CN106832001B - A kind of insecticidal fusion protein, encoding gene and application thereof - Google Patents

Abstract

The invention discloses an insecticidal fusion protein, which comprises IPD072 protein and Vip3 protein. Compared with the original IPD072 protein and Vip3 protein, the artificial protein molecule formed by the fusion of the IPD072 protein and a Vip3 toxin has the following advantages: broad insecticidal spectrum, and can simultaneously prevent and treat various species in Lepidoptera and Coleoptera. Important pests (such as beet armyworm, armyworm, corn rootworm), the insecticidal efficiency is as high as 50%-100%; it is beneficial to use proteins with the above different functions in combination with other insect-resistant proteins (such as ICPs) to further expand insecticidal spectrum, delaying the development of pest resistance.

Description

A kind of insecticidal fusion protein, encoding gene and application thereof

(1) Technical field

The present invention relates to an insecticidal fusion protein, an encoding gene of the insecticidal fusion, and the application of the fusion protein.

(2) Background technology

Pests bring huge losses to global agricultural production. At present, pest control mainly relies on chemical pesticides, but the use of pesticides increases production costs, and pesticide residues bring serious harm to human health. Therefore, the use of genetic engineering methods to control pests has great economic, environmental and social value.

The key to obtaining transgenic insect-resistant crops is to clone excellent insecticidal proteins. There are many insecticidal proteins, the most widely used is a paracellular crystal protein (insecticidal crystal proteins, ICP) secreted by Bacillus thuringiensis (Bt) during cell production, such as Cry1Ab, Cry1C, etc. Lepidoptera, Diptera, Coleoptera and other insects (such as diamondback moth, corn borer) have a strong killing effect (Schnepf, E., Crickmore, N., Van, R.J., Lereclus, D., Baum, J. , & Feitelson, J., et al. 1998, 62 (3), 775-806.), has been in-depth research and widely used, corn, soybeans, potatoes, cotton and other Bt genetically modified crops have been large-scale commercial planting . During vegetative growth, Bacillus thuringiensis also secretes a protein that has no amino acid sequence homology with ICPs and has a completely different insecticidal mechanism, namely vegetative insecticidal proteins (VIPs), such as Vip3A, Vip3B, Vip3C, Vip3D, Vip3H, also have insecticidal activity against some pests that are not sensitive to ICPs, and do not cross-resistance (Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., & Koziel, M.G. 1996, Proceedings of the National Academy of Sciences of the United States of America, 93(11), 5389-94.).

As a new type of insecticidal protein, Vip3 protein is a good supplement to the most widely used ICP protein in terms of insecticidal mechanism, insecticidal spectrum and insecticidal activity. For example, the killing effect of ICPs on Spodoptera litura and armyworm is weak, while Vip3 has a very good control effect on Spodoptera litura and armyworm. Nevertheless, there are still some urgent technical problems to be solved in the practical operation of genetically modified crop research and development. For example, the researchers found that plants with high expression of the Vip3 gene alone were stunted and prone to bleaching. This may be due to the fact that after the direct expression of Vip3 protein in transgenic plants, the protein aggregates and binds to the plant cell membrane and forms pores due to the secretion of signal peptides, thereby causing damage to cells and affecting their growth and development.

Corn is the most productive food crop in the world, but it also faces many threats from pests. And of these pests, the root firefly beetle has a particularly chilling name—one of the most destructive corn pests. After the larvae of the root firefly beetle invade the rhizome of the host, they will devour the tissue, causing root rot and plant lodging, resulting in reduced yield and even death of the plant. Adults also feed on the leaves, flowers, and seeds of their hosts. Before 1955, they were only distributed in a limited number of states in the United States, but after that, they invaded the northern part of the United States, which was not suitable for them to survive. In 1990, the turmoil in Eastern Europe, this pest was accidentally introduced into Serbia at this time, so the European continent, especially Eastern Europe, also became their territory. In recent years, it has also spread to the territory of China. The root firefly beetle is the collective name of the root firefly beetle (Diabrotica sp.) insects. In the United States, several types of root beetles that damage corn are called Corn Rootworm, and the three most harmful are called "Western Corn Rootworm", "Northern Corn Rootworm" and "Southern Corn Rootworm" ". The proteins with the killing effect of corn rootworm found at present are mainly Cry protein and Vip protein from Bacillus thuringiensis. Among them, mCry3A, Cry3Bb1, eCry3.1Ab and the protein complex Cry34Ab1/Cry35Ab1 have been used in commercial transgenic lines, and have produced different degrees of resistance (Lefko S A, Nowatzki T M, Thompson S D, et al.2008, Journal of Applied Entomology, 132(3), 189-204; Zhao, J.Z., Oneal, M.A., Richtman, N.M., Thompson, S.D., Cowart, M.C., & Nelson, M.E., et al. 2016, Journal of Economic Entomology (3)). Studies have also shown that RNAi technology can be used for the control of coleopteran pests such as corn rootworm (Baum, J.A., Bogaert, T., Clinton, W., Heck, G.R., Feldmann, P., & Ilagan, O., et al. 2007, Nature Biotechnology, 25(11), 1322-6.). Recently, a gene IPD072Aa cloned by DuPont from Pseudomonas has the effect of killing corn rootworm (Schellenberger, U., Oral, J., Rosen, B.A., Wei, J.Z., Zhu, G., &Xie, W., et al. 2016, Science, 354(6312), 635-637.).

Most of the first-generation transgenic crops only have the characteristics of resistance to lepidopteran pests, while the second-generation transgenic crops are developing towards complex traits that are resistant to multiple pests at the same time. There are many ways to obtain transgenic crops with complex traits, such as by crossing transgenic crops with a single trait; by constructing multiple gene expression cassettes in the same expression cassette; Agrobacterium is mixed together for mixed transformation, and transgenic plants with multiple genes integrated with two or more plasmid T-DNAs at the same time are screened out. However, the above methods still have some problems in practical operation and application. Therefore, obtaining a simpler and more efficient method for cultivating transgenic crops with complex traits is a problem that needs to be practically solved at present.

