CN110938610B - A kind of transposase mutant, fusion protein, its preparation method and application - Google Patents

Abstract

The invention discloses a transposase mutant, a fusion protein, a preparation method and an application thereof, and relates to the field of biotechnology. The transposase mutant has at least one mutation form compared with a wild-type transposase, and the amino acid sequence of the wild-type transposase is shown in SEQ ID No. 1. The transposase mutant has high stability and can be stored at -20°C for a long time, which can significantly reduce the storage cost, make the use of the transposase or the fusion protein constructed thereof more convenient, and simplify the experimental process.

Description

A kind of transposase mutant, fusion protein, its preparation method and application

technical field

The present invention relates to the field of biotechnology, in particular, to a transposase mutant, a fusion protein, a preparation method and application thereof.

Background technique

With the extensive application of in vitro transposition-related technologies, the development and related applications of new transposases are also increasing, and there are endless cases of replacing old technologies with various new technologies mediated by transposases.

Transposases can not only be used in the general next-generation sequencing library construction process, but also more and more widely used in multi-omics research. However, the existing transposase proteins have some defects, and their protein stability is poor. When stored at -20 °C, the activity is likely to be significantly reduced or lost, and relatively harsh storage conditions are required. The nucleic acid co-incubation of the sequence increases the stability of the complex to form a complex. This limitation on the use conditions usually increases the cost of the experiment itself and increases the complexity of the overall experimental process.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The object of the present invention is to provide transposase mutants, fusion proteins, isolated nucleic acids, vectors, host cells, methods for preparing fusion proteins, kits for constructing sequencing libraries, transposase mutants or fusion proteins in DNA construction Applications in libraries and fusion proteins in chromatin co-precipitation.

The present invention is realized in this way:

In a first aspect, the embodiments provide a transposase mutant having at least one of the following mutations compared to the wild-type transposase: R27T, M56G, Y41R, E190A, K120Q and E146N ;

The amino acid sequence of the wild-type transposase is shown in SEQ ID No.1.

In a second aspect, the embodiment provides a fusion protein comprising the above-mentioned transposase mutant.

In a third aspect, the embodiments provide an isolated nucleic acid encoding the transposase mutant or fusion protein as described in the preceding embodiments.

In a fourth aspect, the embodiments provide a vector comprising the isolated nucleic acid as described in the preceding embodiments.

In a fifth aspect, the embodiments provide a host cell comprising the vector described in the preceding embodiments.

In a sixth aspect, the Examples provide a method of making a transposase mutant or fusion protein, comprising culturing the host cell as described in the preceding examples.

In a seventh aspect, the embodiment provides a kit for constructing a sequencing library, which includes the transposase mutant or fusion protein as described in the preceding embodiments.

In an eighth aspect, the embodiment provides an application of the transposase mutant or fusion protein described in the preceding embodiment in DNA library construction.

In a ninth aspect, the examples provide applications of the transposase mutants or fusion proteins described in the preceding examples in analyzing the interaction between proteins and chromatin.

The present invention has the following beneficial effects:

The embodiment of the present invention provides a transposase mutant, the transposase mutant has high stability, can be stored at -20°C for a long time, can significantly reduce the storage cost of the transposase, and make the transposase The use of the fusion protein or the constructed fusion protein is more convenient and the experimental process is simplified.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the stability detection result of transposase 1 and wild-type transposase in verification example 1 of the present invention;

FIG. 2 shows the binding effect of different fusion proteins and various enzyme-labeled antibodies in Validation Example 3 of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The present invention analyzes the function, protein structure and key sites of the wild-type Tn5 sequence, and designs a large number of mutations for stability transformation, and finally obtains the following stable transposase mutant, and the transposase mutant It does not affect its stability after it is constructed into a fusion protein with protein A.

The embodiment of the present invention provides a transposase mutant, the transposase mutant has at least one or a combination of the following mutations compared to the wild-type transposase: R27T, M56G, Y41R, E190A , K120Q and E146N; the amino acid sequence of the wild-type transposase is shown in SEQ ID No.1. The transposase mutant has high stability, can significantly reduce storage costs, and simplify experimental procedures.

It should be noted that the above-mentioned “combination of at least one or more of the following mutations” is selected from: any one of the six mutations, any two mutations, any three mutations, any four mutations, any 5 mutations and combinations of the above 6 mutations.

In an optional embodiment, the transposase mutant has any one of the following combinations of mutations (1) to (3) compared to the wild-type transposase;

(1) R27T and M56G;

(2) Y41R and M56G;

(3) K120Q and E146N.

In alternative embodiments, the transposase mutant also has mutations E54K and/or K120L compared to the wild-type transposase. It should be noted that "it also has mutations E54K and/or K120L" means that the transposase mutant has any one of the above six mutations, any two mutations, any three On the basis of any kind of mutation, any 4 kinds of mutations, any 5 kinds of mutations and the combination of the above 6 kinds of mutations, it also has mutations E54K and/or K120L.

In an optional embodiment, the transposase mutant has any one of the following combinations of mutations (4) to (5) compared to the wild-type transposase:

(4) Y41R, E54K and M56G;

(5) K120L and E190A.

