440 445

 

Glu Glu Lys Glu Glu Arg Arg Arg Thr Glu Asp Glu Gln Lys Glu Lys

450 455 460

 

Glu Arg Asp Arg Arg Arg His Arg Glu Met Ser Lys Leu Leu Ala Thr

465 470 475 480

 

Val Val Ser Gly Gln Lys Gln Asp Arg Gln Gly Gly Glu Arg Arg Arg

485 490 495

 

Ser Gln Leu Asp Arg Asp Gln Cys Ala Tyr Cys Lys Glu Lys Gly His

500 505 510

 

Trp Ala Lys Asp Cys Pro Lys Lys Pro Arg Gly Pro Arg Gly Pro Arg

515 520 525

 

Pro Gln Thr Ser Leu Leu Thr Leu Asp Asp Gly Gly Gln Gly Gln Glu

530 535 540

 

Pro Pro Pro Glu Pro Arg Ile Thr Leu Lys Val Gly Gly Gln Pro Val

545 550 555 560

 

Thr Phe Leu Val Asp Thr Gly Ala Gln His Ser Val Leu Thr Gln Asn

565 570 575

 

Pro Gly Pro Leu Ser Asp Lys Ser Ala Trp Val Gln Gly Ala Thr Gly

580 585 590

 

Gly Lys Arg Tyr Arg Trp Thr Thr Asp Arg Lys Val His Leu Ala Thr

595 600 605

 

Gly Lys Val Thr His Ser Phe Leu His Val Pro Asp Cys Pro Tyr Pro

610 615 620

 

Leu Leu Gly Arg Asp Leu Leu Thr Lys Leu Lys Ala Gln Ile His Phe

625 630 635 640

 

Glu Gly Ser Gly Ala Gln Val Met Gly Pro Met Gly Gln Pro Leu Gln

645 650 655

 

Val Leu Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser

660 665 670

 

Lys Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro

675 680 685

 

Gln Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala

690 695 700

 

Pro Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys

705 710 715 720

 

Gln Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile

725 730 735

 

Gln Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp

740 745 750

 

Asn Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg

755 760 765

 

Pro Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His

770 775 780

 

Pro Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser

785 790 795 800

 

His Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu

805 810 815

 

Arg Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp

820 825 830

 

Pro Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln

835 840 845

 

Gly Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp

850 855 860

 

Leu Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr

865 870 875 880

 

Val Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln

885 890 895

 

Gly Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala

900 905 910

 

Ser Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly

915 920 925

 

Tyr Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu

930 935 940

 

Thr Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu

945 950 955 960

 

Phe Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala

965 970 975

 

Glu Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe

980 985 990

 

Asn Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala

995 1000 1005

 

Leu Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro

1010 1015 1020

 

Phe Glu Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val

1025 1030 1035

 

Leu Thr Gln Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu

1040 1045 1050

 

Ser Lys Lys Leu Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu

1055 1060 1065

 

Arg Met Val Ala Ala Ile Ala Val Leu Thr Lys Asp Ala Gly Lys

1070 1075 1080

 

Leu Thr Met Gly Gln Pro Leu Val Ile Leu Ala Pro His Ala Val

1085 1090 1095

 

Glu Ala Leu Val Lys Gln Pro Pro Asp Arg Trp Leu Ser Asn Ala

1100 1105 1110

 

Arg Met Thr His Tyr Gln Ala Leu Leu Leu Asp Thr Asp Arg Val

1115 1120 1125

 

Gln Phe Gly Pro Val Val Ala Leu Asn Pro Ala Thr Leu Leu Pro

1130 1135 1140

 

Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu Asp Ile Leu Ala

1145 1150 1155

 

Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln Pro Leu Pro

1160 1165 1170

 

Asp Ala Asp His Thr Trp Tyr Thr Asp Gly Ser Ser Leu Leu Gln

1175 1180 1185

 

Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr Glu

1190 1195 1200

 

Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg

1205 1210 1215

 

Ala Glu Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly

1220 1225 1230

 

Lys Lys Leu Asn Val Tyr Thr Asp Ser Arg Tyr Ala Phe Ala Thr

1235 1240 1245

 

Ala His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr

1250 1255 1260

 

Ser Glu Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu

1265 1270 1275

 

Leu Lys Ala Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys

1280 1285 1290

 

Pro Gly His Gln Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg

1295 1300 1305

 

Met Ala Asp Gln Ala Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro

1310 1315 1320

 

