KR20100093959A - Nucleotide sequence of prophenoloxidase-activating protease gene from the venom of the bumblebee bombus ignitus - Google Patents

Abstract

PURPOSE: A nucleotide sequence encoding pophenoloxidase-activating protease(PAP) of Bombus ignites venom is provided to prepare recombinant baculovirus with improved insecticidal activity. CONSTITUTION: A cDNA encoding prophenoloxidase-activating protease(PAP) of Bombus ignites venom has a nucleotide sequence of sequence number 1. The length of the cDNA is 1083bp. A genomic DNA encoding the PAP has a base sequence of sequence number 2. The genomic DNA comprises 6 exons and 5 introns. A polypeptide is encoded by cDNA sequence.

Description

Nucleotide sequence of prophenoloxidase-activating protease gene from the venom of the bumblebee Bombus ignitus}

The present invention relates to a nucleotide sequence encoding a prophenoloxidase-activating protease (PAP) of a bumblebee ( Bombus ignitus ) venom.

The primary innate immune response in insects is melanization of pathogens or wound tissue. This innate defense is regulated by a prophenoloxidase (proPO) activation system that regulates melanin formation [Cerenius & Soderhall, Immunol. Rev., 198: 116-126 (2004). The mechanism of immune response in wounds and infections is the active form of prophenol oxidase active protease (PAP), a prophenol oxidase active protease (PAP) which converts prophenoloxidase (proPO) into phenoloxidase (phenoloxidase, PO) to induce melanin formation. Melanization in these immune defenses only occurs to treat wounds and kill pathogens locally or in infected areas [Cerenius & Sderderkll, Immunol. Rev., 198: 116-126 (2004). However, since excessive melanization is fatal to the insect itself, the activity of the prophenoloxidase system is thoroughly regulated in a local fashion [Yu et al ., Insect Biochem. Mol. Biol., 33: 197-208 (2003); Zhao et al ., J. Biol. Chem., 280: 24744-24751 (2005)].

For successful parasites in host-parasitic relationships, parasites have venom that inhibits host immunity [Beck & Strand, Proc. Natl. Acad. Sci. USA 104: 19267-19272 (2007). Such venom circumvents the host's immune defenses by inhibiting the phenol oxidase (PO) activity by inhibiting the host's prophenol oxidase active protease (PAP).

Bees have bee stings, which are effective weapons to inject bee venom to defend themselves from predators. Bee venom is composed of several types of venom proteins or peptides, and mellittin [Gauldie et al ., Eur. J. Biochem., 61: 369-376 (1976)], phospholipase A ₂ (PLA ₂ ) [Six & Dennis, Biochim. Biophys. Acta 1488: 1-19 (2000)], apamin [Banks et al ., Nature 282: 415-417 (1979), bee venom hyaluronidase (Kreil, Protein Sci., 4: 1666-1669 (1995)) and the like.

However, bee venom, which is an effective and important defense, contains prophenol oxidase-activated protease (PAP), and when the prophenol oxidase-activated protease (PAP) is injected through bee sting, the predatory insects can be melanized quickly. It is not yet known to cause the death of predatory insects by causing them, and the synthesis and mechanism of action of bee venom prophenol oxidase active protease and poison in the glands are still unknown.

Therefore, the present inventors have studied and sequentially analyzed the sequencing of the bee venom prophenol oxidase active protease gene causing melanization of predator insects, and as a result, the gene sequence encoding the prophenol oxidase active protease of the bumblebee venom solution The present invention has been completed by finding out.

Accordingly, an object of the present invention is to provide a nucleotide sequence encoding a prophenoloxidase active protease of bumblebee venom.

It is another object of the present invention to provide a polypeptide encoded by the nucleotide sequence.

The present invention is characterized by the cDNA of SEQ ID NO: 1 encoding the prophenoloxidase active protease of bumblebee venom.

The present invention also features the genomic DNA of SEQ ID NO: 2 encoding the prophenoloxidase active protease of bumblebee venom.

In addition, the present invention is characterized by a polypeptide encoded by the cDNA sequence of SEQ ID NO: 1.

Gene sequences encoding the prophenoloxidase active protease of bumblebee venom can be introduced into insect baculoviruses currently used as insecticides by recombinant DNA technology to produce recombinant baculoviruses with improved insecticidal properties. Therefore, using the base sequence of the present invention will be able to contribute to the development of pest control agents such as fast-acting and highly toxic recombinant baculovirus preparations.

The present invention relates to a nucleotide sequence encoding the prophenoloxidase active protease of bumblebee venom.