(3) Contents of the invention

The purpose of the present invention is to provide a method for making the same insecticidal fusion protein have killing effect on lepidopteran and coleopteran pests at the same time through gene fusion.

The technical scheme adopted in the present invention is:

The present invention provides an insecticidal fusion protein, wherein the insecticidal fusion protein is IPD072 protein and Vip3 toxin from N-terminus to C-terminus, respectively.

Further, the IPD072 protein is one of IPD072Aa protein, IPD072Bb protein, IPD072Ca protein or IPD072Da protein; the Vip toxin is one of Vip3A toxin, Vip3B toxin, Vip3C toxin, Vip3D toxin or Vip3H toxin.

Further, the insecticidal fusion protein is formed by fusion of IPD072Aa protein and Vip3A toxin, and the amino acid sequence is shown in SEQ ID NO: 2.

Further, the insecticidal fusion protein is formed by fusion of IPD072Aa protein and Vip3H toxin, and the amino acid sequence is shown in SEQ ID NO: 4.

The present invention also provides a gene encoding the insecticidal fusion protein, the genes are the nucleotide sequence encoding the IPD072 protein and the nucleotide sequence encoding the Vip3 toxin in order from 5' to 3'; and the above two The nucleotide sequences are in the same open reading frame.

Further, the gene encoding the IPD072 protein is one of IPD072Aa, IPD072Bb, IPD072Ca, and IPD072Da; the gene encoding the Vip3 toxin is one of Vip3A, Vip3B, Vip3C, Vip3D or Vip3H. More preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 1 or SEQ ID NO: 3.

The present invention also provides an application of the insecticidal fusion protein in the preparation of transgenic insect-resistant crops, transgenic insecticidal microorganisms or antibodies.

Further, the transgenic insect-resistant crop is prepared by transforming the fusion protein and BT crystal toxin.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the artificial protein molecule fused into the IPD072 protein designed by the present invention and a Vip3 toxin has the following advantages compared with the original IPD072 protein and the Vip3 protein: Wide spectrum, can simultaneously control a variety of important pests in Lepidoptera and Coleoptera (such as beet armyworm, armyworm, corn rootworm), the insecticidal efficiency is as high as 50%-100%; Other insect-resistant proteins (such as ICPs) are used in combination to further expand the insecticidal spectrum and delay the development of pest resistance.

(4) Description of drawings

Fig. 1 is a structural diagram of the insecticidal fusion protein of the present invention. In the insecticidal fusion protein, the N-terminal is IPD072 protein, and the C-terminal is Vip3 toxin.

Fig. 2 is an expression block diagram of the insecticidal fusion protein gene introduced into plants in the present invention; pUBI is a maize ubiquitin promoter, and the insecticidal fusion protein gene is an IPD072-Vip3 toxin fusion gene.

Fig. 3 is the structure diagram of the expression box in which the insecticidal fusion protein and ICPs are combined into plants in the present invention; pUBI is the maize ubiquitin promoter, the insecticidal fusion protein gene is the IPD072-Vip3 toxin fusion gene, and p35S is the cauliflower mosaic virus 35S promoter, ICPs is the gene encoding paracytosine crystal protein.

(5) Specific implementation manner

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1. Construction of IPD072Aa-Vip3A insecticidal fusion protein expression vector

The genes of IPD072Aa and Vip3A insecticidal proteins were both synthesized by Shanghai Shenggong, and their DNA sequences were SEQ ID NO: 5 and SEQ ID NO: 7 in Chinese Patent 200610049611.0 respectively, and were cloned into the restriction endonucleases BamHI and BamHI of the pET28a expression vector. between SacI sites. The constructed vectors were named pET28a-IPD072Aa and pET28a-Vip3A, respectively.

The specific steps for the synthesis of IPD072Aa-Vip3A are as follows:

1. Using Vip3A (SEQ ID NO: 7 in Chinese Patent No. 200610049611.0) as a template to go through PCR to obtain a Vip3A gene fragment. The primers were: Vip3A-F: 5'CCCGGGAAGGGTGGAGGAATGAACAAGAACAACACCAAG and Vip3A-R: 5'CGAGCTCCTACTTGATGCTCACGTCGTAGAACTTCACGA. These two primers contain restriction endonuclease sites XmalI and ScaI sites, respectively.

2. The PCR product was treated with XmalI and ScaI, and ligated with the pET28a-IPD072Aa vector treated with the same enzymes.

3. Into E. coli, the obtained plasmid is pET28a-IPD072Aa-Vip3A. The fusion protein gene IPD072Aa-Vip3A (SEQ ID NO: 1) was cloned into the pET28a expression vector, and the fusion protein IPD072Aa-Vip3A (SEQ ID NO: 2) encoded by the fusion protein gene has a molecular weight of about 98kD.

Example 2. Construction of IPD072Aa-Vip3H fusion protein expression vector

The genes of IPD072Aa and Vip3H insecticidal proteins were both synthesized by Shanghai Shenggong, their DNA sequences are SEQ ID NO: 5 and SEQ ID NO: 6, respectively, and cloned between the restriction endonucleases BamHI and SacI sites of pET28a expression vector . The constructed vectors were named pET28a-IPD072Aa and pET28a-Vip3H, respectively.

The specific steps of IPD072Aa-Vip3H synthesis are as follows:

4. Using Vip3H (SEQ ID NO: 6) as a template to go through PCR to obtain the Vip3A gene fragment. The primers were: Vip3H-F: 5'CCCGGGAAGGGTGGAGGAATGAACAAGAACAACAGTAAG and Vip3H-R: 5'CGAGCTCCTACTTGATGCTGAAGTCCCTGAAGGTGATGT. These two primers contain restriction endonuclease sites XmalI and ScaI sites, respectively.

5. The PCR product was treated with XmalI and ScaI and ligated with the pET28a-IPD072Aa vector treated with the same enzymes.

