CN104818263A

CN104818263A - Protease used for normal- and constant-temperature nucleic acid large fragment in-vitro amplification and amplification method

Info

Publication number: CN104818263A
Application number: CN201510231110.3A
Authority: CN
Inventors: 程奇; 周国辉; 李贤祯
Original assignee: ZHEJIANG TAIJING BIOTECHNOLOGY CO Ltd
Current assignee: Zhejiang Shan Shi Wo Knight Biotechnology Co., Ltd.
Priority date: 2015-05-07
Filing date: 2015-05-07
Publication date: 2015-08-05

Abstract

The invention discloses a protease used for normal- and constant-temperature nucleic acid large fragment in-vitro amplification, which includes: (1) a recombinase (Seq ID No.35); (2) a single-chain binding protein (Seq ID No.36); (3) a DNA polymerase (Seq ID No.37); and (4) an accessory protein (Seq ID No.38). The invention also discloses a reagent used for the normal-temperature isothermal nucleic acid large fragment in-vitro amplification and an amplification method. The amplification method is carried out, through a synergistic effect between various engineering enzymes and related chemical components and under a normal and constant temperature, to quickly complete specific amplification of a nucleic acid large fragment in vitro. The method, in the future, can be widely used in the fields of gene sequencing, cyclic plasmid amplification and whole genome amplification, and has a huge application prospect in the fields of simplifying molecular operation processes and promoting researching progress.

Description

For proteolytic enzyme and the amplification method of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature

Technical field

The present invention is a new opplication of nucleic acid isothermal amplification technology, relate to the mutual synergy utilizing multiple engineering enzyme and corresponding chemical ancillary component, at normal temperature and under steady temperature, external rapid amplifying is realized to long segment nucleic acid, thus make the method can be widely used in order-checking, cyclic plasmid amplification from now on, even in the middle of the field such as whole genome amplification.

Background technology

After auto-polymerization polymerase chain reaction (PCR) achieves external nucleic acid amplification, scientists is devoted to obtain longer nucleic acid fragment in vitro always, expect to carry out more deep research to the encoding gene with complete function, such as protein expression research, structural research etc.Or obtain the whole genome sequence of certain specific species, more how useful information can be decoded, thus promote developing fast and deeply of each field of biology.Because PCR opens DNA double chain based on high-temperature denatured principle thus obtains single-stranded template, but Taq archaeal dna polymerase optimal reactive temperature is wherein between 60-80 degree, high temperature (>90 degree) can make its DNA composite reactive lose, and (such as 95 degree of incubations are after 40 minutes, only keep the activity of 50%), when therefore carrying out nucleic acid in vitro amplification under varying temperature environment, PCR success obtains complete lengthy motion picture and breaks, and depends on the catalytic activity of archaeal dna polymerase, processivity, check and correction ability to a great extent and extends the factors such as efficiency.Based on this, carry out genetic engineering modified to several mode below the many employings of Taq archaeal dna polymerase at present, such as hot start Taq polymerase, by suppressing active under its low temperature, thus avoids at the initial a small amount of non-specific amplification produced of reaction the impact of succeeding target sequence amplification; Or by obtaining high heat-resisting Taq enzyme, strengthening its at high temperature transformation period, thus improving its catalytic activity; Or the archaeal dna polymerase mixing several Various Functions reacts the ability in the amplification of overlength segment to improve PCR.

Although by above several strategy, PCR also obtains good progress in lengthy motion picture breaks amplification, and the reaction system optimization process of complexity still makes scientific effort waste time and energy.The nucleic acid isothermal amplification technology occurred in recent years is that external nucleic acid amplification indicates a new outlet, they are no longer only confined to use Taq archaeal dna polymerase, but start to adopt different enzymes, such as there is the Bst archaeal dna polymerase of strand-displacement activity, or the totally different enzyme of multiple function combines mutually, thus go the effective amplification realizing nucleic acid under isothermal conditions.But on the one hand due to the difference of respective reaction principle, on the other hand owing to being main research purpose mainly with field quick detection, the nucleic acid isothermal amplification technology of current maturation is still main mainly with the short and small nucleic acid fragment of amplification.Certainly wherein also exception is had, such as rolling circle amplification (RCA) once utilized the random primer of phi29DNA polysaccharase and many nuclease resistant, going out the DNA of average product length more than 10kb from being less than accurate amplification 10 human cells, achieving normal temperature complete genome DNA to a certain extent and detecting.But because using random primer, so the product obtained is the template tandem sequence repeats double-stranded DNA that length differs.In addition, rely on and untwist enzyme dna isothermal amplification technique (HDA) after technological improvement, utilize T7 phage replication system to establish a kind of method that simultaneously can realize amplification complete plasmid and long target segment, cHDA method.Although the method can complete the insertion sequence amplification reaching 10kb under room temperature (25 degree), but the required reaction times longer (being generally 6 hours), and cHDA can only increase cyclic DNA, can not increase linear DNA, thus makes its application surface be subject to certain limitation.

Current, mainly rely on round pcr to the amplification of the long target segment of length-specific, early stage, system optimization process added a lot of workload to scientific effort for a long time.In addition, the acquisition of recombinant plasmid always needs, through the loaded down with trivial details step such as conversion, cultivation and extraction, to realize a large amount of accumulation of plasmid in vitro; If realize copying of recombinant plasmid in a faster way will save a large amount of working hours, and simplify existing experiment flow.

Summary of the invention

The present invention aim to provide a kind of can in vitro, under normal temperature isothermal condition, realize lengthy motion picture fast to break the proteolytic enzyme of nucleic acid amplification and new amplification method.