The prophenol oxidase active protease gene in bee venom was first isolated from bumble bee venom. Since the characteristics of the bee venom prophenol oxidase protease have not been reported so far, for the first time in the present invention, the sequence of the gene encoding the bee venom venom prophenol oxidase active protease is disclosed.

The prophenoloxidase active protease gene of the bumblebee venom was registered on September 23, 2008 under the approval numbers FJ159443 (cDNA) and FJ159442 (Genomic DNA) in the NCBI GenBank database.

The prophenoloxidase active protease gene of the bumblebee venom solution of the present invention is composed of the base sequences of cDNA and genomic DNA. The cDNA is isolated using a commercially available kit from bumblebee venom, and the base sequence of the cDNA is obtained. Primers can be prepared based on the genomic DNA isolated by PCR amplification. The cDNA of the prophenol oxidase active protease gene of the resulting bumblebee venom has a nucleotide sequence of SEQ ID NO: 1, which is 1083 bp in length. In addition, the genomic DNA has a nucleotide sequence of SEQ ID NO: 2, and consists of six exons and five introns. The length of the start codon and the stop codon is 4505 bp.

The prophenol oxidase active protease gene of the bumblebee venom is an amino acid number or the sequence itself except for the conservation region characterized by the insect-derived prophenol oxidase active protease genes and the prophenol oxidase active protease. It was confirmed that there was a significant difference.

In another aspect, the present invention is characterized by a polypeptide encoded by the prophenol oxidase active protease gene sequence of the bumblebee venom.

The polypeptide consists of 360 amino acids as shown in SEQ ID NO: 3.

The invention can be understood in more detail by the following examples. However, the following examples are intended to illustrate the invention and are not intended to limit the protection scope of the invention.

[Example]

1. Cloning of Prophenoloxidase Activity Protease Gene from Bumble Bee Venom

Total RNA was extracted using SV Total RNA Isolation System Kit (Promega, USA) commercialized from the venom of the bumblebee ( Bombus ignitus ) worker bee that was distributed from the National Institute of Agricultural Science, RDA. PolyATtract® commercialized from all RNAs extracted Poly (A) + mRNA extracted according to the method proposed by Promega using the mRNA Isolation System (Promega, USA) by Uni-ZAP XR vector and Gigapack III Gold Packing Extract (Stratagene, USA) cDNA library was constructed and analyzed for the expression sequence tags (ESTs).

DNA extraction was performed using the Wizard mini-preparation kit (Promega, USA). DNA sequencing was sequenced by an automated DNA sequencer (Applied Biosystems, USA). The sequences were compared by NCBI () 's BLAST program. As a result, the cDNA having the nucleotide sequence of SEQ ID NO: 1 of the bleach bee venom solution, which encodes 360 amino acids, was cloned by bumble bee venom ESTs analysis (FIG. 1). As a result of a database analysis of the amino acid sequence of SEQ ID NO: 3 deduced from the prophenoloxidase active protease cDNA of bumble bee venom, Holotrichia diomphalia PPAF-I (GenBank No. BAA34642), H. diomphalia PPAF-III (GenBank No. BAC15604), Bombyx mori PPAF-3 (GenBank No. AAL31707), Drosophila melanogaster MP1 (GenBank No. NP_649560), D. melanogaster easter (GenBank No. NP_524362) and Manduca sexta PAP-I ( GenBank No. AAX18636), histidine (H), aspartic acid (D), and serine (S) in cysteine residues and serine protease regions in the clip region Residues were well preserved. Through this analysis, the prophenoloxidase active protease of bumble bee venom is composed of 26 amino acid signal sequence and 334 amino acid mature protein, and mature protein is divided into 87 amino acid clip region and 247 amino acid serine protease region. It confirmed that it became.

In addition, the primers of Table 1 were prepared based on the nucleotide sequence of the prophenol oxidase active protease cDNA of bumble bee venom, and genomic DNA of the prophenol oxidase active protease gene of bumble bee venom was isolated by PCR amplification.

primer Sequence location Forward primer 1 5'-ATG ACG GGC TCC AAG ATG CTG TTC-3 '(SEQ ID NO: 4) 1-24 Reverse primer 1 5'-TAC AGC TGG CTT ACC ACC GAC CAC-3 '(SEQ ID NO: 5) 363-340 Forward primer 2 5'-GTG GTC GGT GGT AAG CCA GCT GTA-3 '(SEQ ID NO: 6) 340-363 Reverse primer 2 5'-TTA TTG CAT CGC TGG GAG AAT AAA-3 '(SEQ ID NO: 7) 1083-1060

DNA was isolated from the bumblebee using Wizard Genomic DNA Purification Kit (Promega, USA), a commercialization kit, and the primers and PCR premix kit (Bionia, Daejeon) were used to determine the prophenoloxidase active protease gene of bumblebee venom. Genomic DNA was amplified. At this time, after denaturing at 95 ° C for 5 minutes, a series of reactions of annealing reaction at 60 ° C for 1 minute and polymerization reaction at 72 ° C for 3 minutes were performed 35 times.