Asp Thr Ser Thr Leu Leu Ile Glu Asn Ser Ser Pro Tyr Thr Ser

1325 1330 1335

 

Glu His Phe His Tyr Thr Val Thr Asp Ile Lys Asp Leu Thr Lys

1340 1345 1350

 

Leu Gly Ala Ile Tyr Asp Lys Thr Lys Lys Tyr Trp Val Tyr Gln

1355 1360 1365

 

Gly Lys Pro Val Met Pro Asp Gln Phe Thr Phe Glu Leu Leu Asp

1370 1375 1380

 

Phe Leu His Gln Leu Thr His Leu Ser Phe Ser Lys Met Lys Ala

1385 1390 1395

 

Leu Leu Glu Arg Ser His Ser Pro Tyr Tyr Met Leu Asn Arg Asp

1400 1405 1410

 

Arg Thr Leu Lys Asn Ile Thr Glu Thr Cys Lys Ala Cys Ala Gln

1415 1420 1425

 

Val Asn Ala Ser Lys Ser Ala Val Lys Gln Gly Thr Arg Val Arg

1430 1435 1440

 

Gly His Arg Pro Gly Thr His Trp Glu Ile Asp Phe Thr Glu Ile

1445 1450 1455

 

Lys Pro Gly Leu Tyr Gly Tyr Lys Tyr Leu Leu Val Phe Ile Asp

1460 1465 1470

 

Thr Phe Ser Gly Trp Ile Glu Ala Phe Pro Thr Lys Lys Glu Thr

1475 1480 1485

 

Ala Lys Val Val Thr Lys Lys Leu Leu Glu Glu Ile Phe Pro Arg

1490 1495 1500

 

Phe Gly Met Pro Gln Val Leu Gly Thr Asp Asn Gly Pro Ala Phe

1505 1510 1515

 

al Ser Lys Val Ser Gln Thr Val Ala Asp Leu Leu Gly Ile Asp

1520 1525 1530

 

Trp Lys Leu His Cys Ala Tyr Arg Pro Gln Ser Ser Gly Gln Val

1535 1540 1545

 

Glu Arg Met Asn Arg Thr Ile Lys Glu Thr Leu Thr Lys Leu Thr

1550 1555 1560

 

Leu Ala Thr Gly Ser Arg Asp Trp Val Leu Leu Leu Pro Leu Ala

1565 1570 1575

 

Leu Tyr Arg Ala Arg Asn Thr Pro Gly Pro His Gly Leu Thr Pro

1580 1585 1590

 

Tyr Glu Ile Leu Tyr Gly Ala Pro Pro Pro Leu Val Asn Phe Pro

1595 1600 1605

 

Asp Pro Asp Met Thr Arg Val Thr Asn Ser Pro Ser Leu Gln Ala

1610 1615 1620

 

His Leu Gln Ala Leu Tyr Leu Val Gln His Glu Val Trp Arg Pro

1625 1630 1635

 

Leu Ala Ala Ala Tyr Gln Glu Gln Leu Asp Arg Pro Val Val Pro

1640 1645 1650

 

His Pro Tyr Arg Val Gly Asp Thr Val Trp Val Arg Arg His Gln

1655 1660 1665

 

Thr Lys Asn Leu Glu Pro Arg Trp Lys Gly Pro Tyr Thr Val Leu

1670 1675 1680

 

Leu Thr Thr Pro Thr Ala Leu Lys Val Asp Gly Ile Ala Ala Trp

1685 1690 1695

 

Ile His Ala Ala His Val Lys Ala Ala Asp Pro Gly Gly Gly Pro

1700 1705 1710

 

Ser Ser Arg Leu Thr Trp Arg Val Gln Arg Ser Gln Asn Pro Leu

1715 1720 1725

 

Lys Ile Arg Leu Thr Arg Glu Ala Pro

1730 1735

 

<210> 18

<211> 393

<212> PRT

<213> artificial sequence

 

<400> 18

 

Met Ser Ile Ala Asp Leu Lys Ser Arg Leu Ile Lys Ala Ser Thr Ser

1 5 10 15

 

Lys Met Thr Ala Glu Leu Thr Thr Ser Lys Phe Phe Asn Glu Lys Asp

20 25 30

 

Val Ile Arg Thr Lys Ile Pro Met Leu Asn Ile Ala Ile Ser Gly Ala

35 40 45

 