Compared with other mutation combinations, the stability of the transposase mutant having any of the above-mentioned mutations (1) to (5) is further improved. It should be noted that "the transposase mutant has (1) the combination of mutations described in the wild-type transposase" means that the amino acid sequence of the transposase mutant is: on the amino acid sequence of the wild-type transposase When the 27th arginine is mutated to threonine, the 56th methionine in the amino acid sequence of the wild-type transposase is mutated to glycine, and so on.

The embodiment of the present invention provides a fusion protein comprising the transposase mutant described in any one of the foregoing embodiments.

In an alternative embodiment, the fusion protein further comprises a binding protein for binding to the Fc fragment of an immunoglobulin, and the binding protein is linked to the transposase mutant.

In alternative embodiments, the binding proteins include Staphylococcus aureus Protein A (Protein A, PA) or a variant thereof, Streptococcus Protein G (Protein G) or a variant thereof, and Streptococcus Protein L (Protein L) or at least one of its variants.

In alternative embodiments, the binding protein comprises Staphylococcus aureus protein A or a variant thereof.

In an alternative embodiment, the amino acid sequence of the binding protein is shown in SEQ ID No. 2 or 3.

In an alternative embodiment, the amino acid sequence of the binding protein is located at the N-terminus of the amino acid sequence of the fusion protein, and the amino acid sequence of the transposase mutant is located at the C-terminus of the amino acid sequence of the fusion protein.

In an optional embodiment, the binding protein and the transposase mutant are linked through a linking peptide, and the fusion protein has the structure: binding protein-linking peptide-transposase mutant.

In alternative embodiments, the linker peptide is a rigid linker peptide or a flexible linker peptide.

In an alternative embodiment, the linker peptide is a flexible linker peptide.

In an alternative embodiment, the length of the linking peptide is 6-59 amino acids.

In an alternative embodiment, the linker peptide is 20-35 amino acids in length.

In an alternative embodiment, the sequence of the linking peptide is shown in SEQ ID No.4.

The embodiments of the present invention provide an isolated nucleic acid encoding the transposase mutant according to any of the foregoing embodiments or the fusion protein according to any of the foregoing embodiments.

Embodiments of the present invention provide a vector containing the isolated nucleic acid as described in the preceding embodiments.

The embodiment of the present invention provides a host cell, which contains the vector described in the previous embodiment.

The embodiment of the present invention provides a method for preparing the transposase mutant or fusion protein described in any of the foregoing embodiments, which comprises culturing the host cell described in any of the foregoing embodiments.

The embodiments of the present invention provide a kit for constructing a sequencing library, which includes the transposase mutant described in any of the foregoing embodiments or the fusion protein described in any of the foregoing embodiments.

In an optional embodiment, the kit further includes Tn5 transposase buffer and/or PCR reaction solution.

The embodiment of the present invention provides the application of the transposase mutant according to any of the foregoing embodiments or the fusion protein according to any of the foregoing embodiments in DNA library construction.

In an optional embodiment, the application includes embedding the transposase mutant or the transposase mutant in the fusion protein with an adapter to form a Tn5 transposome, and the obtained transposase The transposome is mixed and incubated with the target gene, and the transposome is activated to fragment the target gene to obtain a DNA library with adapters at both ends.

In an optional embodiment, it also includes using PCR technology to amplify the target gene after the fragment by PCR.

The embodiment of the present invention provides the application of the transposase mutant according to any of the foregoing embodiments or the fusion protein according to any of the foregoing embodiments in analyzing the interaction between proteins and chromatin.

In an alternative embodiment, the application comprises incubating a mixture containing the target protein to be analyzed, the target chromatin, and the transposase mutant.

In an optional embodiment, the mixture system further comprises an antibody to a target protein, the transposase is the fusion protein bound with a binding protein for binding to the Fc segment of an immunoglobulin, and the application comprises firstly combining the target protein , The target chromatin is combined with the antibody of the target protein, and then the fusion protein is added for co-incubation.

In the mixed system, the target protein binds to the target chromatin, the antibody of the target protein binds to the target protein, the binding protein in the fusion protein binds to the Fc segment of the antibody of the target protein, and after the transposase mutant in the fusion protein is activated, It is used to cut the target chromatin to obtain the fragmented target sequence for sequencing.

In an alternative embodiment, the transposase mutant is activated using a metal ion, preferably calcium or magnesium.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

This example provides five transposase mutants, as follows.

The amino acid sequence of transposase 1 is shown in SEQ ID No.5, and the nucleic acid sequence is shown in SEQ ID No.6;

The amino acid sequence of transposase 2 is shown in SEQ ID No.7, and the nucleic acid sequence is shown in SEQ ID No.8;

The amino acid sequence of transposase 3 is shown in SEQ ID No.9, and the nucleic acid sequence is shown in SEQ ID No.10;

The amino acid sequence of transposase 4 is shown in SEQ ID No.11, and the nucleic acid sequence is shown in SEQ ID No.12;

The amino acid sequence of transposase 5 is shown in SEQ ID No.13, and the nucleic acid sequence is shown in SEQ ID No.14.

Example 2

This example provides a fusion protein, which includes the transposase mutant provided in Example 1, a binding protein for binding to the Fc segment of an immunoglobulin, and a linking peptide. The amino acid sequence of the binding protein and the transposase passes through The connecting peptide is connected, and the structure of the fusion protein is: binding protein-connecting peptide-transposase mutant.

Among them, the binding protein is protein A (PA), which includes PA1 and PA2.