First object of the present invention is to provide the proteolytic enzyme for isothermal nucleic acid large fragment amplification in vitros such as normal temperature.

For the proteolytic enzyme of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature, it is characterized in that, described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; The aminoacid sequence of described recombinase is as shown in SEQ IDNo.35; The aminoacid sequence of single strand binding protein is as shown in SEQ ID No.36; The aminoacid sequence of archaeal dna polymerase is as shown in SEQ ID No.37; The aminoacid sequence of accessory protein is as shown in SEQ ID No.38.

Further, in amplification system, recombinase concentration range is 0.5 ~ 0.8mg/ml; Single strand binding protein concentration range is 0.9 ~ 1.2mg/ml; Archaeal dna polymerase concentration range is 0.3 ~ 0.6mg/ml; Accessory protein concentration range is 0.1 ~ 0.3mg/ml.

Described proteolytic enzyme also comprises: (5) topoisomerase or helicase; The aminoacid sequence of topoisomerase is as SEQ ID No.39; The aminoacid sequence of helicase is as shown in SEQ ID No.40.

In amplification system, topoisomerase concentration range is 0.1 ~ 0.5U/ul; Helicase concentration range is 0.1 ~ 0.5U/ul.

Normal temperature isothermal of the present invention, refer to and to hatch under a certain steady temperature, temperature is lower is 37 DEG C, close to normal temperature, and is homogeneous constant temp.

Above-mentioned proteolytic enzyme (engineering enzyme) is multiple through genetic modification, and there is the proteolytic enzyme of high purity and high vigor, having following characteristics: the proteolytic enzyme 1) comprising 4 or 5 kind of Various Functions, is recombinase, single strand binding protein, archaeal dna polymerase, accessory protein and topoisomerase or helicase respectively; 2) all proteolytic enzyme all derives from nature, after to produce through the step such as restructuring, fermentation, cytoclasis, purifying and enzyme activity determination and obtain; 3) recombinase, be responsible for forming initiation complex with primer, the optimal concentration scope in system is 0.5 ~ 0.8mg/ml; 4) single strand binding protein, is responsible for the strand in conjunction with DNA profiling, helps to form Bubble Region, local, so that initiation complex carries out the scanning of matching area in the strand district opened, the optimal concentration scope in system is 0.9 ~ 1.2mg/ml; 5) archaeal dna polymerase, because having strand-displacement activity, therefore except the 3 ' end be responsible for from primer extends and synthesizes new subchain in amplification procedure, also responsiblely substitute a fundamental chain wherein by new subchain, form double-strand with another fundamental chain, the optimal concentration scope in system is 0.3 ~ 0.6mg/ml; 6) accessory protein, help initiation complex affine with DNA single chain better with suitable bonding force, can realize scanning, also well can depart from finding matching area relief recombinase and primer, the optimal concentration scope in system is 0.1 ~ 0.3mg/ml; 7) topoisomerase, help to open DNA superhelix in amplification procedure, in system, belong to optional addO-on therapy, its optimal concentration scope is 0.1 ~ 0.5U/ul; 8) helicase, can assist single strand binding protein to open DNA double chain better, is also optional addO-on therapy.

Second object of the present invention is to provide the reagent for isothermal nucleic acid large fragment amplification in vitros such as normal temperature.

For the reagent of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature, it is characterized in that, described reagent mainly comprises Tris alkali, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT) and proteolytic enzyme, and described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; Preferably also comprise (5) topoisomerase or helicase.

Described reagent is lyophilized powder, the concentration of each component in lyophilized powder is: Tris alkali 20 ~ 30mM, dNTP 200 ~ 250uM, ATP 2 ~ 3mM, disodium creatine phosphate 45 ~ 55mM, creatine phosphokinase 90 ~ 110ng/ μ l, potassium acetate 90 ~ 110mM, trehalose 5.5 ~ 6.5%, N.F,USP MANNITOL 6 ~ 10%, polyoxyethylene glycol 2 ~ 3%, dithiothreitol (DTT) 4 ~ 6mM, recombinase 0.5 ~ 0.8mg/ml, single strand binding protein 0.9 ~ 1.2mg/ml, archaeal dna polymerase 0.3 ~ 0.6mg/ml, accessory protein 0.1 ~ 0.3mg/ml, topoisomerase 0.1 ~ 0.5U/ul, helicase 0.1 ~ 0.5U/ul, described lyophilized powder is made up of following methods: now by reagent lyophilize 4-10 hour at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.

For the various chemical compositions in the isothermal nucleic acid large fragment amplification in vitro reagent such as normal temperature, a series of for reacting the chemical composition providing the suitableeest buffer environment, there is following characteristics: 1) function of each component in system is different, or provide reaction desired raw material, such as deoxyribonucleoside triphosphate (dNTP); Or provide energy, such as Triphosaden (ATP), disodium creatine phosphate (PCr) and creatine phosphokinase (CK) for reacting; Or provide appropriate salt concentration, such as potassium acetate (KAc); Or composition activity is not lost in guarantee freezing dry process, such as trehalose (Trehalose), N.F,USP MANNITOL (Mannitol) and polyoxyethylene glycol (PEG); 2) optimal concentration of each reactive component in freeze-drying system respectively: PEG 2 ~ 3%, Tris alkali 20 ~ 30mM, KAc90 ~ 110mM, DTT (dithiothreitol (DTT)) 4 ~ 6mM, ATP 2 ~ 3mM, PCr 45 ~ 55mM, CK 90 ~ 110ng/ μ l, dNTP 200 ~ 250uM, trehalose 5.5 ~ 6.5%, N.F,USP MANNITOL 6 ~ 10%.