Ile Asp Gly Gly Met Gln Ser Gly Leu Thr Ile Phe Ala Gly Pro Ser

50 55 60

 

Lys His Phe Lys Ser Asn Met Ser Leu Thr Met Val Ala Ala Tyr Leu

65 70 75 80

 

Asn Lys Tyr Pro Asp Ala Val Cys Leu Phe Tyr Asp Ser Glu Phe Gly

85 90 95

 

Ile Thr Pro Ala Tyr Leu Arg Ser Met Gly Val Asp Pro Glu Arg Val

100 105 110

 

Ile His Thr Pro Ile Gln Ser Val Glu Gln Leu Lys Ile Asp Met Val

115 120 125

 

Asn Gln Leu Glu Ala Ile Glu Arg Gly Glu Lys Val Ile Val Phe Ile

130 135 140

 

Asp Ser Ile Gly Asn Met Ala Ser Lys Lys Glu Thr Glu Asp Ala Leu

145 150 155 160

 

Asn Glu Lys Ser Val Ala Asp Met Thr Arg Ala Lys Ser Leu Lys Ser

165 170 175

 

Leu Phe Arg Ile Val Thr Pro Tyr Phe Ser Leu Lys Asn Ile Pro Cys

180 185 190

 

Val Ala Val Asn His Thr Ile Glu Thr Ile Glu Met Phe Ser Lys Thr

195 200 205

 

Val Met Thr Gly Gly Thr Gly Val Met Tyr Ser Ala Asp Thr Val Phe

210 215 220

 

Ile Ile Gly Lys Arg Gln Ile Lys Asp Gly Ser Asp Leu Gln Gly Tyr

225 230 235 240

 

Gln Phe Val Leu Asn Val Glu Lys Ser Arg Thr Val Lys Glu Lys Ser

245 250 255

 

Lys Phe Phe Ile Asp Val Lys Phe Asp Gly Gly Ile Asp Pro Tyr Ser

260 265 270

 

Gly Leu Leu Asp Met Ala Leu Glu Leu Gly Phe Val Val Lys Pro Lys

275 280 285

 

Asn Gly Trp Tyr Ala Arg Glu Phe Leu Asp Glu Glu Thr Gly Glu Met

290 295 300

 

Ile Arg Glu Glu Lys Ser Trp Arg Ala Lys Asp Thr Asn Cys Thr Thr

305 310 315 320

 

Phe Trp Gly Pro Leu Phe Lys His Gln Pro Phe Arg Asp Ala Ile Lys

325 330 335

 

Arg Ala Tyr Gln Leu Gly Ala Ile Asp Ser Asn Glu Ile Val Glu Ala

340 345 350

 

Glu Val Asp Glu Leu Ile Asn Ser Lys Val Glu Lys Phe Lys Ser Pro

355 360 365

 

Glu Ser Lys Ser Lys Ser Ala Ala Asp Leu Glu Thr Asp Leu Glu Gln

370 375 380

 

Leu Ser Asp Met Glu Glu Phe Asn Glu

385 390

 

<210> 19

<211> 298

<212> PRT

<213> artificial sequence

 

<400> 19

 

Met Phe Lys Arg Lys Ser Thr Ala Asp Leu Ala Ala Gln Met Ala Lys

1 5 10 15

 

Leu Asn Gly Asn Lys Gly Phe Ser Ser Glu Asp Lys Gly Glu Trp Lys

20 25 30

 

Leu Lys Leu Asp Ala Ser Gly Asn Gly Gln Ala Val Ile Arg Phe Leu

35 40 45

 

Pro Ala Lys Thr Asp Asp Ala Leu Pro Phe Ala Ile Leu Val Asn His

50 55 60

 

Gly Phe Lys Lys Asn Gly Lys Trp Tyr Ile Glu Thr Cys Ser Ser Thr

65 70 75 80

 

His Gly Asp Tyr Asp Ser Cys Pro Val Cys Gln Tyr Ile Ser Lys Asn

85 90 95

 

Asp Leu Tyr Asn Thr Asn Lys Thr Glu Tyr Ser Gln Leu Lys Arg Lys

100 105 110

 

Thr Ser Tyr Trp Ala Asn Ile Leu Val Val Lys Asp Pro Gln Ala Pro

115 120 125

 

Asp Asn Glu Gly Lys Val Phe Lys Tyr Arg Phe Gly Lys Lys Ile Trp

130 135 140

 