Specifically, the sequence of PA1 is shown in SEQ ID No. 2, the sequence of PA2 is shown in SEQ ID No. 3, and the sequence of the connecting peptide is shown in SEQ ID No. 4.

The preparation method of the fusion protein is as follows.

The above-mentioned transposase mutants (transposases 1 to 5 in Example 1) and wild-type transposase Tn5 were respectively constructed as fusion proteins with PA1 and PA2. All sequences were synthesized by artificial genes, and the above sequences were synthesized. The fragments were respectively connected to plasmid vectors for constructing expression vectors, and fusion proteins 1-12 were obtained respectively.

Fusion protein 1: PA1-linking peptide-transposase 1;

Fusion protein 2: PA1-connecting peptide-transposase 2;

Fusion protein 3: PA1-linking peptide-transposase 3;

Fusion protein 4: PA1-linking peptide-transposase 4;

Fusion protein 5: PA1-connecting peptide-transposase 5;

Fusion protein 6: PA1-connecting peptide-wild-type transposase Tn5;

Fusion protein 7: PA2-linking peptide-transposase 1;

Fusion protein 8: PA2-linking peptide-transposase 2;

Fusion protein 9: PA2-linking peptide-transposase 3;

Fusion protein 10: PA2-linking peptide-transposase 4;

Fusion protein 11: PA2-linking peptide-transposase 5;

Fusion protein 12: PA2-linking peptide-wild-type transposase Tn5.

The constructed expression vector was transformed into a competent Escherichia coli expression strain, the expression strain was added to 1 mL of LB medium, and cultured at 37°C for 30 min. After culturing, 200 μl of bacterial solution was taken and coated on LB solid containing kanamycin. On the plate, placed in a 37 °C incubator overnight for 16 h, and the monoclonal strains of white colonies were selected and inoculated into 3 ml of liquid LB medium containing kanamycin, and placed in a constant temperature shaker for 16 h overnight at 37 °C.

The next day, the cultured bacterial liquid was taken and placed in a 500ml triangular flask at a ratio of 1:100 to 200ml of liquid LB medium containing kanamycin, and cultured in a constant temperature shaker at 37°C at OD600 When it reached about 0.6, IPTG with a final concentration of 0.25 mM was added for induction, and cultured at 18°C overnight for 16 h. For the genetically engineered bacteria liquid expressing the target protein obtained by fermentation, take 500 ml of centrifuge (4°C, 12000 rpm, 5 min) with a low-temperature high-speed centrifuge, discard the supernatant, and add 50 ml of lysis buffer to the collected cells. (50mM HEPES-KOH at PH 7.5, 800mM NaCl, 1mM EDTA, 0.25% Tween-20) to resuspend the cells, sonicate the cells (30s sonication, 10 times), centrifuge again under the same conditions, collect the centrifuge obtained supernatant. The supernatant was purified to obtain the target protein (fusion protein) with a purity higher than 90%.

Verification Example 1

The stability of the transposase mutant provided in Example 1 was verified.

Take 3 × 100 μl of transposases 1 to 5 and wild-type transposase provided in Example 1 into a 1.5 mL EP tube respectively, and set up 8 groups of test examples as a control. For the wild-type transposase, it has the following mutations in order: R27T, M56G, Y41R, E54K, K120L, E190A, K120Q and E146N;

Then, transposases 1 to 5, wild-type transposase, and transposase mutants provided in test examples 1 to 8 were placed at 25 °C, 4 °C, and -20 °C for 7 days, respectively, to carry out the target protein. Stability check.

When the target protein was placed for 7 days under the conditions of each examination temperature, the DNA fragmentation activity was detected, and the detection method was as follows.

Prepare Adapter Mix

Mix 100 μM Primer M with equal volumes of 100 μM Primer T1 and 100 μM Primer T2, respectively, and then anneal them, then mix the annealed products of M-T1 and M-T2 in equal volumes, and the resulting mixture is the Adapter Mix. The Primer sequence is as follows:

Primer M: 5'-pCTGTCTCTTATACACATCT-3' (p refers to phosphorylation modification);

Primer T1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3';

Primer T2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3';

Preparation of transposomes

30 μM samples (transposase 1-5 and wild-type transposase) were respectively mixed with 60 μM annealed Adapter Mix in Coupling buffer (50 mM Tris-Ac at PH8.2, 400 mM NaCl, 0.1 mM EDTA, 0.25% Tween-20, 25% glycerol) was mixed and incubated at room temperature for 1 h to obtain transposomes.

DNA fragmentation

Take 1 μl of the above transposome, add it to the system containing 2×Tnp buffer (50mM TAPS at PH7.9, 5mM MgCl ₂ , 2mM trehalose, 0.1% NP-40) and 200ng human genome, and add water to 20 μl, Then incubate at 55 °C for 10 min in a thermal cycler (Lid 99 °C), take 10 μl of the reaction product, and detect the distribution range of the fragmented products by agarose gel electrophoresis. Please refer to Figure 1 and Table 1 for the results.

Table 1 Transposase stability results

Remarks: 1. +++ indicates a high degree of fragmentation, the band dispersion is mainly 100-500kb, and there are no fragments above 2000kb;

2. ++ indicates a high degree of fragmentation, the band dispersion is mainly 500-1000kb, and a small amount of fragments above 2000kb;

3. + means that the degree of fragmentation is low, and the bands are mainly scattered above 2000kb;

4. - means that it is basically impossible to fragment, and the bands are not diffused.