The DNA sequence of the amplified DNA sequence was analyzed by automated DNA sequencing. The genomic DNA of the prophenoloxidase activating protease gene of bumblebee venom had the nucleotide sequence of SEQ ID NO: 2, 6 exons and 5 introns. It was composed of (intron), it was confirmed that the length of the start codon and the stop codon is 4505 bp (Fig. 3).

2. Poison gland specific expression, cleavage of bumblebee venom prophenoloxidase active protease O - Sugar chain

RNA was isolated from the bumblebee worker's fat body, midgut, muscle and venom gland using Total RNA Separation Kit (Promega, USA). The isolated RNA was electrophoresed on a 1.0% formaldehyde agarose gel at 5 μg per lane and then transferred to a nylon membrane (nylon blotting membrane, Schleicher & Schuell, Germany), and Prime-It II Random Primer Labeling. Using a kit (Stratagene, USA), hybridization was carried out at 42 ° C. using a probe of prophenoloxidase active protease cDNA of bumble bee venom labeled with [α- ³² P] dCTP (Amersham, USA). Northern blot analysis was performed. As a result, the transcript of the prophenoloxidase active protease gene of bumblebee venom was specifically detected in the venom gland (FIG. 4).

To prepare an antibody of a bumble bee venom prophenol oxidase active protease, a restriction enzyme of the insect baculovirus ( Autographa californica nucleopolyhedrovirus) transfer vector pBAC1 (Clontech, USA) was prepared. After insertion into the Bam H I and Xho I sites, the transfection vector (100 ng) and the bA GOZA virus DNA (500 ng) [Je et al ., Biotechnol. Lett., 23: 575-582 (2001)] was co-transfected into the insect cell line Sf9 ( Spodoptera frugiperda 9) using Lipofectin (Clonetech). After 5 days, the culture was collected to prepare recombinant baculovirus expressing the prophenoloxidase active protease of bumblebee venom. Recombinant baculovirus was propagated in Sf9 cell line, and recombinant prophenoloxidase active protease was isolated using HisTrap column (Amersham Bioscience). Polyclonal antibodies were prepared by injecting the isolated recombinant prophenoloxidase active protease into Balb / c mice [Choo et al. al ., Mol. Cell. Neurisci., 38: 224-235 (2008). 5 shows a photograph of Western blot analysis using the prophenol oxidase active protease and the antibody of the isolated bumblebee venom solution.

Protein samples were prepared from venom gland, venom sac, and emitted venom, followed by 15% SDS-PAGE electrophoresis, followed by Western blot analysis using the antibody. . As a result, in the venom gland, there were prophenol oxidase-activated protease (BiVn-proPAP) of bumblebee venom and activated phenol bee venom (PiVn-PAP). In the activated form of bumblebee venom there was a prophenoloxidase active protease (BiVn-PAP) (FIG. 6). From these results, it was confirmed that the prophenol oxidase protease of the bumble bee venom was expressed in the venom gland, cleaved into an activated form, stored in the sac and radiated.

34 kDa bumblebee venom prophenol in a radiated venom to assay sites cleaved from the prophenoloxidase active protease (BiVn-proPAP) of bumble bee venom into the active form of prophenoloxidase active protease (BiVn-PAP) N-terminal sequences were analyzed by Edman degradation method by transferring oxidase active protease (BiVn-PAP) to polyvinylidene difluoride (PVDF) membrane (Applied Biosystems). As a result, as shown in FIG. 2, the bleach venom prophenol oxidase active protease (BiVn-proPAP) was cleaved between 113 th Arg and 114 th Val to form an activating form consisting of 247 amino acid serine proteaase regions. It can be seen that it is converted to prophenol oxidase active protease (BiVn-PAP).

The estimated molecular weight, calculated by calculation of the active form of prophenoloxidase active protease (BiVn-PAP) consisting of 247 amino acids, is 27 kDa or 34 kDa on SDS-PAGE electrophoresis. This is because the presence of the N -sugar chain portion of the prophenol oxidase active protease (BiVn-PAP) of bumblebee venom solution, but the O -sugar chain portion. To confirm this, glycoprotein staining of the active form of bumblebee venom prophenoloxidase active protease (BiVn-PAP) was performed using Gel / Code glycoprotein staining kit (Pierce). As a result, it was confirmed that the bumble bee venom prophenol oxidase active protease (BiVn-PAP) of the activated form is an O -sugar chained glycoprotein (FIG. 7).