Proliferation time freezing dry process, is a kind of flow process of reaction system being carried out low temperature drying and other treatment, has following characteristics: 1) comprise dry and dry two steps again of trunk, trunk is dry is-30 ~-35 DEG C, 4 ~ 10 hours, then drying is 0 ~ 20 DEG C, 30 minutes ~ 1 hour; 2) actual temperature often walked and duration depend on the sample total amount of each freeze-drying; 3) reaction system after process can carry out experimental implementation at normal temperatures, can not cause losing or reducing of any detection effect; 4) response sample 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; 5) after freeze-drying, the detection vigor of reaction system can maintain half a year.

3rd object of the present invention is to provide the isothermal nucleic acid large fragment amplification in vitro method such as a kind of normal temperature.

The isothermal nucleic acid large fragment amplification in vitro method such as a kind of normal temperature, it is characterized in that, described amplification method is after adding template and amplimer with described amplifing reagent, is placed in a temperature control device and hatches 30 minutes to 4 hours, then carry out Visual retrieval by agarose gel electrophoresis; The temperature of described normal temperature is 37 DEG C, keeps homogeneous steady temperature in amplification procedure.Described reagent mainly comprises Tris alkali, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT) and proteolytic enzyme, and described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; Preferably also comprise (5) topoisomerase or helicase.

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) the present invention utilizes the mutual synergy of multiple engineering enzyme and corresponding chemical ancillary component can realize specific amplification under normal temperature isothermal condition: can under normal temperature isothermal condition (such as 37 DEG C), by amplification and agarose gel electrophoresis after, can the specific location of glue figure observe one special, become clear band.Its light levels is enough to distinguish with other non-specific amplification band.

2) simple fast: break for below 3kb lengthy motion picture, incubation is enough to for 40 minutes; Amplification is greater than the nucleic acid fragment of 3kb, needs to be advisable by 4 hours for 40 minutes.

3) hi-fi: owing to being the situation at utmost simulating the amplification of biological nucleic acid in vivo, the mispairing rate in amplification procedure is very low, should be less than 1/10000kb.

4) difficult expansion sample is applicable to: for difficult extension increasing sequence, the such as sample of high GC content or high AT content, amplification method of the present invention has some superiority.

5) amplification method of the present invention under steady temperature, realizes external rapid amplifying to long segment nucleic acid at normal temperature, can be widely used in order-checking, cyclic plasmid amplification, even in the middle of the field such as whole genome amplification.

Accompanying drawing explanation

Fig. 1 is 1 ~ 0-1.6kb nucleic acid fragment amplification figure.In figure, swimming lane 1 product size is 1132bp, and swimming lane 2 product size is 1246bp, and swimming lane 3 product size is 1346bp, and swimming lane 4 product size is 1446bp, and swimming lane 5 product size is 1550bp, and swimming lane 6 is positive controls, and its size is 1000bp, M is DNA marker thing.

Fig. 2 is 1.6 ~ 2.0kb nucleic acid fragment amplification figure.In figure, swimming lane 1 product size is 1636bp, and swimming lane 2 product size is 1739bp, and swimming lane 3 product size is 1823bp, and swimming lane 4 product size is 1950bp, and swimming lane 5 product size is 2020bp, and swimming lane 6 is positive controls, and its size is 1550bp, M is DNA marker thing.

Fig. 3 is 2.0 ~ 3.4kb nucleic acid fragment amplification figure.In figure, swimming lane 1 product size is 2134bp, swimming lane 2 product size is 2220bp, swimming lane 3 product size is 2350bp, swimming lane 4 product size is 2435bp, swimming lane 5 product size is 2548bp, swimming lane 6 product size is 2505bp, and swimming lane 7 product size is 2761bp, and swimming lane 8 product size is 3053bp, swimming lane 9 product size is 3364bp, swimming lane 10 product size is 2476bp, and swimming lane 11 product size is 2768bp, and swimming lane 12 product size is 3080bp, swimming lane 13 product size is 2641bp, M is DNA marker thing.

Fig. 4 is 1.0 ~ 3.3kb nucleic acid fragment amplification figure (after freeze-drying reaction system).In figure, swimming lane 1 product size is 1132bp, swimming lane 2 product size is 1246bp, swimming lane 3 product size is 1346bp, swimming lane 4 product size is 1446bp, swimming lane 5 product size is 1550bp, swimming lane 6 product size is 1636bp, swimming lane 7 product size is 1739bp, swimming lane 8 product size is 1823bp, swimming lane 9 product size is 1950bp, swimming lane 10 product size is 2020bp, swimming lane 11 product size is 2134bp, swimming lane 12 product size is 2220bp, swimming lane 13 product size is 2350bp, swimming lane 14 product size is 2435bp, swimming lane 15 product size is 2548bp, swimming lane 16 product size is 2641bp, swimming lane 17 product size is 2768bp, swimming lane 18 product size is 3080bp, swimming lane 19 product size is 3364bp, swimming lane 20 and 21 is negative controls, M is DNA marker thing.

Fig. 5 is linear die lambda DNA cloning figure.In figure, swimming lane 1 is that forward primer 1000F increases, and swimming lane 2 is that reverse primer 1000R increases, and swimming lane 3 is that forward and reverse primer 1000F/R increases, and specificity segment size is 1000bp.

Fig. 6-1 and 6-2 is ring plasmid template amplification figure.In Fig. 6-1, swimming lane 1 is that forward primer 400F increases, and swimming lane 2 is that reverse primer 400R increases, and swimming lane 3 is that forward and reverse primer 400F/R increases, and specificity segment size is 400bp; In Fig. 6-2, swimming lane 1 is that forward primer 425F increases, and swimming lane 2 is that reverse primer 425R increases, and swimming lane 3 is that forward and reverse primer 425F/R increases, and specificity segment size is 425bp;

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. linear die lambda DNA buys in precious biotechnology (Dalian) company limited.