Figure 1 shows the stability test results of transposase 1 and wild-type transposase, wherein 1 to 3 are the detection of wild-type transposase stored at -20°C, -4°C, and 25°C for 7 days, respectively. As a result, 4 to 6 are the results of storing transposase 1 under conditions of -20°C, -4°C, and 25°C for 7 days, respectively.

It can be seen from Table 1 that the stability of the single mutations K120Q, E146N, E190A, Y41R, M56G and R27T in the test example has been improved compared to the wild-type transposase;

Combined with Table 1 and Figure 1, the stability of the transposase mutant provided in Example 1 of the present invention is significantly better than that of the wild type and the transposase mutants (single mutation) in Test Examples 1 to 8, which can be in -20 Long-term storage at ℃, and even short-term storage at 4℃, avoids the inconvenience caused by storage at -80℃, and there is no need to construct it as a transposome for storage, which greatly improves the efficiency of storage. Improve the convenience of use and system adjustment.

Verification example 2

The stability of the fusion protein prepared in Example 2 was tested.

The detection method is the same as the verification example 1.

Please refer to Table 2 for the test results.

Table 2 Stability evaluation results

It can be seen from Table 2 that the stability of the fusion protein constructed by the transposase mutant is significantly higher than that of the fusion protein constructed by the wild-type transposase.

Verification example 3

The ability of the fusion protein prepared in Example 2 to bind to the antibody was verified.

The enzyme-linked immune reaction (ELISA) principle was used to detect the ability of different fusion proteins to bind to various enzyme-labeled antibodies (IgG). The specific detection process is as follows.

The fusion proteins 1-12 provided in Example 2 were all diluted to a uniform concentration (0.8 nmol/ml, ie 0.6 mg/ml) with a diluent. Add 100 μl of the diluted fusion protein to the corresponding wells of the ELISA plate. Each sample is set up with 24 parallel groups. At the same time, 3 reaction wells correspond to one enzyme-labeled antibody to ensure the accuracy of the data. Replace with ddH ₂ O, place the plate to incubate at 37°C for 30 min after sealing; remove the liquid in the wells and wash with washing solution 4 times to completely remove the residual washing solution in the wells, then add 250 μl blocking solution to each well, and then seal the plate. Incubate at 37°C for 1h, dilute various enzyme-labeled antibodies (mouse, rabbit, dog, sheep, cattle, horse, human, pig) to (0.4nmol/ml) with diluent, and add them to the corresponding reaction wells respectively. 250 μl of the plate was sealed and incubated at 37°C for 30 min; the liquid in the well was removed and washed 4 times with washing solution and the residual washing solution in the well was completely removed, then 100 μl of chromogenic solution was added to each well, and the plate was placed at room temperature after sealing. (20-25°C) Incubate in the dark for 15 min, then add 50 μl of stop solution to each well, and measure the OD450 value with a microplate reader immediately after termination.

Taking the value of OD450 as the ordinate and the types of various enzyme-labeled antibodies as the abscissa, it can be seen that the binding effect of different fusion proteins and various enzyme-labeled antibodies is shown in Figure 2.

It can be seen from Figure 2 that fusion proteins 7-12 can effectively bind to different types of antibodies. It can be seen that the fusion proteins constructed by PA2 have no obvious bias in the binding ability of antibodies from different species, and their application scenarios are relatively wider.

Verification Example 4

The protein-chromatin interaction analysis (ChIP-Seq technology) was performed using the fusion protein provided in Example 2, which included the following steps.

Prepare Adapter Mix

Mix 100 μM Primer M with equal volumes of 100 μM Primer T1 and 100 μM Primer T2, respectively, and then anneal them. Then mix the annealed products of M-T1 and M-T2 in equal volumes. The resulting mixture is the Adapter Mix. The Primer sequence is as follows:

Primer M: 5'-pCTGTCTCTTATACACATCT-3' (p refers to phosphorylation modification);

Primer T1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3';

Primer T2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'.

Preparation of transposomes

Mix 30 μM target protein (fusion protein 1-5) with 60 μM Adapter Mix in Couplingbuffer (50 mM Tris-Ac at PH8.2, 400 mM NaCl, 0.1 mM EDTA, 0.25% Tween-20, 25% glycerol). Incubate at room temperature for 1 h to obtain transposomes.

DNA fragmentation

Take 1 μl of the above transposome, add it to a system containing 2×Tnp buffer (50mM TAPS at PH7.9, 5mM MgCl ₂ , 2mM trehalose, 0.1% NP-40), 50ng human genome, and add water to 20 μl, Then incubate in a thermocycler for 10 min (Lid 99°C), add 1 μl of digestion buffer to the reaction system, and then incubate at 55°C in a thermocycler for 5 min (Lid 99°C) to terminate.

The reaction system was then filled with water to 50 μl and purified with 1×FP Beads. The purified product was amplified using the FP NM008 kit, and the amplified product was subjected to two rounds of fragment sorting with 0.6×/0.15× FP Beads to obtain the final The library was sequenced with illumina Hiseq, and the sequencing strategy was PE150. The sequencing data comparison is shown in Table 3.