3. Activated forms of bumblebee venom prophenoloxidase active protease ( BiVn - PAP Prophenol oxidase proPO Activation and Melanization ( melanization ) Judo

Activated forms of bumblebee venom prophenoloxidase active protease (BiVn-PAP) activate prophenol oxidase (proPO) as phenoloxidase (PO) and determine if this causes melanization In order to see the proPO genes were performed on a known silkworm ( Bombyx mori ) [Satoh et al ., J. Biol. Chem., 274: 7441-7453 (1999); Asano & Ashida, J. Biol. Chem., 276: 11100-11112 (2001). Silkworm prophenol oxidase B. mori PPO-1 (BmPPO-1, GenBank No. NM_001043870) and B. mori PPO-2s (BmPPO-2s, GenBank No. NM_001112747) were prepared using RT- Amplification by PCR.

Primers for cloning the silkworm BmPPO-1 and BmPPO-2s cDNAs were prepared as shown in Table 2 based on the sequence registered in GenBank.

primer Sequence BmPPO-1 Forward Primer 5'-GGA TCC ATG CTG ACG CCA AGA ACA AC-3 '(SEQ ID NO: 8) BmPPO-1 Reverse Primer 5'-CTC GAG CCC CTG CTG GCC GCG CTG GCG-3 '(SEQ ID NO: 9) BmPPO-2s Forward Primer 5'-GGA TCC ATG GCT GAC GTT TTT GAA AGC-3 '(SEQ ID NO: 10) BmPPO-2s Reverse Primer 5'-GCG GCC GCA ACT GAC ATG GGA GGG TTC CG-3 '(SEQ ID NO: 11)

RNA isolated from the primer was amplified silkworm prophenol oxidase cDNA using a PCR premix kit (Bionia, Daejeon). At this time, after denaturing at 95 ° C. for 5 minutes, a series of reactions of annealing reaction at 60 ° C. for 1 minute and polymerization reaction at 72 ° C. for 5 minutes were performed 35 times. The amplified PCR product was analyzed for DNA sequence by automated DNA sequencing. Amplified silkworm prophenol oxidase cDNAs were inserted into the baculovirus (Autographa californica nucleopolyhedrovirus) transition vector pBAC1 (Clontech, USA), as shown in Example 2, and then to be transgenic vector (100 ng). (bAcGOZA) viral DNA (500 ng) [Je et al., Biotechnol. Lett., 23: 575-582 (2001)] was used to co-transfection the insect cell line Sf9 (Spodoptera frugiperda 9) using Lipofectin (Clonetech) to produce each recombinant baculovirus. . Recombinant baculovirus was propagated in Sf9 cell line and each recombinant silkworm phenol oxidase was isolated using HisTrap column (Amersham Bioscience).

Recombinant recombinant BmPPO-1 (1 μg) and BmPPO-2s (1 μg), respectively, were treated with 20 mM Tris-HCl buffer (pH 8.0) of BiVn-proPAP (0.5 μg) or BiVn-PAP (0.5 μg) and 5 mM CaCl ₂ . ) After 20 ° C. at 30 ° C. for 10 minutes [Kim et al ., J. Biol. Chem., 277: 39999-40004 (2002)], His-tag antibody (Clontech) to Western blot analysis for activation. In addition, PO activity was measured at 490 nm by adding 980 μl of substrate solution [containing 20 mM L-DOPA in 20 mM Tris-HCl buffer (pH 8.0)] to the sample [Ligoxygakis et al ., EMBO J., 21: 6330-6337 (2002). Of Ca ²⁺ when viewed measurement results, BmPPO-1 and BmPPO-2s is cut only by the BiVn-PAP was to find out enters the activated form BmPO-1 and BmPO-2s, compared with the case CaCl ₂ is not present It was found that presence promotes the conversion of the activated form (FIG. 8). The results indicate that the bumble bee venom prophenoloxidase active protease (BiVn-PAP) in the activated form activates proPO to PO.

Then, BiVn-proPAP (5 μg) or BiVn-PAP (5 μg) Injection was performed to observe whether the blood caused melanization and PO activity analysis was performed. As a control, the same amount of PBS buffer was injected. The silkworm blood was collected by time after injection and centrifuged at 800 ° C. for 5 minutes to collect supernatant, and then PO activity was examined at 490 nm by the method described above. Photographed at 12 hours. PO activity increased rapidly after BiHn-PAP injection 1 hour after injection, and blood melanization was observed. On the other hand, the injection of BiVn-proPAP and PBS buffer did not show PO activity and melanization, suggesting that activated bumblebee venom prophenoloxidase-activated protease (BiVn-PAP) activates proPO to PO and induces melanization. Confirmation (FIG. 9).