2., containing recombinant plasmid pM18-EF1b and pUC57-A of exogenous sequences, all utilize genetic engineering technique to build in this laboratory, recombinate and complete preparation.

3. all primers are used in amplification, all entrust Shanghai Sheng Gong biotechnology limited-liability company to synthesize.

Embodiment 1

The present embodiment is for illustration of the specificity long segment normal temperature isothermal duplication carried out on linear die lambda DNA.

1. from database NCBI GenBank, finding the gene order of lambda DNA, (Reference sequence number is NC_001416.1, overall length is 48.502kb), recycling software Vector NTI7.0 carries out the design of primers of nucleic acid fragment different in size for gene order.

2. by each primer pair of design, 1132-F/R (SEQ ID No.1 & 2) respectively, 1246F/R (SEQ ID No.3 & 4), 1346F/R (SEQ ID No.5 & 6), 1446F/R (SEQ ID No.7 & 8), 1550F/R (SEQ ID No.9 & 10), 1636F/R (SEQ ID No.11 & 12), 1739F/R (SEQ ID No.13 & 14), 1823F/R (SEQ ID No.15 & 16), 1950F/R (SEQ ID No.17 & 18), 2020F/R (SEQ ID No.19 & 20), 2134F/R (SEQ ID No.21 & 22), 2220F/R (SEQ ID No.23 & 24), 2350F/R (SEQ ID No.25 & 26), 2435F/R (SEQ ID No.27 & 28), 2548F/R (SEQ ID No.29 & 30), Shanghai Sheng Gong biotechnology limited-liability company is entrusted to synthesize.

The gene order of lambda DNA: the Reference sequence number in ncbi database is NC_001416.1, overall length is 48.502kb;

1132-F：5’-GTTTCCTTTCTCTGTTTTTGTCCGTGGAAT-3’(SEQ ID No.1)

1132-R：5’-GAGAGCCTTCCTGTTCAATATCATCATCAA-3’(SEQ ID No.2)

1246-F：5’-GTTTAAGGCGTTTCCGTTCTTCTTCGTCAT-3’(SEQ ID No.3)

1246-R：5’-TCGGAGAGCCTTCCTGTTCAATATCATCAT-3’(SEQ ID No.4)

1346-F：5’-AGATCTCCAGCCAGGAACTATTGAGTACGAA-3’(SEQ ID No.5)

1346-R：5’-CGTATCCCCTTTCGTTTTCATCCAGTCTTT-3’(SEQ ID No.6)

1446-F：5’-GTTTCCTTTCTCTGTTTTTGTCCGTGGAAT-3’(SEQ ID No.7)

1446-R：5’-CTTTTCGCAGATATAACGGGCATCAGTAAA-3’(SEQ ID No.8)

1550-F：5’-GCGTTTCCGTTCTTCTTCGTCATAACTTAA-3’(SEQ ID No.9)

1550-R：5’-CTTTTCGCAGATATAACGGGCATCAGTAAA-3’(SEQ ID No.10)

1636-F：5’-CGTTAACGATTTGCTGAACACACCAGTGTAA-3’(SEQ ID No.11)

1636-R：5’-AGTTTCATCCGTGTCATCAAGCTCCTCTTT-3’(SEQ ID No.12)

1739-F：5’-TGGTGTACCGGCTGTCTGGTATGTATGAGTT-3’(SEQ ID No.13)

1739-R：5’-GTAAGCCTTCTGCGCCTCTTCGGTATATTT-3’(SEQ ID No.14)

1823-F：5’-GACGTTTGAGCAGAATAACCATGTGGTGAT-3’(SEQ ID No.15)

1823-R：5’-CTCTCTCGTTTGCTCAGTTGTTCAGGAATA-3’(SEQ ID No.16)

1950-F：5’-ATCAGCGTGGTCTGAGTGTGTTACAGAGGTT-3’(SEQ ID No.17)

1950-R：5’-AGTGGGCTTTTCTGTTCGTTTCATCCATTA-3’(SEQ ID No.18)

2020-F：5’-AATTCCGTTGCAGATGTTCTTGAATACCTT-3’(SEQ ID No.19)

2020-R：5’-GAGCGTCTAAAACTGGTAGATAAGCCTAAA-3’(SEQ ID No.20)

2134-F：5’-TTAAAATTAGAGTTGTGGCTTGGCTCTGCTA-3’(SEQ ID No.21)

2134-R：5’-AGTGCATTTGATCCTTTTACTCCTCCTAAA-3’(SEQ ID No.22)

2220-F：5’-AATGACCTGCCTAGGAATTGGTTAGCAAGTT-3’(SEQ ID No.23)

2220-R：5’-TTGAAAATGAAAGCGTCCTTAACACCTCAT-3’(SEQ ID No.24)

2350-F：5’-ATCCCTCTGAAAAAATCTTCCGAGTTTGCTA-3’(SEQ ID No.25)

2350-R：5’-TATCTTACTGTCTTTGATGAGCATGGTGAA-3’(SEQ ID No.26)

2435-F：5’-CCTGCCTCCAAACGATACCTGTTAGCAATA-3’(SEQ ID No.27)

2435-R：5’-GTGGGTTATCCAAAAGGAAGCAGAAAGCTA-3’(SEQ ID No.28)