6. Transform into E. coli to obtain plasmid pET28a-IPD072Aa-Vip3H. The fusion protein gene IPD072Aa-Vip3H (SEQ ID NO: 3) was cloned into the pET28a expression vector, and the fusion protein IPD072Aa-Vip3H (SEQ ID NO: 4) encoded by the fusion protein gene has a molecular weight of about 98kD.

Embodiment 3, the expression of insecticidal protein

The above-mentioned expression vectors containing fusion protein genes (pET28a-IPD072Aa-Vip3A, pET28a-IPD072Aa-Vip3H, pET28a-IPD072Aa, pET28a-Vip3A and pET28a-Vip3H) were transferred into Escherichia coli BL21star respectively using a general standard method. Pick a single clone colony and inoculate it into 100ml of LB bacterial culture solution, shake it at 37°C to OD ₆₀₀ =0.6, add IPTG to 0.5mM, and continue to incubate for 4 hours under the same conditions. The culture medium was collected and centrifuged at 5000g for 10 minutes to precipitate E. coli cells, and then the supernatant was discarded to collect the pellet. Add 30 ml of pH 7.5, 20 mM Tris-HCL buffer to the precipitate, and it can be used for the determination of insecticidal activity after ultrasonication.

Example 4. IPD072Aa-Vip3A and IPD072Aa-Vip3H have killing effects on various lepidopteran and coleopteran pests

The insecticidal proteins obtained in Example 3 (IPD072Aa-Vip3A, IPD072Aa-Vip3H, IPD072Aa, Vip3A and Vip3H) were coated on the surface of artificial insect feeds, respectively, and the first-instar larvae were used for insecticidal assay. The negative control was pET28a empty vector, and the preparation method was the same as that in Example 3. The results of insecticidal activity are shown in Table 1:

Table 1. Insecticidal efficiency of fusion proteins

armyworm Beet armyworm Western corn rootworm IPD072Aa 0% 0% 100% Vip3A 100% 100% 0% Vip3H 100% 100% 0% IPD072Aa-Vip3A 100% 100% 100% IPD072Aa-Vip3H 100% 100% 100% negative control 0% 0% 0%

Example 5. Construction of IPD072Aa-Vip3A plant transformation vector

The IPD072Aa-Vip3A gene fragment was obtained by PCR using IPD072Aa-Vip3A (SEQ ID NO: 1) as a template. The primers were: IVA-F: 5'AGGATCCAACAATGGGCATCACCGTGACCAAC and IVA-R: 5'CGAGCTCCTACTTGATGCTCACGTCGTAGAACTTC. These two primers contain restriction endonuclease sites BamH1 and ScaI sites, respectively. The artificially synthesized terminator sequence ter (SEQ ID NO: 8) is provided with ScaI and KpnI sites at the 5' and 3' ends, respectively. The IPD072Aa-Vip3A digested with BamH1 and ScaI and the terminator digested with ScaI and KpnI were ligated into the pCambia1300 vector digested with BamH1 and KpnI to obtain pCambia1300-IPD072Aa-Vip3A-ter.

pUBI is a maize ubiquitin protein promoter, obtained by PCR. pUBI was obtained by PCR amplification using the genomic DNA of the commercial maize variety Zhengdan 958 as a template. PCR reaction conditions were: 95°C for 3 minutes; 95°C for 15 seconds, 68°C for 15 seconds, 72°C for 2 minutes, repeating 32 cycles; then 72°C for 10 minutes. The obtained PCR product of approximately 2.0 Kb was cloned into the T-vector pMD19. A clone with the correct sequence was obtained for the following experiments. The PCR primers were pUBI-F (5'GAAGCTTGCATGCCTACAGTGCAGCGTGACCC) and pUBI-R (5'GGGTGGATCCTCTAGAGTCGACCTGCAGAAGTAAC), and HindIII and BamHI restriction endonuclease sites were set on the primers, respectively.

The pUBI was digested with HindIII and BamHI, and the digested fragment was ligated into the pCambia1300-IPD072Aa-Vip3A-ter vector digested with the same enzyme to obtain the final vector. This vector was named: pCambia1300-pUBI-IPD072Aa-Vip3A.

Finally, the above-mentioned T-DNA plasmid was transferred into Agrobacterium LBA4404 by electroporation, and the positive clones were screened out by YEP solid medium containing 15 μg/ml tetracycline and 50 μg/mL kanamycin, and the bacteria were preserved for inoculation. down the plant transformation.

Example 6. Construction of IPD072Aa-Vip3H plant transformation vector

The IPD072Aa-Vip3H gene fragment was obtained by PCR using IPD072Aa-Vip3H (SEQ ID NO: 3) as a template. The primers were: IVH-F: 5'AGGATCCAACAATGGGCATCACCGTGACCAAC and IVH-R: 5'CGAGCTCCTACTTGATGCTGAAGTCCCTGAAGG. These two primers contain restriction endonuclease sites BamH1 and ScaI sites, respectively. The artificially synthesized terminator sequence ter (SEQ ID NO: 8) was provided with ScaI and KpnI sites at the 5' and 3' ends, respectively. The IPD072Aa-Vip3H digested with BamH1 and ScaI and the terminator digested with ScaI and KpnI were ligated into the pCambia1300 vector digested with BamH1 and KpnI to obtain pCambia1300-IPD072Aa-Vip3H-ter.

The method for obtaining pUBI as a maize ubiquitin protein promoter is the same as that in Example 5.

The pUBI was digested with HindIII and BamHI, and the digested fragment was ligated into the pCambia1300-IPD072Aa-Vip3H-ter vector digested with the same enzyme to obtain the final vector. This vector was named: pCambia1300-pUBI-IPD072Aa-Vip3H.

Finally, the above-mentioned T-DNA plasmid was transferred into Agrobacterium LBA4404 by electroporation, and the positive clones were screened out by YEP solid medium containing 15 μg/ml tetracycline and 50 μg/mL kanamycin, and the bacteria were preserved for inoculation. down the plant transformation.