4. Prophenol oxidase (proPO) activation and melanization of bumble bee venom deficient in the active form of prophenol oxidase active protease (BiVn-PAP)

To analyze whether bumble bee venom deficient in the active form of prophenoloxidase active protease (BiVn-PAP) is involved in the activation and melanization of prophenol oxidase (proPO), FPLC from spun bumble bee venom BiVn-PAP was isolated and the remaining fractions were combined to deplete BiVn-PAP from bumble bee venom and subjected to electrophoresis and Western blot analysis, and injected into silkworms to observe PO activity and melanization. (FIG. 10). As in Example 3, silkworm blood was collected by time after injection, and the supernatant was collected by centrifugation at 4 ° C. and 800 g for 5 minutes, and then PO activity was investigated at 490 nm. Photographed 12 hours after injection. As a result, unlike bumble bee venom containing BiVn-PAP, PO activity and melanization were not observed in BiVn-PAP deficient venom, which caused BiVn-PAP to activate proPO as PO in the bumble bee venom component, thereby causing melanization. Was confirmed.

5. Green chestnut moth Spodoptera exigua ) And Chinese Cabbage Butterfly Pieris rapae Activated and melanization of prophenol oxidase (BiVn-PAP) prophenol oxidase (BiVn-PAP) in larvae

Activated form of bumble bee venom prophenoloxidase activating protease (BiVn-PAP) is active and melanization of prophenol oxidase (proPO) in the insect pests Spodoptera exigua and Pieris rapae as well as silkworms. BiVn-PAP (2 μg) was injected into the larvae of the early ages of brown chestnut moth and cabbage butterfly, which were distributed from the Ministry of Agriculture and Biology, National Academy of Agricultural Science, RDA. As a control, the same amount of PBS buffer was injected. PO activity and melanization as in Example 3, the silkworm blood was collected by time after injection and centrifuged for 5 minutes at 4 ℃, 800 g and the supernatant was collected and the PO activity was examined at 490 nm. Melanization pictures of the carcass were taken 12 hours after injection. As a result, as in silkworms, the PO activity was rapidly increased in the blood of Pabam moth and Chinese cabbage larvae from 1 hour after BiVn-PAP injection, and melanization was confirmed (FIG. 11).

6. Bombamot by injection of bumble bee venom prophenoloxidase active protease (BiVn-PAP) in activated form Spodoptera exigua Insecticidal effect of larva

To investigate the insecticidal effect of prophenol oxidase (proPO) activation and melanization of Spodoptera exigua larvae following the injection of bumblebee venom prophenol oxidase active protease (BiVn-PAP) (1.0 [mu] g or 2.0 [mu] g) was injected into the 5 night early moth larvae. Insecticidal effect was expressed as cumulative mortality rate by averaging 11 repetitions per concentration and cumulative number of insects. As a result, melanization was observed from 1 hour after injection without significant difference between 1.0 μg and 2.0 μg of BiVn-PAP injection, and the mortality rate increased rapidly from 18 hours after injection to 100% mortality rate at 36 hours (FIG. 12). ).

1 is a cDNA sequence of the prophenol oxidase active protease of bumblebee venom

It is shown. The square ATG represents the initiation codon and the underlined TAA represents the termination codon.

2 is an amino acid sequence deduced from the cDNA of the prophenol oxidase active protease of bumblebee venom. The open triangle (▽) distinguishes the signal sequence from the clip region, and the closed triangle (▼) distinguishes between the clip region and the serine protease region.

Figure 3 shows the genomic DNA sequence of the prophenoloxidase active protease of bumblebee venom.

Figure 4 is a northern blot analysis of the prophenol oxidase active protease cDNA extracted from RNA from the fat body, midgut, muscle and venom gland of bumblebee worker bees with a probe .

Figure 5 is an electrophoresis picture of the prophenol oxidase active protease of the isolated bumble bee venom (left) and Western blot analysis using an antibody prepared by injecting the recombinant prophenol oxidase active protease into Balb / c mice (Right).

FIG. 6 is a photograph of protein electrophoresis and Western blot analysis of venom gland, venom sac and spun venom solution. BiVn-proPAP represents the prophenoloxidase active protease of bumblebee venom, and BiVn-PAP represents the prophenoloxidase active protease of activated bumblebee venom.

Figure 7 is a protein electrophoresis and glycoprotein staining of the prophenol oxidase active protease (BiVn-PAP) of bumblebee venom. Soybean trypsin inhibitor is a negative control and horseradish peroxidase is a positive control.