2548-F：5’-GTACCTCATCTACTGCGAAAACTTGACCTTT-3’(SEQ ID No.29)

2548-R：5’-ACTACGACCTGCATAACCAGTAAGAAGATA-3’(SEQ ID No.30)

3. use the primer pair of the lyophilized powder reaction system for preparing and synthesis or mix and take primer pair and carry out normal temperature isothermal amplification respectively.Lyophilized powder reaction system is:

Tris-Ac	5ul
		E-mix	2.5ul
CK	2.5ul
		20％PEG35000	12ul
Recombinase	0.75mg/ml
		Accessory protein	0.22mg/ml
Single strand binding protein	1.15mg/ml
		Archaeal dna polymerase	0.51mg/ml
Topoisomerase	0.25U/ul
		Helicase	0.2U/ul
dNTP	4ul
		Trehalose	6％
N.F,USP MANNITOL	7％

Wherein, Tris-Ac is dissolved in 10ml distilled water by the 2M DTT of 0.6gTris alkali, 0.98g potassium acetate and 250ul, and pH regulator obtains to 8.3 preparations.E-mix is dissolved in by 0.3g ATP and 2.55g PCr and prepares in 10ml distilled water and obtain.

Complete the preparation of freeze-drying reaction system on ice after, be dispensed in 200ul reaction tubes and carry out follow-up freeze-drying preparation: lyophilize 4-10 hour at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.

Amplification reaction system is formulated as follows:

Lyophilized powder reaction reagent	1 pipe
		PEG	25μl
Distilled water	17.5μl
		Primer-F	2.0μl
Primer-R	2.0μl
		Template	1μl
Magnesium acetate	2.5μl

4. amplified reaction program: 37 DEG C of isothermals, 40 minutes.

5. agarose gel electrophoresis carry out result detection (Fig. 1., Fig. 2., Fig. 3. and Fig. 4 .).

Fig. 1 is using linear lambda DNA as template, and the nucleotide fragment different for length between 1000-1600bp increases, and the bright single band of corresponding size confirms specific amplification.Fig. 2, equally using linear lambda DNA as template, carries out the amplification of Nucleotide large fragment for 1600-2000bp, and the bright single band of same corresponding size confirms its specific amplification.Fig. 3 is the amplification of the long segment for 2000-3400bp, and indivedual amplifications exist the interference of non-specific amplification, but do not affect the difference of specific band.Fig. 4 carries out the result figure tested that increases again after system is carried out frozen dried, clip size contains 1.0-3.3kb, and most swimming lane presents with single bright amplified fragments, confirms the feasibility of the isothermal nucleic acid large fragment amplifications such as normal temperature.

Embodiment 2

The present embodiment for illustration of in circular template, the normal temperature isothermal duplication that recombinant plasmid pM18-EF1b and pUC57-A carries out.

1. utilize genetic engineering technique to construct two recombinant plasmid pM18-EF1b and pUC57-A in vitro, as annular template.

2. respectively for corresponding templates, carry out the normal temperature isothermal duplication of single primer and decoding for DTMF with the Auele Specific Primer EF1b-F/R (SEQ ID No.31 & 32) designed and A-F/R (SEQ ID No.33 & 34).

EF1b-F：5’-TCACACACACACATATAGAAAGAGAGAGAC-3’(SEQ ID No.31)

EF1b-R：5’-CTGCTCCAGTTAGTTCATATAAGAGATAGG-3’(SEQ ID No.32)

A-F：5’-AGCATCAACTTCTACCGTTCTACATAGCAT-3’(SEQ ID No.33)

A-R：5’-CCGCTATAATACTGCATTTACACTTGGCAT-3’(SEQ ID No.34)

3. lyophilized powder reaction system is:

Tris-Ac

5ul

E-mix	2.5ul
		CK	2.5ul
20％PEG35000	12ul
		Recombinase	0.75mg/ml
Accessory protein	0.22mg/ml
		Single strand binding protein	1.15mg/ml
Archaeal dna polymerase	0.51mg/ml
		Topoisomerase	0.25U/ul
Helicase	0.2U/ul
		dNTP	4ul
Trehalose	6％
		N.F,USP MANNITOL	7％

4. wherein, Tris-Ac is dissolved in 10ml distilled water by the 2M DTT of 0.6gTris alkali, 0.98g potassium acetate and 250ul, and pH regulator obtains to 8.3 preparations.E-mix is dissolved in by 0.3g ATP and 2.55g PCr and prepares in 10ml distilled water and obtain.

5. complete the preparation of freeze-drying reaction system on ice after, be dispensed in 200ul reaction tubes and carry out follow-up freeze-drying preparation: lyophilize 4-10 hour at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.

6. amplification reaction system is formulated as follows:

7. amplified reaction program: 37 DEG C of isothermals, 4 hours.

8. utilize subsequently agarose gel electrophoresis carry out result detection (Fig. 5. with Fig. 6-1,6-2.).

Fig. 5 is the single primer amplification test carried out with linear die lambda DNA, and swimming lane 1 is forward primer, and swimming lane 2 is reverse primers, and swimming lane 3 is positive/negative to primer.Result confirms in linear die, carry out single primer amplification can only unidirectional acquisition long segment, and this may be relevant to the lasting amplification ability of the position of primer, reaction system.Fig. 6 is with circular template, and the single primer amplification test that recombinant plasmid carries out, swimming lane 1 is forward primer, and swimming lane 2 is reverse primers, and swimming lane 3 is positive/negative to primer.Result confirms that in circular template, carry out single primer amplification can all obtain long or annular amplified production in both direction, illustrates the ability tentatively possessing and carry out plasmid amplification in vitro.