Example 7. Construction of IPD072Aa-Vip3A and ICPs protein plant transformation vector

The gene of the insecticidal protein of Cry1Ab (SEQ ID NO: 7) was synthesized by Shanghai Shenggong, and the 5' and 3' ends were respectively provided with BamH1 and ScaI sites. The terminator sequence ter (SEQ ID NO: 8) was synthesized artificially, and ScaI and EcoRI sites were set at the 5' and 3' ends, respectively. The Cry1Ab digested with BamHI and ScaI and the terminator digested with ScaI and EcoRI were ligated into the pCambia1300 vector digested with BamH1 and EcoRI to obtain pCambia1300-Cry1Ab-ter.

The p35S promoter is the cauliflower mosaic virus CaMV 35S promoter, which was synthesized by Shanghai Shenggong.

The pCambia1300-Cry1Ab-ter was digested with BamH1 and EcoRI, and p35S was digested with KpnI and BamHI. The above two fragments were ligated in three sections with the vector pCambia1300-IPD072Aa-Vip3A-ter digested with KpnI and EcoRI to obtain the final vector. This vector was named: pCambia1300-pUBI-IPD072Aa-Vip3A-p35S-Cry1Ab.

Finally, the above-mentioned T-DNA plasmid was transferred into Agrobacterium LBA4404 by electroporation, and the positive clones were screened out by YEP solid medium containing 15 μg/ml tetracycline and 50 μg/mL kanamycin, and the bacteria were preserved for inoculation. down the plant transformation.

Example 8. Construction of IPD072Aa-Vip3H and ICPs protein plant transformation vector

The gene of the insecticidal protein of Cry1Ab (SEQ ID NO: 7) was synthesized by Shanghai Shenggong, and the 5' and 3' ends were respectively provided with BamH1 and ScaI sites. The terminator sequence ter (SEQ ID NO: 8) was synthesized artificially, and ScaI and EcoRI sites were set at the 5' and 3' ends, respectively. The Cry1Ab digested with BamHI and ScaI and the terminator digested with ScaI and EcoRI were ligated into the pCambia1300 vector digested with BamH1 and EcoRI to obtain pCambia1300-Cry1Ab-ter.

The p35S promoter is the cauliflower mosaic virus CaMV 35S promoter, which was synthesized by Shanghai Shenggong.

The pCambia1300-Cry1Ab-ter was digested with BamH1 and EcoRI, and p35S was digested with KpnI and BamHI. The above two fragments were ligated in three sections with the vector pCambia1300-IPD072Aa-Vip3H-ter digested with KpnI and EcoRI to obtain the final vector. This vector was named: pCambia1300-pUBI-IPD072Aa-Vip3H-p35S-Cry1Ab.

Finally, the above-mentioned T-DNA plasmid was transferred into Agrobacterium LBA4404 by electroporation, and the positive clones were screened out by YEP solid medium containing 15 μg/ml tetracycline and 50 μg/mL kanamycin, and the bacteria were preserved for inoculation. down the plant transformation.

Example 9. Transgenic corn

The transformation technology of maize is relatively mature. References such as: Vladimir Sidorov & David Duncan (in M. Paul Scott (ed.), Methods in Molecular Biology: Transgenic Maize, vol: 526; YujiIshida, Yukoh Hiei & Toshihiko Komari (2007) Agrobacterium-mediated transformation of maize. Nature Protocols 2: 1614-1622. Basic Methods as below:

Hi-II corn ears 8-10 days after pollination were taken and all immature embryos (1.0-1.5 mm in size) were collected. The recombinant Agrobacterium and immature embryos obtained by transforming Agrobacterium with the T-DNA vector constructed in Example 8 were cultured on a co-cultivation medium (MS+2mg/L 2,4-D+30g/L sucrose+3g/L Agar (sigma 7921) + 40 mg/L acetosyringone) was co-cultured for 2-3 days (22°C). Transfer immature embryos to callus induction medium (MS+2mg/L 2,4-D+30 g/L sucrose+2.5g/Lgelrite+5mg/L AgNO3+200mg/L acetosyringone), dark at 28°C Culture for 10-14 days. All calli were transferred to selection medium (same as callus induction medium) with 2 mM glyphosate and cultivated in the dark at 28°C for 2-3 weeks. All tissues were transferred to fresh selection medium containing 2 mM glyphosate and incubated in the dark at 28°C for 2-3 weeks. Then, transfer all the viable embryogenic tissues after selection to regeneration medium (MS+30g/L sucrose+0.5mg/L kinetin+2.5g/L gelrite+200mg/L acetosyringone), and culture at 28°C in the dark for 10- 14 days, one strain per dish. Embryogenic tissue was transferred to fresh regeneration medium and incubated in the light at 26°C for 10-14 days. All fully developed plants were transferred to rooting medium (1/2MS+20g/L sucrose+2.5g/Lgelrite+200mg/L acetosyringone) and incubated in the light at 26°C until roots were fully developed. Transgenic maize plants containing the above transformation vector are obtained.

Example 10. Transgenic corn can kill insects

The TO generation plants of the transgenic maize plants prepared in Example 9 were transplanted into a greenhouse, pollinated with the pollen of the female parent of the commercial variety "Zhengdan 958" and "Zheng 58" (Z58), and the TO generation seeds were harvested. These lines were then backcrossed with the female parent of the commercial variety "Zheng Dan 958", "Zheng 58" (Z58), to obtain a Z58 near-allelic line. The insect resistance of these near-allelic lines was then compared.

We tested the insect resistance of 98 transgenic lines transformed with pCambia1300-IPD072Aa-Vip3A-ter vector (named IV3A) and 105 transgenic lines transformed with pCambia1300-IPD072Aa-Vip3H-ter vector (named IV3H). , its insecticidal effect is shown in Table 2:

Table 2*:

*Note: Table 2 shows the insecticidal effects of different pests on the corn leaf extract 30 days after sowing. At least 3 replicates were set up for each experiment.