FIG. 8A shows silkworm prophenoloxidase (BmPPO-1) activation by treatment of bleach venom with prophenoloxidase active protease (BiVn-proPAP) and activated bumble bee venom with prophenoloxidase active protease (BiVn-PAP) Western blot analysis and graph showing PO activity.

FIG. 8B shows silkworm prophenoloxidase (BmPPO-2s) activation by treatment of bumble bee venom with prophenoloxidase active protease (BiVn-proPAP) and activated bumble bee venom with prophenoloxidase active protease (BiVn-PAP) Western blot analysis and PO activity graph.

FIG. 9 is a graph of PO activity in silkworm blood when injected with silkworm silkworms injected with prophenoloxidase active protease (BiVn-proPAP) of bumblebee venom and prophenoloxidase active protease (BiVn-PAP) of activated bumblebee venom And a picture of melanization of the blood and the umbilical cord.

10A shows protein electrophoresis and westernization of bumble bee venom, bumble bee venom (venomΔBiVn-PAP) lacking prophenol oxidase active protease, and isolated bumble bee venous prophenol oxidase active protease (BiVn-PAP) Blot analysis picture.

FIG. 10B is a graph of PO activity in silkworm blood and a melaninization picture of blood and worms upon injection of five-stage silkworms from a bumblebee venom (venomΔBiVn-PAP) lacking a bumblebee venom and a prophenoloxidase active protease .

FIG. 11A is a graph of PO activity and blood and caramelization photographs of Babam moth larvae upon injection of Bumblebee Venom Prophenoloxidase Activating Protease (BiVn-PAP) into Babam moth larvae. FIG.

FIG. 11B is a graph of PO activity in the cabbage butterfly butterfly larvae and a picture of the melanization of blood and worms with the bumblebee venom prophenol oxidase active protease (BiVn-PAP).