SEQUENCE LISTING

<110> Zhejiang Taijing Biotechnology Co., Ltd.

<120> is used for proteolytic enzyme and the amplification method of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature

<130>

<160> 40

<170> PatentIn version 3.3

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<211> 30

<212> DNA

<213> artificial sequence

<400> 14

gtaagccttc tgcgcctctt cggtatattt 30

<210> 15

<211> 30

<212> DNA

<213> artificial sequence

<400> 15

gacgtttgag cagaataacc atgtggtgat 30

<210> 16

<211> 30

<212> DNA

<213> artificial sequence

<400> 16

ctctctcgtt tgctcagttg ttcaggaata 30

<210> 17

<211> 31

<212> DNA

<213> artificial sequence

<400> 17

atcagcgtgg tctgagtgtg ttacagaggt t 31

<210> 18

<211> 30

<212> DNA

<213> artificial sequence

<400> 18

agtgggcttt tctgttcgtt tcatccatta 30

<210> 19

<211> 30

<212> DNA

<213> artificial sequence

<400> 19

aattccgttg cagatgttct tgaatacctt 30

<210> 20

<211> 30

<212> DNA

<213> artificial sequence

<400> 20

gagcgtctaa aactggtaga taagcctaaa 30

<210> 21

<211> 31

<212> DNA

<213> artificial sequence

<400> 21

ttaaaattag agttgtggct tggctctgct a 31

<210> 22

<211> 30

<212> DNA

<213> artificial sequence

<400> 22

agtgcatttg atccttttac tcctcctaaa 30

<210> 23

<211> 31

<212> DNA

<213> artificial sequence

<400> 23

aatgacctgc ctaggaattg gttagcaagt t 31

<210> 24

<211> 30

<212> DNA

<213> artificial sequence

<400> 24

ttgaaaatga aagcgtcctt aacacctcat 30

<210> 25

<211> 31

<212> DNA

<213> artificial sequence

<400> 25

atccctctga aaaaatcttc cgagtttgct a 31

<210> 26

<211> 30

<212> DNA

<213> artificial sequence

<400> 26

tatcttactg tctttgatga gcatggtgaa 30

<210> 27

<211> 30

<212> DNA

<213> artificial sequence

<400> 27

cctgcctcca aacgatacct gttagcaata 30

<210> 28

<211> 30

<212> DNA

<213> artificial sequence

<400> 28

gtgggttatc caaaaggaag cagaaagcta 30

<210> 29

<211> 31

<212> DNA

<213> artificial sequence

<400> 29

gtacctcatc tactgcgaaa acttgacctt t 31

<210> 30

<211> 30

<212> DNA

<213> artificial sequence

<400> 30

actacgacct gcataaccag taagaagata 30

<210> 31

<211> 30

<212> DNA

<213> artificial sequence

<400> 31

tcacacacac acatatagaa agagagagac 30

<210> 32

<211> 30

<212> DNA

<213> artificial sequence

<400> 32

ctgctccagt tagttcatat aagagatagg 30

<210> 33

<211> 30

<212> DNA

<213> artificial sequence

<400> 33

agcatcaact tctaccgttc tacatagcat 30

<210> 34

<211> 30

<212> DNA

<213> artificial sequence

<400> 34

ccgctataat actgcattta cacttggcat 30

<210> 35

<211> 393

<212> PRT

<213> artificial sequence

<400> 35

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> 36

<211> 298

<212> PRT

<213> artificial sequence

<400> 36

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> 37

<211> 876

<212> PRT

<213> artificial sequence

<400> 37

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

Po 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> 38

<211> 140

<212> PRT

<213> artificial sequence

<400> 38

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

<210> 39

<211> 865

<212> PRT

<213> artificial sequence

<400> 39

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

1 5 10 15

Ile Asn Lys Tyr Leu Gly Ser Asp Tyr Val Val Lys Ser Ser Val Gly

20 25 30

His Ile Arg Asp Leu Pro Thr Ser Gly Ser Ala Ala Lys Lys Ser Ala

35 40 45

Asp Ser Thr Ser Thr Lys Thr Ala Lys Lys Pro Lys Lys Asp Glu Arg

50 55 60

Gly Ala Leu Val Asn Arg Met Gly Val Asp Pro Trp His Asn Trp Glu

65 70 75 80

Ala His Tyr Glu Val Leu Pro Gly Lys Glu Lys Val Val Ser Glu Leu

85 90 95

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

100 105 110

Asp Arg Glu Gly Glu Ala Ile Ala Trp His Leu Arg Glu Val Ile Gly

115 120 125

Gly Asp Asp Ala Arg Tyr Ser Arg Val Val Phe Asn Glu Ile Thr Lys

130 135 140

Asn Ala Ile Arg Gln Ala Phe Asn Lys Pro Gly Glu Leu Asn Ile Asp

145 150 155 160

Arg Val Asn Ala Gln Gln Ala Arg Arg Phe Met Asp Arg Val Val Gly

165 170 175

Tyr Met Val Ser Pro Leu Leu Trp Lys Lys Ile Ala Arg Gly Leu Ser

180 185 190

Ala Gly Arg Val Gln Ser Val Ala Val Arg Leu Val Val Glu Arg Glu

195 200 205

Arg Glu Ile Lys Ala Phe Val Pro Glu Glu Phe Trp Glu Val Asp Ala

210 215 220

Ser Thr Thr Thr Pro Ser Gly Glu Ala Leu Ala Leu Gln Val Thr His

225 230 235 240

Gln Asn Asp Lys Pro Phe Arg Pro Val Asn Lys Glu Gln Thr Gln Ala