Asp Lys Ile Asn Ala Met Ile Ala Val Asp Thr Glu Met Gly Glu Thr

145 150 155 160

 

Pro Val Asp Val Thr Cys Pro Trp Glu Gly Ala Asn Phe Val Leu Lys

165 170 175

 

Val Lys Gln Val Ser Gly Phe Ser Asn Tyr Asp Glu Ser Lys Phe Leu

180 185 190

 

Asn Gln Ser Ala Ile Pro Asn Ile Asp Asp Glu Ser Phe Gln Lys Glu

195 200 205

 

Leu Phe Glu Gln Met Val Asp Leu Ser Glu Met Thr Ser Lys Asp Lys

210 215 220

 

Phe Lys Ser Phe Glu Glu Leu Asn Thr Lys Phe Asn Gln Val Leu Gly

225 230 235 240

 

Thr Ala Ala Leu Gly Gly Ala Ala Ala Ala Ala Ala Ser Val Ala Asp

245 250 255

 

Lys Val Ala Ser Asp Leu Asp Asp Phe Asp Lys Asp Met Glu Ala Ser

260 265 270

 

Ser Ala Lys Thr Glu Asp Asp Phe Met Ser Ser Ser Ser Ser Asp Asp

275 280 285

 

Gly Asp Leu Asp Asp Leu Leu Ala Gly Leu

290 295

 

<210> 20

<211> 876

<212> PRT

<213> artificial sequence

 

<400> 20

 

Val Asn Lys Leu Val Leu Ile Asp Gly Asn Ser Leu Ser Phe Arg Ala

1 5 10 15

 

Phe Tyr Ala Leu Pro Leu Leu Ser Asn Lys Ala Gly Ile His Thr Asn

20 25 30

 

Ala Val Tyr Gly Phe Ala Met Leu Leu Glu Lys Ile Leu Lys Glu Glu

35 40 45

 

Lys Pro Asn His Phe Leu Val Ala Phe Asp Ala Gly Lys Thr Thr Phe

50 55 60

 

Arg His Glu Lys Tyr Ser Glu Tyr Lys Gly Gly Arg Gln Lys Thr Pro

65 70 75 80

 

Pro Glu Leu Ser Glu Gln Phe Pro Tyr Ile Arg Gln Leu Leu Asp Ala

85 90 95

 

Tyr His Ile Lys Arg Tyr Glu Leu Asp Asn Tyr Glu Ala Asp Asp Ile

100 105 110

 

Ile Gly Thr Leu Ser Lys Glu Ala Asp Lys Ala Gly Phe Gln Thr Ile

115 120 125

 

Ile Ile Thr Gly Asp Arg Asp Leu Thr Gln Leu Ala Thr Asp Asn Val

130 135 140

 

Thr Ile Tyr Tyr Thr Lys Lys Gly Val Thr Asp Val Asp His Tyr Thr

145 150 155 160

 

Pro Asp Phe Ile Ala Glu Lys Tyr Asn Gly Lys Thr Pro Asn Gln Ile

165 170 175

 

Ile Asp Met Lys Gly Leu Met Gly Asp Thr Ser Asp Asn Ile Pro Gly

180 185 190

 

Val Ala Gly Val Gly Glu Lys Thr Ala Ile Lys Leu Leu Asn Gln Phe

195 200 205

 

Asp Thr Val Glu Gly Val Tyr Glu His Leu Asp Glu Ile Ser Gly Lys

210 215 220

 

Lys Leu Lys Glu Lys Leu Gln Asn Ser Lys Glu Asp Ala Leu Met Ser

225 230 235 240

 

Lys Glu Leu Ala Thr Ile Asn Val Asp Ser Pro Ile Glu Val Lys Leu

245 250 255

 

Glu Asp Thr Leu Met Thr His Gln Asp Glu Gln Gln Glu Lys Ile Glu

260 265 270

 

 

Leu Phe Lys Lys Leu Glu Phe Lys Gln Leu Leu Ala Asp Ile Asp Gln

275 280 285

 

Ser Ala Ser Val Glu Asp Ala Ile Glu Lys Thr Phe Glu Ile Glu Thr

290 295 300

 

Ser Phe Asp Asn Ile Asp Phe Thr Ser Leu Lys Glu Ala Ala Ile His

305 310 315 320

 

Phe Glu Leu Asp Gly Gly Asn Tyr Leu Arg Asn Asn Ile Leu Lys Phe

325 330 335

 