We obtained 69 transgenic lines (named IV3A1Ab) transformed with pCambia1300-IPD072Aa-Vip3A-p35S-Cry1Ab vector and 73 transgenic lines (named IV3H1Ab) transformed with pCambia1300-IPD072Aa-Vip3H-p35S-Cry1Ab vector Insect resistance was measured, and its insecticidal effect was shown in Table 3:

table 3*:

pest insecticidal rate corn borer 100% bollworm 100% armyworm 100% Beet armyworm 100% Western corn rootworm 100%

*Note: Table 3 shows the insecticidal effects of different pests on the corn leaf extract 30 days after sowing. At least 3 replicates were set up for each experiment.

Finally, it should also be noted that the above enumeration is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and many modifications are possible. All deformations that those of ordinary skill in the art can directly derive or associate from the disclosure of the present invention shall be considered as the protection scope of the present invention.

SEQUENCE LISTING

<110> Zhejiang University

<120> An insecticidal fusion protein, encoding gene and application thereof

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ctgaagcaga acctgcagct ggacagcttc agcacctaca gggtgtactt cagcgtgagc 2460

ggcgacgcca acgtgaggat caggaacagc agggaggtgc tgttcgagaa gaggtacatg 2520

agcggcgcca aggacgtgag cgagatcttc accaccaagc tgggcaagga caacttctac 2580

atcgagctga gccagggcaa caacctgaac ggcggcccga tcgtgaagtt ctacgacgtg 2640

agcatcaagt ag 2652

<210> 2

<211> 883

<212> PRT

<213> unknown

<220>

<223> Artificial sequences

<400> 2

Met Gly Ile Thr Val Thr Asn Asn Ser Ser Asn Pro Ile Glu Val Ala

1 5 10 15

Ile Asn His Trp Gly Ser Asp Gly Asp Thr Ser Phe Phe Ser Val Gly

20 25 30

Asn Gly Lys Gln Glu Thr Trp Asp Arg Ser Asp Ser Arg Gly Phe Val

35 40 45

Leu Ser Leu Lys Lys Asn Gly Ala Gln His Pro Tyr Tyr Val Gln Ala

50 55 60

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

65 70 75 80

Leu Ile Glu Pro Leu Ser Arg Gly Pro Gly Lys Gly Gly Gly Met Asn

85 90 95

Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Ile Asp

100 105 110

Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile Met

115 120 125

Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu Asp Glu

130 135 140

Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys Leu Asp

145 150 155 160

Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn Leu Asn

165 170 175

Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln Asn Gln

180 185 190

Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met Leu

195 200 205

Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val Met Lys

210 215 220

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

225 230 235 240

Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val Leu Ile

245 250 255

Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys Tyr

260 265 270

Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser Ser

275 280 285

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

290 295 300

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

305 310 315 320

Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly Asn Asn

325 330 335

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

340 345 350

Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn Ser

355 360 365

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

370 375 380

Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser Ile

385 390 395 400

Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val Asn Ile

405 410 415

Pro Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala Lys Val

420 425 430

Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro Gly

435 440 445

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

450 455 460

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

465 470 475 480

Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu Gly Pro

485 490 495

Asp Gln Ser Gly Pro Ile Tyr Tyr Pro Asn Asn Ile Val Phe Pro Asn

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val Ile Ser

565 570 575

Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala Asp Glu

580 585 590

Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu Leu

595 600 605

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

610 615 620

Pro Ser Gly Phe Ile Lys Asn Ile Val Glu Asn Gly Ser Ile Glu Glu

625 630 635 640

Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr Val Asp

645 650 655

His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His Lys Asp

660 665 670

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

675 680 685

Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His Leu Lys

690 695 700

Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn Leu

705 710 715 720

Glu Asp Tyr Gln Thr Ile Thr Lys Arg Phe Thr Thr Gly Thr Asp Leu

725 730 735

Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu Ala Trp

740 745 750

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

755 760 765

Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly Ser Thr

770 775 780

Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg Gly Ile

785 790 795 800

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

805 810 815

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

820 825 830

Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val Ser Glu