245 250 255

Ala Val Ser Leu Leu Glu Lys Ala Arg Tyr Ser Val Leu Glu Arg Glu

260 265 270

Asp Lys Pro Thr Thr Ser Lys Pro Gly Ala Pro Phe Ile Thr Ser Thr

275 280 285

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

290 295 300

Met Met Met Ala Gln Arg Leu Tyr Glu Ala Gly Tyr Ile Thr Tyr Met

305 310 315 320

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

325 330 335

Gly Tyr Ile Ser Asp Asn Phe Gly Lys Lys Tyr Leu Pro Glu Ser Pro

340 345 350

Asn Gln Tyr Ala Ser Lys Glu Asn Ser Gln Glu Ala His Glu Ala Ile

355 360 365

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

370 375 380

Ala Asp Ala Gln Lys Leu Tyr Gln Leu Ile Trp Arg Gln Phe Val Ala

385 390 395 400

Cys Gln Met Thr Pro Ala Lys Tyr Asp Ser Thr Thr Leu Thr Val Gly

405 410 415

Ala Gly Asp Phe Arg Leu Lys Ala Arg Gly Arg Ile Leu Arg Phe Asp

420 425 430

Gly Trp Thr Lys Val Met Pro Ala Leu Arg Lys Gly Asp Glu Asp Arg

435 440 445

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

450 455 460

Thr Pro Ala Gln His Phe Thr Lys Pro Pro Ala Arg Phe Ser Glu Ala

465 470 475 480

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

485 490 495

Tyr Ala Ser Ile Ile Ser Thr Ile Gln Asp Arg Gly Tyr Val Arg Val

500 505 510

Glu Asn Arg Arg Phe Tyr Ala Glu Lys Met Gly Glu Ile Val Thr Asp

515 520 525

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

530 535 540

Gln Met Glu Asn Ser Leu Asp Gln Val Ala Asn His Glu Ala Glu Trp

545 550 555 560

Lys Ala Val Leu Asp His Phe Phe Ser Asp Phe Thr Gln Gln Leu Asp

565 570 575

Lys Ala Glu Lys Asp Pro Glu Glu Gly Gly Met Arg Pro Asn Gln Met

580 585 590

Val Leu Thr Ser Ile Asp Cys Pro Thr Cys Gly Arg Lys Met Gly Ile

595 600 605

Arg Thr Ala Ser Thr Gly Val Phe Leu Gly Cys Ser Gly Tyr Ala Leu

610 615 620

Pro Pro Lys Glu Arg Cys Lys Thr Thr Ile Asn Leu Val Pro Glu Asn

625 630 635 640

Glu Val Leu Asn Val Leu Glu Gly Glu Asp Ala Glu Thr Asn Ala Leu

645 650 655

Arg Ala Lys Arg Arg Cys Pro Lys Cys Gly Thr Ala Met Asp Ser Tyr

660 665 670

Leu Ile Asp Pro Lys Arg Lys Leu His Val Cys Gly Asn Asn Pro Thr

675 680 685

Cys Asp Gly Tyr Glu Ile Glu Glu Gly Glu Phe Arg Ile Lys Gly Tyr

690 695 700

Asp Gly Pro Ile Val Glu Cys Glu Lys Cys Gly Ser Glu Met His Leu

705 710 715 720

Lys Met Gly Arg Phe Gly Lys Tyr Met Ala Cys Thr Asn Glu Glu Cys

725 730 735

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

740 745 750

Glu Asp Pro Val Pro Leu Pro Glu Leu Pro Cys Glu Lys Ser Asp Ala

755 760 765

Tyr Phe Val Leu Arg Asp Gly Ala Ala Gly Val Phe Leu Ala Ala Asn

770 775 780

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

785 790 795 800

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

805 810 815

Ala Pro Gln Gln Asp Pro Glu Gly Asn Lys Thr Met Val Arg Phe Ser

820 825 830

Arg Lys Thr Lys Gln Gln Tyr Val Ser Ser Glu Lys Asp Gly Lys Ala

835 840 845

Thr Gly Trp Ser Ala Phe Tyr Val Asp Gly Lys Trp Val Glu Gly Lys

850 855 860

Lys

865

<210> 40

<211> 471

<212> PRT

<213> artificial sequence

<400> 40

Met Ala Gly Asn Lys Pro Phe Asn Lys Gln Gln Ala Glu Pro Arg Glu

1 5 10 15

Arg Asp Pro Gln Val Ala Gly Leu Lys Val Pro Pro His Ser Ile Glu

20 25 30

Ala Glu Gln Ser Val Leu Gly Gly Leu Met Leu Asp Asn Glu Arg Trp

35 40 45

Asp Asp Val Ala Glu Arg Val Val Ala Asp Asp Phe Tyr Thr Arg Pro

50 55 60

His Arg His Ile Phe Thr Glu Met Ala Arg Leu Gln Glu Ser Gly Ser

65 70 75 80

Pro Ile Asp Leu Ile Thr Leu Ala Glu Ser Leu Glu Arg Gln Gly Gln

85 90 95

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

100 105 110

Thr Pro Ser Ala Ala Asn Ile Ser Ala Tyr Ala Asp Ile Val Arg Glu

115 120 125

Arg Ala Val Val Arg Glu Met Ile Ser Val Ala Asn Glu Ile Ala Glu

130 135 140

Ala Gly Phe Asp Pro Gln Gly Arg Thr Ser Glu Asp Leu Leu Asp Leu

145 150 155 160

Ala Glu Ser Arg Val Phe Lys Ile Ala Glu Ser Arg Ala Asn Lys Asp

165 170 175

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

180 185 190

Ile Glu Gln Leu Phe Gln Gln Pro His Asp Gly Val Thr Gly Val Asn

195 200 205

Thr Gly Tyr Asp Asp Leu Asn Lys Lys Thr Ala Gly Leu Gln Pro Ser

210 215 220

Asp Leu Ile Ile Val Ala Ala Arg Pro Ser Met Gly Lys Thr Thr Phe

225 230 235 240

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

245 250 255

Leu Ile Phe Ser Leu Glu Met Pro Ser Glu Gln Ile Met Met Arg Ser

260 265 270