Ser Leu Phe Thr Gly Glu Lys His Ile Val Ile Asn Ala Asp Asp Ile

340 345 350

 

Asn Asn Tyr Val Glu Leu Val Ser Trp Leu Glu Asn Pro Asn Ser Lys

355 360 365

 

Lys Val Val Tyr Asp Ala Lys Lys Thr Tyr Val Ala Ser His Arg Leu

370 375 380

 

Gly Ile Asp Ile Gln Asn Ile Ser Phe Asp Ile Met Leu Ala Ser Tyr

385 390 395 400

 

Ile Ile Asp Pro Ser Arg Thr Ile Ser Asp Val Gln Ser Val Val Ser

405 410 415

 

Leu Tyr Gly Gln Ser Phe Val Lys Asp Asp Val Ser Ile Tyr Gly Lys

420 425 430

 

Gly Lys Lys Phe Lys Val Pro Glu Asp Asp Val Leu Asn Pro Tyr Val

435 440 445

 

Ala Ser Ile Thr Asp Ala Ile Tyr Phe Ala Lys Pro Asn Met Asp Lys

450 455 460

 

Gln Leu Glu Glu Tyr Asn Gln Val Glu Leu Leu Ala Asp Leu Glu Leu

465 470 475 480

 

Pro Leu Ala Lys Ile Leu Ser Glu Met Glu Glu Ile Gly Ile Phe Thr

485 490 495

 

Asp Val His Asp Leu Glu Glu Met Glu Lys Glu Ile Gln Glu Lys Leu

500 505 510

 

Asp Val Leu Ile Arg Asn Ile His Asp Ala Ala Gly Glu Asp Phe Asn

515 520 525

 

Ile Asn Ser Pro Lys Gln Leu Gly Val Val Leu Phe Glu Thr Leu Gln

530 535 540

 

Leu Pro Val Ile Lys Lys Thr Lys Thr Gly Tyr Ser Thr Ala Val Asp

545 550 555 560

 

Val Leu Glu Gln Leu Gln Gly Glu His Pro Ile Ile Asp Tyr Ile Leu

565 570 575

 

Glu Tyr Arg Gln Leu Ser Lys Leu Gln Ser Thr Tyr Val Glu Gly Leu

580 585 590

 

Gln Lys Val Ile Ser Asp Asp Gln Arg Ile His Thr Arg Phe Asn Gln

595 600 605

 

Thr Leu Ala Gln Thr Gly Arg Leu Ser Ser Val Asp Pro Asn Leu Gln

610 615 620

 

Asn Ile Pro Val Arg Leu Glu Glu Gly Arg Lys Ile Arg Lys Ala Phe

625 630 635 640

 

Lys Pro Thr Ser Lys Asp Ser Val Ile Leu Ser Ala Asp Tyr Ser Gln

645 650 655

 

Ile Glu Leu Arg Val Leu Ala His Ile Thr Gln Asp Glu Ser Met Lys

660 665 670

 

Glu Ala Phe Ile Asn Gly Asp Asp Ile His Thr Ala Thr Ala Met Lys

675 680 685

 

Val Phe Gly Val Glu Ala Asp Gln Val Asp Ser Leu Met Arg Arg Gln

690 695 700

 

Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly Ile Ser Asp Tyr Gly

705 710 715 720

 

Leu Ser Gln Ser Leu Gly Ile Thr Arg Lys Lys Ala Lys Ala Phe Ile

725 730 735

 

Asp Asp Tyr Leu Ala Ser Phe Pro Gly Val Lys Gln Tyr Met Ser Asp

740 745 750

 

Ile Val Lys Asp Ala Lys Ala Leu Gly Tyr Val Glu Thr Leu Leu His

755 760 765

 

Arg Arg Arg Tyr Ile Pro Asp Ile Thr Ser Arg Asn Phe Asn Leu Arg

770 775 780

 

Gly Phe Ala Glu Arg Thr Ala Met Asn Thr Pro Ile Gln Gly Ser Ala

785 790 795 800

 

Ala Asp Ile Ile Lys Leu Ala Met Val Lys Phe Ala Gln Lys Met Lys

805 810 815

 

Glu Thr Thr Tyr Gln Ala Lys Leu Leu Leu Gln Val His Asp Glu Leu

820 825 830

 

Ile Phe Glu Val Pro Lys Ser Glu Val Asp Ser Phe Ser Glu Phe Val

835 840 845

 