835 840 845

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

850 855 860

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

865 870 875 880

Ser Ile Lys

<210> 3

<211> 2646

<212> DNA

<213> unknown

<220>

<223> Artificial sequences

<400> 3

atgggcatca ccgtgaccaa caacagcagc aacccgatcg aggtggccat caaccactgg 60

ggcagcgacg gcgacaccag cttcttcagc gtgggcaacg gcaagcagga gacctgggac 120

aggagcgaca gcaggggctt cgtgctgagc ctgaagaaga acggcgccca gcacccgtac 180

tacgtgcagg ccagcagcaa gatcgaggtg gacaacaacg ccgtgaagga ccagggcagg 240

ctgatcgagc cgctgagccg aggccccggg aagggtggag gaatgaacaa gaacaacagt 300

aagctctcca cccgcgccct cccgtccttc atcgactact tcaacggcat ctacggcttc 360

gccaccggca tcaaggacat catgaacatg atcttcaaga ccgacaccgg cggcaacgtc 420

accctcgacg agatcctcaa gaaccagcag ctcctcaacg agatcagcgg caagctcgac 480

ggcgtgaacg gctccctcaa cgagctgatc gcccaggtca acctcaacac cgagctgtcc 540

aaggagatcc tcaagatctc caacgagcag aaccaggtgc tcaacgacgt gaacaacaag 600

ctggacgcca tcaacaccat gctgcacatc tacctcccga agatcacctc catgctctcc 660

gacgtgatga agcagaacta cgccctctcc ctccagatcg agtacctctc caagcagctc 720

caggagatca gcgacaagct cgacatcatc aacgtgaacg tgctcatcaa ctccaccctc 780

accgagatca ccccggccta ccagcgcatc aagtacgtga acgagaagtt cgaggagctg 840

accttcgcca ccgagaccac cctcaaggtg aagaaggact cctccccggc cgacatcctc 900

gacgagctga ccgagctgac cgagctggcc aagtccgtga ccaagaacga cgtggacggc 960

ttcgagttct acctcaacac cttgcacgac gtgatggtgg gcaacaacct cttcggccgc 1020

tccgccctca agaccgcctc cgagctgatc gccaaggaga acgtgaagac ctccggctcc 1080

gaggtgggca acgtgtacaa cttcctcatc gtgctcaccg ccctgcaggc caaggccttc 1140

ctcaccctca ccacctgccg caagctcctc ggcctcgccg gcatcgacta cacctccatc 1200

atgaacgagc acctcaacaa ggagaaggag gagttccgcg tgaacatcct cccgaccctc 1260

tccaacacct tctccaaccc gaactacgcc aaggtgaagg gctccgacga ggacgccaag 1320

atgatcgtgg aggccaagcc gggccacgcc ctcgtgggct tcgagatgtc caacgactcc 1380

atcaccgtgc tcaaggtgta cgaggccaag ctcaagcaga actaccaggt ggacaaggac 1440

tccctctccg aggtgatcta cggcgacacc gacaagctct tctgcccgga ccagtccgag 1500

cagatatact acaccaacaa catcgtgttc ccgaacgagt acgtgatcac caagatcgac 1560

ttcaccaaga agatgaagac cctccgctac gaggtgaccg ccaacttcta cgactcctcc 1620

accggcgaga tcgacctcaa caagaagaag gtggagtcct ccgaggccga gtaccgcacc 1680

ctctccgcca acgacgacgg cgtgtacatg ccgctcggcg tgatctccga aaccttcctc 1740

accccgatca acggcttcgg cctccaggcc gacgagaact cccgcctcat caccctcacc 1800

tgcaagtcct acctccgcga gctgctcctc gccaccgacc tctccaacaa ggagaccaag 1860

ctcatcgtgc cgccgtccgg cttcatctcc aacatcgtgg agaacggcgg catcgaggag 1920

gacaacctcg agccgtggaa ggccaacaac aagaacgcct acgtggacca caccggcggc 1980

gtgaacggca ccaaggccct ctacgtgcac aaggacggcg gcttctccca gttcatcggc 2040

gacaagctca agccgaagac cgagtacgtg atccagtaca ccgtgaaggg caaggccagc 2100

atctacctga aggacgagaa gaacaacgag ggcatctacg aggagatcaa caacgacctg 2160

gaggacttcc agaccgtgac caagaggttc atcaccggca ccgacagcag cggcgtgcac 2220

ctgatcttca ccagccagaa cggcgacgag gccttcggcg gcaacttcat catcagcgag 2280

atcaggagca gcgaggagct gctgagcccg gagctgatca agagcgacgc ctgggtgggc 2340

agccagggca cctggatcag cggcaacagc ctgaccatca acagcaacgc caacggcacc 2400

ttcaggcaga acctgccgct ggagagctac agcacctaca gcatgaactt caacgtgaac 2460

ggcttcgcca aggtgaccgt gaggaacagc agggaggtgc tgttcgagaa gaacttcagc 2520

cagctgagcc cgaaggacta cagcgagaag ttcaccaccg ccgccaacaa caccggcttc 2580

tacgtggagc tgagcagggg cacccagggc ggcaacatca ccttcaggga cttcagcatc 2640

aagtag 2646

<210> 4

<211> 881

<212> PRT

<213> unknown

<220>

<223> Artificial sequences

<400> 4

Met Gly Ile Thr Val Thr Asn Asn Ser Ser Asn Pro Ile Glu Val Ala

1 5 10 15

Ile Asn His Trp Gly Ser Asp Gly Asp Thr Ser Phe Phe Ser Val Gly

20 25 30

Asn Gly Lys Gln Glu Thr Trp Asp Arg Ser Asp Ser Arg Gly Phe Val

35 40 45

Leu Ser Leu Lys Lys Asn Gly Ala Gln His Pro Tyr Tyr Val Gln Ala

50 55 60

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

65 70 75 80

Leu Ile Glu Pro Leu Ser Arg Gly Pro Gly Lys Gly Gly Gly Met Asn

85 90 95

Lys Asn Asn Ser Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Ile Asp

100 105 110

Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile Met

115 120 125

Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asn Val Thr Leu Asp Glu

130 135 140

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

145 150 155 160

Gly Val Asn Gly Ser Leu Asn Glu Leu Ile Ala Gln Val Asn Leu Asn

165 170 175

Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ser Asn Glu Gln Asn Gln

180 185 190

Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met Leu

195 200 205

His Ile Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val Met Lys

210 215 220

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

225 230 235 240

Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val Leu Ile

245 250 255

Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys Tyr

260 265 270

Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Thr Leu

275 280 285

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

290 295 300

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

305 310 315 320

Phe Glu Phe Tyr Leu Asn Thr Leu His Asp Val Met Val Gly Asn Asn

325 330 335

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

340 345 350

Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn Phe

355 360 365

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

370 375 380

Thr Cys Arg Lys Leu Leu Gly Leu Ala Gly Ile Asp Tyr Thr Ser Ile

385 390 395 400

Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val Asn Ile

405 410 415

Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala Lys Val

420 425 430

Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro Gly

435 440 445

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

450 455 460

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

465 470 475 480

Ser Leu Ser Glu Val Ile Tyr Gly Asp Thr Asp Lys Leu Phe Cys Pro

485 490 495

Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe Pro Asn

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val Ile Ser

565 570 575

Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala Asp Glu

580 585 590

Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu Leu

595 600 605

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

610 615 620

Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Gly Ile Glu Glu

625 630 635 640

Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr Val Asp

645 650 655

His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His Lys Asp

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

Glu Asp Phe Gln Thr Val Thr Lys Arg Phe Ile Thr Gly Thr Asp Ser

725 730 735

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

740 745 750

Gly Gly Asn Phe Ile Ile Ser Glu Ile Arg Ser Ser Glu Glu Leu Leu

755 760 765

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

770 775 780

Trp Ile Ser Gly Asn Ser Leu Thr Ile Asn Ser Asn Ala Asn Gly Thr

785 790 795 800

Phe Arg Gln Asn Leu Pro Leu Glu Ser Tyr Ser Thr Tyr Ser Met Asn

805 810 815

Phe Asn Val Asn Gly Phe Ala Lys Val Thr Val Arg Asn Ser Arg Glu

820 825 830

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

835 840 845

Glu Lys Phe Thr Thr Ala Ala Asn Asn Thr Gly Phe Tyr Val Glu Leu

850 855 860

Ser Arg Gly Thr Gln Gly Gly Asn Ile Thr Phe Arg Asp Phe Ser Ile

865 870 875 880

Lys

<210> 5

<211> 261

<212> DNA

<213> unknown

<220>

<223> Artificial sequences

<400> 5

atgggcatca ccgtgaccaa caacagcagc aacccgatcg aggtggccat caaccactgg 60

ggcagcgacg gcgacaccag cttcttcagc gtgggcaacg gcaagcagga gacctgggac 120

aggagcgaca gcaggggctt cgtgctgagc ctgaagaaga acggcgccca gcacccgtac 180

tacgtgcagg ccagcagcaa gatcgaggtg gacaacaacg ccgtgaagga ccagggcagg 240

ctgatcgagc cgctgagcta g 261

<210> 6

<211> 2364

<212> DNA

<213> unknown

<220>

<223> Artificial sequences

<400> 6

atgaacaaga acaacagtaa gctctccacc cgcgccctcc cgtccttcat cgactacttc 60

aacggcatct acggcttcgc caccggcatc aaggacatca tgaacatgat cttcaagacc 120

gacaccggcg gcaacgtcac cctcgacgag atcctcaaga accagcagct cctcaacgag 180

atcagcggca agctcgacgg cgtgaacggc tccctcaacg agctgatcgc ccaggtcaac 240

ctcaacaccg agctgtccaa ggagatcctc aagatctcca acgagcagaa ccaggtgctc 300

aacgacgtga acaacaagct ggacgccatc aacaccatgc tgcacatcta cctcccgaag 360

atcacctcca tgctctccga cgtgatgaag cagaactacg ccctctccct ccagatcgag 420

tacctctcca agcagctcca ggagatcagc gacaagctcg acatcatcaa cgtgaacgtg 480

ctcatcaact ccaccctcac cgagatcacc ccggcctacc agcgcatcaa gtacgtgaac 540

gagaagttcg aggagctgac cttcgccacc gagaccaccc tcaaggtgaa gaaggactcc 600

tccccggccg acatcctcga cgagctgacc gagctgaccg agctggccaa gtccgtgacc 660

aagaacgacg tggacggctt cgagttctac ctcaacacct tgcacgacgt gatggtgggc 720

aacaacctct tcggccgctc cgccctcaag accgcctccg agctgatcgc caaggagaac 780

gtgaagacct ccggctccga ggtgggcaac gtgtacaact tcctcatcgt gctcaccgcc 840

ctgcaggcca aggccttcct caccctcacc acctgccgca agctcctcgg cctcgccggc 900

atcgactaca cctccatcat gaacgagcac ctcaacaagg agaaggagga gttccgcgtg 960

aacatcctcc cgaccctctc caacaccttc tccaacccga actacgccaa ggtgaagggc 1020

tccgacgagg acgccaagat gatcgtggag gccaagccgg gccacgccct cgtgggcttc 1080

gagatgtcca acgactccat caccgtgctc aaggtgtacg aggccaagct caagcagaac 1140

taccaggtgg acaaggactc cctctccgag gtgatctacg gcgacaccga caagctcttc 1200

tgcccggacc agtccgagca gatatactac accaacaaca tcgtgttccc gaacgagtac 1260

gtgatcacca agatcgactt caccaagaag atgaagaccc tccgctacga ggtgaccgcc 1320

aacttctacg actcctccac cggcgagatc gacctcaaca agaagaaggt ggagtcctcc 1380

gaggccgagt accgcaccct ctccgccaac gacgacggcg tgtacatgcc gctcggcgtg 1440

atctccgaaa ccttcctcac cccgatcaac ggcttcggcc tccaggccga cgagaactcc 1500

cgcctcatca ccctcacctg caagtcctac ctccgcgagc tgctcctcgc caccgacctc 1560

tccaacaagg agaccaagct catcgtgccg ccgtccggct tcatctccaa catcgtggag 1620

aacggcggca tcgaggagga caacctcgag ccgtggaagg ccaacaacaa gaacgcctac 1680

gtggaccaca ccggcggcgt gaacggcacc aaggccctct acgtgcacaa ggacggcggc 1740

ttctcccagt tcatcggcga caagctcaag ccgaagaccg agtacgtgat ccagtacacc 1800

gtgaagggca aggccagcat ctacctgaag gacgagaaga acaacgaggg catctacgag 1860

gagatcaaca acgacctgga ggacttccag accgtgacca agaggttcat caccggcacc 1920

gacagcagcg gcgtgcacct gatcttcacc agccagaacg gcgacgaggc cttcggcggc 1980

aacttcatca tcagcgagat caggagcagc gaggagctgc tgagcccgga gctgatcaag 2040

agcgacgcct gggtgggcag ccagggcacc tggatcagcg gcaacagcct gaccatcaac 2100

agcaacgcca acggcacctt caggcagaac ctgccgctgg agagctacag cacctacagc 2160

atgaacttca acgtgaacgg cttcgccaag gtgaccgtga ggaacagcag ggaggtgctg 2220

ttcgagaaga acttcagcca gctgagcccg aaggactaca gcgagaagtt caccaccgcc 2280