FIG. 12 is a graph showing the cumulative mortality rate of Parknamo larvae when 1.0 μg or 2.0 μg of bumblebee venom prophenoloxidase activating protease (BiVn-PAP) was injected into 5 night larvae.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Nucleotide sequence of prophenoloxidase-activating protease gene          from the venom of the bumblebee Bombus ignitus <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 1083 <212> DNA <213> Bombus ignitus <400> 1 atgacgggct ccaagatgct gttcgcatgt ttggcgttaa ttgctttcct gcatccatta 60 gttcacgtgg cgtcagctca agaatgtacc acaccgaaca ataaagcagg caagtgtctc 120 ggcatcagag tatgtaaacc gctgctggaa atgctgcaga ctcagggcca tgcagctgcc 180 gatttcctga ggcaatcagt gtgtaaatac gagaataata atccgatcgt ttgttgtccg 240 aacgaagaaa gcagggagga cagaggaatt ttggtaggaa acgagtatga gcctttgcgt 300 ccaccacact gtggttttag caacgtctct cacaccaggg tggtcggtgg taagccagct 360 gtacttggtg cttggccatg gattgctgca ttaggttttc gttatccccg aaacccagct 420 cttgaaccac tatggaagtg cggaggttcc ctgatatcgt ctaggcatgt tttaactgca 480 gcacattgtg cagaaatcaa tgaattgtac gtggttcgta tcggtgactt aaatctagta 540 cgaaatgacg acggagcaca tcctgttcaa atagaaatcg aatctaaaat aatacatcct 600 gattatattt ccggagtaac caaacatgat atcgccattc ttaaattggt ggaggaggtg 660 ccattttcgg agtacgtata tcccatttgt cttcccgtag aggataacct tcgaaataac 720 aatttcgagc gctattaccc cttcgttgct ggatggggat cactagcaca tcatggacca 780 ggtagtgacg atttaatgga agtacaagtg ccagtgatta gcaacaccga atgcaagaac 840 tcttatgcca gatttgctgc tgcacatgtt accgatactg tattatgcgc cggatacact 900 caaggcggaa aggatgcttg tcaaggtgac agcggaggac cactgatgct accaaagaaa 960 ttcaccttct atcaaatagg tgttgtgtct tatggtcata agtgcgccgc agctggatat 1020 cccggcgttt acactagggt cacgtcgtac ctcgacgact ttattctccc agcgatgcaa 1080 taa 1083 <210> 2 <211> 4505 <212> DNA <213> Bombus ignitus <400> 2 atgacgggct ccaagatgct gttcgcatgt ttggcgttaa ttgctttcct gcatccatta 60 gttcacgtgg cgtcagctca aggtcttgta cttcgagttg tctttttcca tgcactttaa 120 attccaatac gatctggtca cgtagccttc gcataatttc gaaattttac ctgtttagta 180 atcgtcaatg aaaaaagtat ttatgcgtga ccttaagaca ttgacaaaat ttttgtgatt 240 ttttatttcc cgattggtca ggaagttagt ggaaaaaaac tacttaacta ttcacgttaa 300 tttctttggt ttaacttttg gggaatgctt cgttagcttc gggaatattc caatgattcg 360 ttttacgcac aaaataagcg tgataaatat tacatcatag taatgaaaac tattggcgta 420 atacctaaga taaagatgga aagacattct tagaatttct aatgacacct ttatagattt 480 ctagtttaat gtcactacat ttgtatcact tttacctaac ggtatcatat agtctcgttg 540 aaaatttttc tagctcctca aaatgatcgc tggaaatttt cctaaatttc tacgaattta 600 tttatcgcga ggtaaagaaa atgtgtatta gaaagatgag attgaaggtt tatctttttt 660 tcaagtatgg cgaacgcaat atgtaatcaa ttaaaatttt gtatttttac gttaaaagtt 720 tcttcagata attattatcc agatgatgac cttacgaatg atgatccaaa ttacgaatta 780 atgcgtgtct gtagggcaat ccggtaaact tgatccgctt tttatatcga aagttgagca 840 atcggtgacg cattcttatg caccgaaccg cgaaagcaag ttcatatgac tctcaccatt 900 tcggatatca gtcaaataat aagttcgtgt taatatacac gtttgatttt tgaaaagctt 960 gttcaattta ataagagtct tggtaacagg cttatgtttt tgtacctaat tggttgtttt 1020 catttacaag tatgtcatgt tacagtactt ataaaatgac attaaaatca gaaaactaat 1080 aacagaaaga ttatcatttc tcctctgtag tagtctacat ataacataat gcaactaaca 1140 tgtcacgatt aatgcaaaat aaataaatta ctaatataga tattctttta gtaacttgta 1200 taatcgtgaa acgaattaga ctttttaaag ataatattaa agaaaacacg aaattgattc 1260 caaacctact acaaatatta tttatactac ttacacgaaa ttgattccaa acctactaca 1320 aatattattt atactactta tttgagcagt ttgaagcgaa tcaaattgat aagggtttct 1380 ctgttacata ctaatttaca ttttataaga cataacagac acgttaataa ttttacttaa 1440 gacactttcg agatatcaag ttagacgcgt tttcatgaaa tcatattaat ttatgatgat 1500 atttttctat caaggatgca attataattg tatcgttgat tagtgcacga actacgtaca 1560 tacaattatc tatgtacatt cgtctctcca agtaccgccg ttcaatattg ctaatttcga 1620 attcgttcgg gaatgtagcc tgtatcttgc acaggaaatt gggcaacgaa gtttacacaa 1680 aggcaaatac gtatccttcg ctgtaggttc tgagaaacca gccaatgata acattacttg 1740 atgagaatta acataagcgt tttgatcgta gcaagaatca caggaaattg cgcaacgaaa 1800 gtttacacaa aggcaaatac gtatccttcg ctgtaggttc tgagaaacca gccaatgata 1860 acattacttg