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

275 280 285

Leu Asp Asp Glu Asp Trp Ala Arg Ile Ser Gly Thr Met Gly Ile Leu

290 295 300

Leu Glu Lys Arg Asn Ile Tyr Ile Asp Asp Ser Ser Gly Leu Thr Pro

305 310 315 320

Thr Glu Val Arg Ser Arg Ala Arg Arg Ile Ala Arg Glu His Gly Gly

325 330 335

Ile Gly Leu Ile Met Ile Asp Tyr Leu Gln Leu Met Arg Val Pro Ala

340 345 350

Leu Ser Asp Asn Arg Thr Leu Glu Ile Ala Glu Ile Ser Arg Ser Leu

355 360 365

Lys Ala Leu Ala Lys Glu Leu Asn Val Pro Val Val Ala Leu Ser Gln

370 375 380

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

385 390 395 400

Asp Leu Arg Glu Ser Gly Ser Ile Glu Gln Asp Ala Asp Leu Ile Met

405 410 415

Phe Ile Tyr Arg Asp Glu Val Tyr His Glu Asn Ser Asp Leu Lys Gly

420 425 430

Ile Ala Glu Ile Ile Ile Gly Lys Gln Arg Asn Gly Pro Ile Gly Thr

435 440 445

Val Arg Leu Thr Phe Asn Gly Gln Trp Ser Arg Phe Asp Asn Tyr Ala

450 455 460

Gly Pro Gln Tyr Asp Asp Glu

465 470

Claims

1. for the proteolytic enzyme of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature, it is characterized in that, described proteolytic enzyme comprises: (1) recombinase; (2) single strand binding protein; (3) archaeal dna polymerase; (4) accessory protein; The aminoacid sequence of described recombinase is as shown in SEQID No.35; The aminoacid sequence of single strand binding protein is as shown in SEQ ID No.36; The aminoacid sequence of archaeal dna polymerase is as shown in SEQ ID No.37; The aminoacid sequence of accessory protein is as shown in SEQ ID No.38.

2. proteolytic enzyme as claimed in claim 1, it is characterized in that, in amplification system, recombinase concentration range is 0.5 ~ 0.8mg/ml; Single strand binding protein concentration range is 0.9 ~ 1.2mg/ml; Archaeal dna polymerase concentration range is 0.3 ~ 0.6mg/ml; Accessory protein concentration range is 0.1 ~ 0.3mg/ml.

3. proteolytic enzyme as claimed in claim 1, it is characterized in that, described proteolytic enzyme also comprises: (5) topoisomerase or helicase; The aminoacid sequence of topoisomerase is as SEQ ID No.39; The aminoacid sequence of helicase is as shown in SEQ ID No.40.

4. proteolytic enzyme as claimed in claim 3, it is characterized in that, in amplification system, topoisomerase concentration range is 0.1 ~ 0.5U/ul; Helicase concentration range is 0.1 ~ 0.5U/ul.

5. for the reagent of the isothermal nucleic acid large fragment amplification in vitros such as normal temperature, it is characterized in that, described reagent mainly comprises Tris alkali, dNTP, ATP, disodium creatine phosphate, creatine phosphokinase, potassium acetate, trehalose, N.F,USP MANNITOL, polyoxyethylene glycol, dithiothreitol (DTT) and proteolytic enzyme, and described proteolytic enzyme is the proteolytic enzyme described in one of Claims 1 to 4.

6. reagent as claimed in claim 5, it is characterized in that, described reagent is lyophilized powder, the concentration of each component in lyophilized powder is: Tris alkali 20 ~ 30mM, dNTP 200 ~ 250uM, ATP 2 ~ 3mM, disodium creatine phosphate 45 ~ 5mM, creatine phosphokinase 90 ~ 110ng/ μ l, potassium acetate 90 ~ 110mM, trehalose 5.5 ~ 6.5%, N.F,USP MANNITOL 6 ~ 10%, polyoxyethylene glycol 2 ~ 3%, dithiothreitol (DTT) 4 ~ 6mM, recombinase 0.5 ~ 0.8mg/ml, single strand binding protein 0.9 ~ 1.2mg/ml, archaeal dna polymerase 0.3 ~ 0.6mg/ml, accessory protein 0.1 ~ 0.3mg/ml, topoisomerase 0.1 ~ 0.5U/ul, helicase 0.1 ~ 0.5U/ul, described lyophilized powder is made up of following methods: now by reagent lyophilize 4 ~ 10 hours at-30 ~-35 DEG C, then 0 ~ 20 DEG C of drying 30 minutes ~ 1 hour.

7. the isothermal nucleic acid large fragment amplification in vitro method such as normal temperature, it is characterized in that, described amplification method is after adding template and amplimer with reagent described in claim 5 or 6, is placed in a temperature control device and hatches 30 minutes to 4 hours, then carry out Visual retrieval by agarose gel electrophoresis; The temperature of described normal temperature is 37 DEG C, keeps homogeneous steady temperature in amplification procedure.

8. amplification method as claimed in claim 7, described temperature control device has temperature control modules, set of time function and sample well.