gccaacaaca ccggcttcta cgtggagctg agcaggggca cccagggcgg caacatcacc 2340

ttcagggact tcagcatcaa gtaa 2364

<210> 7

<211> 1848

<212> DNA

<213> unknown

<220>

<223> Artificial sequences

<400> 7

atggacaaca acccaaacat caacgaatgc attccataca actgcttgag taacccagaa 60

gttgaagtac ttggtggaga acgcattgaa accggttaca ctcccatcga catctccttg 120

tccttgacac agtttctgct cagcgagttc gtgccaggtg ctgggttcgt tctcggacta 180

gttgacatca tctggggtat ctttggtcca tctcaatggg atgcattcct ggtgcaaatt 240

gagcagttga tcaaccagag gatcgaagag ttcgccagga accaggccat ctctaggttg 300

gaaggattga gcaatctcta ccaaatctat gcagagagct tcagagagtg ggaagccgat 360

cctactaacc cagctctccg cgaggaaatg cgtattcaat tcaacgacat gaacagcgcc 420

ttgaccacag ctatcccatt gttcgcagtc cagaactacc aagttcctct cttgtccgtg 480

tacgttcaag cagctaatct tcacctcagc gtgcttcgag acgttagcgt gtttgggcaa 540

aggtggggat tcgatgctgc aaccatcaat agccgttaca acgaccttac taggctgatt 600

ggaaactaca ccgaccacgc tgttcgttgg tacaacactg gcttggagcg tgtctggggt 660

cctgattcta gagattggat tagatacaac cagttcagga gagaattgac cctcacagtt 720

ttggacattg tgtctctctt cccgaactat gactccagaa cctaccctat ccgtacagtg 780

tcccaactta ccagagaaat ctatactaac ccagttcttg agaacttcga cggtagcttc 840

cgtggttctg cccaaggtat cgaaggctcc atcaggagcc cacacttgat ggacatcttg 900

aacagcataa ctatctacac cgatgctcac agaggagagt attactggtc tggacaccag 960

atcatggcct ctccagttgg attcagcggg cccgagttta cctttcctct ctatggaact 1020

atgggaaacg ccgctccaca acaacgtatc gttgctcaac taggtcaggg tgtctacaga 1080

accttgtctt ccaccttgta cagaagaccc ttcaatatcg gtatcaacaa ccagcaactt 1140

tccgttcttg acggaacaga gttcgcctat ggaacctctt ctaacttgcc atccgctgtt 1200

tacagaaaga gcggaaccgt tgattccttg gacgaaatcc caccacagaa caacaatgtg 1260

ccacccaggc aaggattctc ccacaggttg agccacgtgt ccatgttccg ttccggattc 1320

agcaacagtt ccgtgagcat catcagagct cctatgttct catggattca tcgtagtgct 1380

gagttcaaca atatcattcc ttcctctcaa atcacccaaa tcccattgac caagtctact 1440

aaccttggat ctggaacttc tgtcgtgaaa ggaccaggct tcacaggagg tgatattctt 1500

agaagaactt ctcctggcca gattagcacc ctcagagtta acatcactgc accactttct 1560

caaagatatc gtgtcaggat tcgttacgca tctaccacaa acttgcaatt ccacacctcc 1620

atcgacggaa ggcctatcaa tcagggtaac ttctccgcaa ccatgtcaag cggcagcaac 1680

ttgcaatccg gcagcttcag aaccgtcggt ttcactactc ctttcaactt ctctaacgga 1740

tcaagcgttt tcacccttag cgctcatgtg ttcaattctg gcaatgaagt gtacattgac 1800

cgtattgagt ttgtgcctgc cgaagttacc ttcgaggctg agtactag 1848

<210> 8

<211> 208

<212> DNA

<213> unknown

<220>

<223> Artificial sequences

<400> 8

gagctctaga tgggccctgt tctgcacaaa gtggagtagt cagtcatcga tcaggaacca 60

gacaccagac ttttattcat acagtgaagt gaagtgaagt gcagtgcagt gagttgctgg 120

ttttttgtaca acttagtatg tatttgtatt tgtaaaatac ttctatcaat aaaatttcta 180

attcctaaaa ccaaaatcca ggggtacc 208

<210> 9

<211> 792

<212> DNA

<213> unknown

<220>

<223> Artificial sequences

<400> 9

ggtacctggt ggagcacgac actctcgtct actccaagaa tatcaaagat acagtctcag 60

aagaccaaag ggctattgag acttttcaac aaagggtaat atcgggaaac ctcctcggat 120

tccattgccc agctatctgt cacttcatca aaaggacagt agaaaaggaa ggtggcacct 180

acaaatgcca tcattgcgat aaaggaaagg ctatcgttca agatgcctct gccgacagtg 240

gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca 300

cgtcttcaaa gcaagtggat tgatgtgata acatggtgga gcacgacact ctcgtctact 360

ccaagaatat caaagataca gtctcagaag accaaagggc tattgagact tttcaacaaa 420

gggtaatatc gggaaacctc ctcggattcc attgcccagc tatctgtcac ttcatcaaaa 480

ggacagtaga aaaggaaggt ggcacctaca aatgccatca ttgcgataaa ggaaaggcta 540

tcgttcaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc acgaggagca 600

tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatatct 660

ccactgacgt aagggatgac gcacaatccc actatccttc gcaagacctt cctctatata 720

aggaagttca tttcatttgg agaggacacg ctgaaatcac cagtctctct ctacaaatct 780

atctctggat cc 792

Claims (5)

1. An insecticidal fusion protein is characterized in that the insecticidal fusion protein is successively IPD072 protein and Vip3 toxin from the N-terminal to the C-terminal, and the insecticidal fusion protein is one of the following: (1) consists of the IPD072Aa protein and the Vip3 toxin. Vip3A toxin is fused, and the amino acid sequence of the insecticidal fusion protein is shown in SEQ ID NO: 2; (2) IPD072Aa protein and Vip3H toxin are fused from the N-terminal to the C-terminal, and the amino acid sequence of the insecticidal fusion protein is: shown in SEQ ID NO:4. 2. the coding gene of the described insecticidal fusion protein of claim 1, it is characterized in that described gene is the nucleotide sequence of coding IPD072 protein and the nucleotide sequence of coding Vip3 toxin successively from 5 '-3 '; And the above two nucleotide sequences are located in the same open reading frame. 3 . The coding gene according to claim 2 , wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO: 1 or SEQ ID NO: 3. 4 . 4. The application of the insecticidal fusion protein of claim 1 in the preparation of transgenic insect-resistant crops, transgenic insecticidal microorganisms or antibodies. 5. The application according to claim 4, wherein the transgenic insect-resistant crop is prepared by transforming the fusion protein in combination with BT crystal toxin.

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