Table 3 Sequencing data comparison results

It can be seen from Table 3 that the fusion proteins provided in the embodiments of the present invention can all be used for library construction and sequencing. Among them, from the analysis of coverage and effective data rate, it can be found that fusion proteins 1 and 3 have better effects.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

SEQUENCE LISTING

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465 470 475

<210> 6

<211> 1428

<212> DNA

<213> Artificial sequences

<400> 6

atgatcacct ctgctctgca ccgtgctgct gactgggcta aatctgtttt ctcttctgct 60

gctctgggtg acccgcgtac caccgctcgt ctggttaacg ttgctgctca gctggctaaa 120

tactctggta aatctatcac catctcttct gaaggttctg aagctggtca ggaaggtgct 180

taccgtttca tccgtaaccc gaacgtttct gctgaagcta tccgtaaagc tggtgctatg 240

cagaccgtta aactggctca ggaatttccg gaactgctgg ctatcgaaga caccacctct 300

ctgtcttacc gtcaccaggt tgctgaagaa ctgggtaaac tgggttctat ccaggacaaa 360

tctcgtggtt ggtgggttca ctctgttctg ctgctggaag ctaccacctt ccgtaccgtt 420

ggtctgctgc accaggaatg gtggatgcgt ccggacgacc cggctgacgc tgacgaaaaa 480

gaatctggta aatggctggc tgctgctgct acctctcgtc tgcgtatggg ttctatgatg 540

tctaacgtta tcgctgtttg cgaccgtgaa gctgacatcc acgcttacct gcaggacaaa 600

ctggctcaca acgaacgttt cgttgttcgt tctaaacacc cgcgtaaaga cgttgaatct 660

ggtctgtacc tgtacgacca cctgaaaaac cagccggaac tgggtggtta ccagatctct 720

atcccgcaga aaggtgttgt tgacaaacgt ggtaaacgta aaaaccgtcc ggctcgtaaa 780

gcttctctgt ctctgcgttc tggtcgtatc accctgaaac agggtaacat caccctgaac 840

gctgttctgg ctgaagaaat caacccgccg aaaggtgaaa ccccgctgaa atggctgctg 900

ctgacctctg aaccggttga atctctggct caggctctgc gtgttatcga catctacacc 960

caccgttggc gtatcgaaga atttcacaaa gcttggaaaa ccggtgctgg tgctgaacgt 1020

cagcgtatgg aagaaccgga caacctggaa cgtatggttt ctatcctgtc tttcgttgct 1080

gttcgtctgc tgcagctgcg tgaatctttc accctgccgc aggctctgcg tgctcagggt 1140

ctgctgaaag aagctgaaca cgttgaatct cagtctgctg aaaccgttct gaccccggac 1200

gaatgccagc tgctgggtta cctggacaaa ggtaaacgta aacgtaaaga aaaagctggt 1260

tctctgcagt gggcttacat ggctatcgct cgtctgggtg gtttcatgga ctctaaacgt 1320

accggtatcg cttcttgggg tgctctgtgg gaaggttggg aagctctgca gtcgaagctg 1380

gacggcttcc tggctgctaa ggacctgatg gctcagggta tcaaaatc 1428

<210> 7

<211> 476

<212> PRT

<213> Artificial sequences

<400> 7

Met Ile Thr Ser Ala Leu His Arg Ala Ala Asp Trp Ala Lys Ser Val

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Glu Gly Ser Glu Ala Gly Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60

Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met

65 70 75 80

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

85 90 95

Asp Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110

Lys Leu Gly Ser Ile Gln Asp Lys Ser Arg Gly Trp Trp Val His Ser

115 120 125

Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130 135 140

Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys

145 150 155 160

Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175

Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Asp Arg Glu Ala Asp

180 185 190

Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205

Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220

Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser

225 230 235 240

Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 250 255

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

260 265 270

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

275 280 285

Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Leu Thr Ser Glu

290 295 300

Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr

305 310 315 320

His Arg Trp Arg Ile Glu Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335

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

340 345 350

Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365

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

370 375 380

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

385 390 395 400

Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415

Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430

Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445

Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450 455 460

Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile

465 470 475

<210> 8

<211> 1428

<212> DNA

<213> Artificial sequences

<400> 8

atgatcacct ctgctctgca ccgtgctgct gactgggcta aatctgtttt ctcttctgct 60

gctctgggtg acccgcgtcg taccgctcgt ctggttaacg ttgctgctca gctggctaaa 120

cgttctggta aatctatcac catctcttct gaaggttctg aagctggtca ggaaggtgct 180

taccgtttca tccgtaaccc gaacgtttct gctgaagcta tccgtaaagc tggtgctatg 240

cagaccgtta aactggctca ggaatttccg gaactgctgg ctatcgaaga caccacctct 300

ctgtcttacc gtcaccaggt tgctgaagaa ctgggtaaac tgggttctat ccaggacaaa 360

tctcgtggtt ggtgggttca ctctgttctg ctgctggaag ctaccacctt ccgtaccgtt 420

ggtctgctgc accaggaatg gtggatgcgt ccggacgacc cggctgacgc tgacgaaaaa 480

gaatctggta aatggctggc tgctgctgct acctctcgtc tgcgtatggg ttctatgatg 540

tctaacgtta tcgctgtttg cgaccgtgaa gctgacatcc acgcttacct gcaggacaaa 600

ctggctcaca acgaacgttt cgttgttcgt tctaaacacc cgcgtaaaga cgttgaatct 660

ggtctgtacc tgtacgacca cctgaaaaac cagccggaac tgggtggtta ccagatctct 720

atcccgcaga aaggtgttgt tgacaaacgt ggtaaacgta aaaaccgtcc ggctcgtaaa 780

gcttctctgt ctctgcgttc tggtcgtatc accctgaaac agggtaacat caccctgaac 840

gctgttctgg ctgaagaaat caacccgccg aaaggtgaaa ccccgctgaa atggctgctg 900

ctgacctctg aaccggttga atctctggct caggctctgc gtgttatcga catctacacc 960

caccgttggc gtatcgaaga atttcacaaa gcttggaaaa ccggtgctgg tgctgaacgt 1020

cagcgtatgg aagaaccgga caacctggaa cgtatggttt ctatcctgtc tttcgttgct 1080

gttcgtctgc tgcagctgcg tgaatctttc accctgccgc aggctctgcg tgctcagggt 1140

ctgctgaaag aagctgaaca cgttgaatct cagtctgctg aaaccgttct gaccccggac 1200

gaatgccagc tgctgggtta cctggacaaa ggtaaacgta aacgtaaaga aaaagctggt 1260

tctctgcagt gggcttacat ggctatcgct cgtctgggtg gtttcatgga ctctaaacgt 1320

accggtatcg cttcttgggg tgctctgtgg gaaggttggg aagctctgca gtcgaagctg 1380

gacggcttcc tggctgctaa ggacctgatg gctcagggta tcaaaatc 1428

<210> 9

<211> 476

<212> PRT

<213> Artificial sequences

<400> 9

Met Ile Thr Ser Ala Leu His Arg Ala Ala Asp Trp Ala Lys Ser Val

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Glu Gly Ser Lys Ala Gly Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60

Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met

65 70 75 80

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

85 90 95

Asp Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110

Lys Leu Gly Ser Ile Gln Asp Lys Ser Arg Gly Trp Trp Val His Ser

115 120 125

Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130 135 140

Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys

145 150 155 160

Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175

Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Asp Arg Glu Ala Asp

180 185 190

Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205

Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220

Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser

225 230 235 240

Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 250 255

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

260 265 270

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

275 280 285

Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Leu Thr Ser Glu

290 295 300

Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr

305 310 315 320

His Arg Trp Arg Ile Glu Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335

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

340 345 350

Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365

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

370 375 380

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

385 390 395 400

Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415

Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430

Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445

Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450 455 460

Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile

465 470 475

<210> 10

<211> 1428

<212> DNA

<213> Artificial sequences

<400> 10

atgatcacct ctgctctgca ccgtgctgct gactgggcta aatctgtttt ctcttctgct 60

gctctgggtg acccgcgtcg taccgctcgt ctggttaacg ttgctgctca gctggctaaa 120

cgttctggta aatctatcac catctcttct gaaggttcta aagctggtca ggaaggtgct 180

taccgtttca tccgtaaccc gaacgtttct gctgaagcta tccgtaaagc tggtgctatg 240

cagaccgtta aactggctca ggaatttccg gaactgctgg ctatcgaaga caccacctct 300

ctgtcttacc gtcaccaggt tgctgaagaa ctgggtaaac tgggttctat ccaggacaaa 360

tctcgtggtt ggtgggttca ctctgttctg ctgctggaag ctaccacctt ccgtaccgtt 420

ggtctgctgc accaggaatg gtggatgcgt ccggacgacc cggctgacgc tgacgaaaaa 480

gaatctggta aatggctggc tgctgctgct acctctcgtc tgcgtatggg ttctatgatg 540

tctaacgtta tcgctgtttg cgaccgtgaa gctgacatcc acgcttacct gcaggacaaa 600

ctggctcaca acgaacgttt cgttgttcgt tctaaacacc cgcgtaaaga cgttgaatct 660

ggtctgtacc tgtacgacca cctgaaaaac cagccggaac tgggtggtta ccagatctct 720

atcccgcaga aaggtgttgt tgacaaacgt ggtaaacgta aaaaccgtcc ggctcgtaaa 780

gcttctctgt ctctgcgttc tggtcgtatc accctgaaac agggtaacat caccctgaac 840

gctgttctgg ctgaagaaat caacccgccg aaaggtgaaa ccccgctgaa atggctgctg 900

ctgacctctg aaccggttga atctctggct caggctctgc gtgttatcga catctacacc 960

caccgttggc gtatcgaaga atttcacaaa gcttggaaaa ccggtgctgg tgctgaacgt 1020

cagcgtatgg aagaaccgga caacctggaa cgtatggttt ctatcctgtc tttcgttgct 1080

gttcgtctgc tgcagctgcg tgaatctttc accctgccgc aggctctgcg tgctcagggt 1140

ctgctgaaag aagctgaaca cgttgaatct cagtctgctg aaaccgttct gaccccggac 1200

gaatgccagc tgctgggtta cctggacaaa ggtaaacgta aacgtaaaga aaaagctggt 1260

tctctgcagt gggcttacat ggctatcgct cgtctgggtg gtttcatgga ctctaaacgt 1320

accggtatcg cttcttgggg tgctctgtgg gaaggttggg aagctctgca gtcgaagctg 1380

gacggcttcc tggctgctaa ggacctgatg gctcagggta tcaaaatc 1428

<210> 11

<211> 476

<212> PRT

<213> Artificial sequences

<400> 11

Met Ile Thr Ser Ala Leu His Arg Ala Ala Asp Trp Ala Lys Ser Val

1 5 10 15

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

20 25 30

Asn Val Ala Ala Gln Leu Ala Lys Tyr Ser Gly Lys Ser Ile Thr Ile

35 40 45

Ser Ser Glu Gly Ser Glu Ala Met Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60

Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met

65 70 75 80

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

85 90 95

Asp Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110

Lys Leu Gly Ser Ile Gln Asp Leu Ser Arg Gly Trp Trp Val His Ser

115 120 125

Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130 135 140

Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys

145 150 155 160

Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175

Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Asp Arg Ala Ala Asp

180 185 190

Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205

Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220

Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser

225 230 235 240

Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 250 255

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

260 265 270

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

275 280 285

Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Leu Thr Ser Glu

290 295 300

Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr

305 310 315 320

His Arg Trp Arg Ile Glu Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335

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

340 345 350

Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365

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

370 375 380

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

385 390 395 400

Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415

Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430

Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445

Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450 455 460

Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile

465 470 475

<210> 12

<211> 1428

<212> DNA

<213> Artificial sequences

<400> 12

atgatcacct ctgctctgca ccgtgctgct gactgggcta aatctgtttt ctcttctgct 60

gctctgggtg acccgcgtcg taccgctcgt ctggttaacg ttgctgctca gctggctaaa 120

tactctggta aatctatcac catctcttct gaaggttctg aagctatgca ggaaggtgct 180

taccgtttca tccgtaaccc gaacgtttct gctgaagcta tccgtaaagc tggtgctatg 240

cagaccgtta aactggctca ggaatttccg gaactgctgg ctatcgaaga caccacctct 300

ctgtcttacc gtcaccaggt tgctgaagaa ctgggtaaac tgggttctat ccaggacctg 360

tctcgtggtt ggtgggttca ctctgttctg ctgctggaag ctaccacctt ccgtaccgtt 420

ggtctgctgc accaggaatg gtggatgcgt ccggacgacc cggctgacgc tgacgaaaaa 480

gaatctggta aatggctggc tgctgctgct acctctcgtc tgcgtatggg ttctatgatg 540

tctaacgtta tcgctgtttg cgaccgtgct gctgacatcc acgcttacct gcaggacaaa 600

ctggctcaca acgaacgttt cgttgttcgt tctaaacacc cgcgtaaaga cgttgaatct 660

ggtctgtacc tgtacgacca cctgaaaaac cagccggaac tgggtggtta ccagatctct 720

atcccgcaga aaggtgttgt tgacaaacgt ggtaaacgta aaaaccgtcc ggctcgtaaa 780

gcttctctgt ctctgcgttc tggtcgtatc accctgaaac agggtaacat caccctgaac 840

gctgttctgg ctgaagaaat caacccgccg aaaggtgaaa ccccgctgaa atggctgctg 900

ctgacctctg aaccggttga atctctggct caggctctgc gtgttatcga catctacacc 960

caccgttggc gtatcgaaga atttcacaaa gcttggaaaa ccggtgctgg tgctgaacgt 1020

cagcgtatgg aagaaccgga caacctggaa cgtatggttt ctatcctgtc tttcgttgct 1080

gttcgtctgc tgcagctgcg tgaatctttc accctgccgc aggctctgcg tgctcagggt 1140

ctgctgaaag aagctgaaca cgttgaatct cagtctgctg aaaccgttct gaccccggac 1200

gaatgccagc tgctgggtta cctggacaaa ggtaaacgta aacgtaaaga aaaagctggt 1260

tctctgcagt gggcttacat ggctatcgct cgtctgggtg gtttcatgga ctctaaacgt 1320

accggtatcg cttcttgggg tgctctgtgg gaaggttggg aagctctgca gtcgaagctg 1380

gacggcttcc tggctgctaa ggacctgatg gctcagggta tcaaaatc 1428

<210> 13

<211> 476

<212> PRT

<213> Artificial sequences

<400> 13

Met Ile Thr Ser Ala Leu His Arg Ala Ala Asp Trp Ala Lys Ser Val

1 5 10 15

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

20 25 30

Asn Val Ala Ala Gln Leu Ala Lys Tyr Ser Gly Lys Ser Ile Thr Ile

35 40 45

Ser Ser Glu Gly Ser Glu Ala Met Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60

Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met

65 70 75 80

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

85 90 95

Asp Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110

Lys Leu Gly Ser Ile Gln Asp Gln Ser Arg Gly Trp Trp Val His Ser

115 120 125

Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130 135 140

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

145 150 155 160

Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175

Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Asp Arg Glu Ala Asp

180 185 190

Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205

Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220

Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser

225 230 235 240

Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 250 255

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

260 265 270

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

275 280 285

Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Leu Thr Ser Glu

290 295 300

Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr

305 310 315 320

His Arg Trp Arg Ile Glu Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335

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

340 345 350

Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365

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

370 375 380

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

385 390 395 400

Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415

Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430

Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445

Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450 455 460

Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile

465 470 475

<210> 14

<211> 1428

<212> DNA

<213> Artificial sequences

<400> 14

atgatcacct ctgctctgca ccgtgctgct gactgggcta aatctgtttt ctcttctgct 60

gctctgggtg acccgcgtcg taccgctcgt ctggttaacg ttgctgctca gctggctaaa 120

tactctggta aatctatcac catctcttct gaaggttctg aagctatgca ggaaggtgct 180

taccgtttca tccgtaaccc gaacgtttct gctgaagcta tccgtaaagc tggtgctatg 240

cagaccgtta aactggctca ggaatttccg gaactgctgg ctatcgaaga caccacctct 300

ctgtcttacc gtcaccaggt tgctgaagaa ctgggtaaac tgggttctat ccaggaccag 360

tctcgtggtt ggtgggttca ctctgttctg ctgctggaag ctaccacctt ccgtaccgtt 420

ggtctgctgc accagaactg gtggatgcgt ccggacgacc cggctgacgc tgacgaaaaa 480

gaatctggta aatggctggc tgctgctgct acctctcgtc tgcgtatggg ttctatgatg 540

tctaacgtta tcgctgtttg cgaccgtgaa gctgacatcc acgcttacct gcaggacaaa 600

ctggctcaca acgaacgttt cgttgttcgt tctaaacacc cgcgtaaaga cgttgaatct 660

ggtctgtacc tgtacgacca cctgaaaaac cagccggaac tgggtggtta ccagatctct 720

atcccgcaga aaggtgttgt tgacaaacgt ggtaaacgta aaaaccgtcc ggctcgtaaa 780

gcttctctgt ctctgcgttc tggtcgtatc accctgaaac agggtaacat caccctgaac 840

gctgttctgg ctgaagaaat caacccgccg aaaggtgaaa ccccgctgaa atggctgctg 900

ctgacctctg aaccggttga atctctggct caggctctgc gtgttatcga catctacacc 960

caccgttggc gtatcgaaga atttcacaaa gcttggaaaa ccggtgctgg tgctgaacgt 1020

cagcgtatgg aagaaccgga caacctggaa cgtatggttt ctatcctgtc tttcgttgct 1080

gttcgtctgc tgcagctgcg tgaatctttc accctgccgc aggctctgcg tgctcagggt 1140

ctgctgaaag aagctgaaca cgttgaatct cagtctgctg aaaccgttct gaccccggac 1200

gaatgccagc tgctgggtta cctggacaaa ggtaaacgta aacgtaaaga aaaagctggt 1260

tctctgcagt gggcttacat ggctatcgct cgtctgggtg gtttcatgga ctctaaacgt 1320

accggtatcg cttcttgggg tgctctgtgg gaaggttggg aagctctgca gtcgaagctg 1380

gacggcttcc tggctgctaa ggacctgatg gctcagggta tcaaaatc 1428

Claims (21)

1. a transposase mutant, it is characterized in that, described transposase mutant has R27T mutation compared with wild-type transposase; The aminoacid sequence of described wild-type transposase is as shown in SEQ ID No.1 Show. 2. The transposase mutant according to claim 1, wherein the transposase mutant has a mutation combination shown in (1) below compared to the wild-type transposase; (1) R27T and M56G. 3. a fusion protein is characterized in that, it comprises the described transposase mutant of claim 1 or 2; The fusion protein also includes a binding protein for binding to the Fc fragment of an immunoglobulin, the binding protein being linked to the transposase mutant. 4. The fusion protein according to claim 3, wherein the binding protein comprises at least one of Staphylococcus aureus protein A, Streptococcus protein G and Streptococcus L protein. 5 . The fusion protein according to claim 3 , wherein the amino acid sequence of the binding protein is shown in SEQ ID No. 2 or 3. 6 . 6. The fusion protein according to claim 3, wherein the binding protein is located at the N-terminus of the amino acid sequence of the fusion protein, and the transposase mutant is located at the C-terminus of the amino acid sequence of the fusion protein . 7. The fusion protein of claim 3, wherein the binding protein is linked to the transposase mutant through a linker peptide. 8. The fusion protein according to claim 7, wherein the connecting peptide is a rigid connecting peptide or a flexible connecting peptide. 9. The fusion protein of claim 7, wherein the connecting peptide is a flexible connecting peptide. 10 . The fusion protein according to claim 7 , wherein the length of the connecting peptide is 6-59 amino acids. 11 . 11. The fusion protein according to claim 7, wherein the length of the connecting peptide is 20-35 amino acids. 12 . The fusion protein according to claim 7 , wherein the sequence of the connecting peptide is shown in SEQ ID No. 4. 13 . 13. An isolated nucleic acid, characterized in that it encodes the transposase mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3-12. 14. A vector comprising the isolated nucleic acid of claim 13. 15. A host cell comprising the vector of claim 14. 16. The method for preparing the transposase mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3-12, characterized in that it comprises culturing the host according to claim 15 cell. 17. A kit for constructing a sequencing library, characterized in that it comprises the transposase mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3-12. 18. Use of the transposase mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 12 in DNA library construction. 19. Use of the transposase mutant according to claim 1 or 2 or the fusion protein according to any one of claims 3 to 12 in analyzing the interaction of proteins with chromatin. 20. The application of claim 19, wherein the application comprises incubating a mixture system containing the target protein to be analyzed, the target chromatin, and the transposase mutant. 21. The application according to claim 20, wherein the mixture system further comprises an antibody to a target protein.

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