Glu Glu Ile Met Glu Asn Ala Leu Gln Leu Asp Val Pro Leu Lys Val

850 855 860

 

Asp Ser Ser Tyr Gly Ala Thr Trp Tyr Asp Ala Lys

865 870 875

 

<210> 21

<211> 140

<212> PRT

<213> artificial sequence

 

<400> 21

 

Met Arg Leu Glu Asp Leu Gln Glu Glu Leu Lys Lys Asp Val Phe Ile

1 5 10 15

 

Asp Ser Thr Lys Leu Gln Tyr Glu Ala Ala Asn Asn Val Met Leu Tyr

20 25 30

 

Ser Lys Trp Leu Asn Lys His Ser Ser Ile Lys Lys Glu Met Leu Arg

35 40 45

 

Ile Glu Ala Gln Lys Lys Val Ala Leu Lys Ala Arg Leu Asp Tyr Tyr

50 55 60

 

Ser Gly Arg Gly Asp Gly Asp Glu Phe Ser Met Asp Arg Tyr Glu Lys

65 70 75 80

 

Ser Glu Met Lys Thr Val Leu Ser Ala Asp Lys Asp Val Leu Lys Val

85 90 95

 

Asp Thr Ser Leu Gln Tyr Trp Gly Ile Leu Leu Asp Phe Cys Ser Glu

100 105 110

 

Leu Leu Met Leu Leu Asn His Val Asp Leu Leu Leu Ser Ile Phe Lys

115 120 125

 

Thr Cys Glu His Leu Arg Leu Gln Asn Asn Glu Ile

130 135 140

Claims (7)

1. for the proteolytic enzyme of normal temperature isothermal rapid detection Yeast Nucleic Acid, it is characterized in that, described proteolytic enzyme comprises: (1) reversed transcriptive enzyme; (2) recombinase; (3) single strand binding protein; (4) archaeal dna polymerase; (5) accessory protein; The aminoacid sequence of described reversed transcriptive enzyme is as shown in SEQ ID No.17; The aminoacid sequence of recombinase is as shown in SEQ ID No.18; The aminoacid sequence of single strand binding protein is as shown in SEQ ID No.19; The aminoacid sequence of archaeal dna polymerase is as shown in SEQ ID No.20; The aminoacid sequence of accessory protein is as shown in SEQ ID No.21.

2. proteolytic enzyme as claimed in claim 1, it is characterized in that, in amplification system, reverse transcriptase concentrations scope is 8-12 unit/ul; Recombinase concentration range is 100-150ng/ul; Single strand binding protein concentration range is 700-1000ng/ul; Archaeal dna polymerase concentration range is 60-90ng/ul; Accessory protein concentration range is 20-40ng/ul.

3. for the reagent of normal temperature isothermal rapid detection Yeast Nucleic Acid, it is characterized in that, described reagent mainly comprises Tris, RNase inhibitor, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT), amplimer and proteolytic enzyme according to claim 1.

4. reagent as claimed in claim 3, it is characterized in that, described reagent comprises: 5%PEG35000,6% trehalose, the recombinase of 115ng/ul, the accessory protein of 35ng/ul, the single strand binding protein of 830ng/ul, the Bsu polysaccharase of 78ng/ul, the reversed transcriptive enzyme of 12 units/ul, the RNase inhibitor of 0.15 unit/ul, the ATP of 3.5mM, the dNTPs of 490uM, the Tris damping fluid of 55mM, the dithiothreitol (DTT) of 12mM, the disodium creatine phosphate of 12mM, the creatine phosphokinase of 114ng/ul, the potassium acetate of 75mM and 7% N.F,USP MANNITOL; The amplimer of 350-450nM; Cumulative volume is 50ul.

5. reagent as claimed in claim 4, is characterized in that, described reagent, through lyophilize process, removes liquid phase, retains solid matter, makes white dry powder.

6. a method for normal temperature isothermal rapid detection Yeast Nucleic Acid, is characterized in that, after detection reagent adds template, being placed in a temperature control device, to maintain incubation temperature be 40-42 DEG C, hatches 20-40 minute, hatch in process and keep homogeneous steady temperature; Visual retrieval is carried out again by agarose gel electrophoresis; Described detection reagent mainly comprises Tris, RNase inhibitor, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT), amplimer and proteolytic enzyme according to claim 1.

7. amplification method as claimed in claim 6, described incubation temperature is 40 DEG C.