atgagaatta acataagcgt tttgatcgta gcaagaattg tttatgacta 1920 cgttattccg agatttcagg atttatcatg catctaaatg taacgacaca agagaacatt 1980 atccagaaat tcacatttgt ataatctcca aagcttttta tttaatgcct aatgaagtta 2040 attctaatta aaggatattc caaagatatg ctacatgctt atatgctgat ttgatatatg 2100 ctatatgatg atttcatatt ttctattttt gtattgtcat catgctaata attatatcgt 2160 gctaaactta taaagacgaa tgaagtatcc gcaagtaact atgttgttaa ttctaggaga 2220 acacgtaatc caatcgtaca acgtgaacga agtatcagca gtaacggtta atatatcatg 2280 Aaaaaatatat atatacatat atatacatta ttacaaattt tatttaacaa gttatacaga 2340 gaagacgaat taattcgtca ctctattgtc aatttcttag ttataataat ttcttttttt 2400 ataaatgtat tattttaaaa tcgtttgatc cagaatgtac cacaccgaac aataaagcag 2460 gcaagtgtct cggcatcaga gtatgtaaac cgctgctgga aatgctgcag actcagggcc 2520 atgcagctgc cgatttcctg aggcaatcag tgtgtaaata cgagaataat aatccgatcg 2580 tttgttgtcc gaacgaagaa agcagggagg acagaggaat tttggtagga aacgagtatg 2640 agcctttgcg tccaccacac tgtggtttta gcaacgtctc tcacaccagg gtggtcggtg 2700 gtaagccagc tgtacttggt acgttttaca tttttctttt cgattaataa taagcgattt 2760 actgcgaaga atgaacttta aaggtactaa acggcacatc aatgtacgtt tcaattagac 2820 taaataataa tacgatttca tcggtagtga ctttacttat taacttgaat tatttctttc 2880 ttttagttca tgttaggaac agtaaccttt tacttgaaat taaaaatcga tataggaata 2940 ataatatgaa tagagatgaa aaactgttag aggagaaatt acatgtttga actttagagt 3000 tcactgtagt tcaagtagaa attatataca attatctgat aatttttatt ccattaaagt 3060 gaaaatttaa tttctctctt aatcaagtct ctatttggga atatttgtat gtatgtactt 3120 atcgtcttct gcatcatcaa ataccggatg gataataata gttttcaact tattacgcga 3180 gaaaaagtta cgtatattca caactatggc tattgcattt taggtgcttg gccatggatt 3240 gctgcattag gttttcgtta tccccgaaac ccagctcttg aaccactatg gaagtgcgga 3300 ggttccctga tatcgtctag gcatgtttta actgcagcac attgtgcaga aatcaatgaa 3360 ttgtacgtgg ttcgtatcgg tgacttaaat ctagtacgaa atgacgacgg agcacatcct 3420 gttcaaatag aaatcgaatc taaaataata catcctgatt atatttccgg agtaaccaaa 3480 catgatatcg ccattcttaa attggtggag gaggtgccat tttcgggtaa gtcttcaaat 3540 attgttgaat tgccaatatt tatactattg aagattagcg aagtatcatt tcttcaaatt 3600 tctgtttcat atattgtatc acaaaatatg ttgaattctt tctcatactt ttcctcattc 3660 gtttcgctca ctattactta ttgccttatt tttcagagta cgtatatccc atttgtcttc 3720 ccgtagagga taaccttcga aataacaatt tcgagcgcta ttaccccttc gttgctggat 3780 ggggatcact agcacatcgt agtgaatcaa ttctccataa aatgaaatag ttcccgtctt 3840 aaatatcttt ctcattttct ttatagatgg accaggtagt gacgatttaa tggaagtaca 3900 agtgccagtg attagcaaca ccgaatgcaa gaactcttat gccagatttg ctgctgcaca 3960 tgttaccgat actgtattat gcgccggata cactcaaggc ggaaaggatg cttgtcaagt 4020 aattaaataa cagatttgtc ataaattata ccgtgtctgg agacacgtca attcgaatca 4080 acatcaaatc gacgtcttaa acattaaaaa taatagataa acacaattta atagttttgc 4140 ccatttctca cataccatta tgatatttaa tctaatttct ttctaatctt agcttttttc 4200 aaaatacata taacatatac attataaatt ggaatatttc aatacataaa ttaataatag 4260 attattactg tatataagta taatttcaat ataattcgat tgaaatattt cagtaacctt 4320 gattaaaaat ttaatgaccg tttcagggtg acagcggagg accactgatg ctaccaaaga 4380 aattcacctt ctatcaaata ggtgttgtgt cttatggtca taagtgcgcc gcagctggat 4440 atcccggcgt ttacactagg gtcacgtcgt acctcgacga ctttattctc ccagcgatgc 4500 aataa 4505 <210> 3 <211> 360 <212> PRT <213> Bombus ignitus <400> 3 Met Thr Gly Ser Lys Met Leu Phe Ala Cys Leu Ala Leu Ile Ala Phe   1 5 10 15 Leu His Pro Leu Val His Val Ala Ser Ala Gln Glu Cys Thr Thr Pro              20 25 30 Asn Asn Lys Ala Gly Lys Cys Leu Gly Ile Arg Val Cys Lys Pro Leu          35 40 45 Leu Glu Met Leu Gln Thr Gln Gly His Ala Ala Ala Asp Phe Leu Arg      50 55 60 Gln Ser Val Cys Lys Tyr Glu Asn Asn Asn Pro Ile Val Cys Cys Pro  65 70 75 80 Asn Glu Glu Ser Arg Glu Asp Arg Gly Ile Leu Val Gly Asn Glu Tyr                  85 90 95 Glu Pro Leu Arg Pro Pro His Cys Gly Phe Ser Asn Val Ser His Thr             100 105 110 Arg Val Val Gly Gly Lys Pro Ala Val Leu Gly Ala Trp Pro Trp Ile         115 120 125 Ala Ala Leu Gly Phe Arg Tyr Pro Arg Asn Pro Ala Leu Glu Pro Leu     130 135 140 Trp Lys Cys Gly Gly Ser Leu Ile Ser Ser Arg His Val Leu Thr Ala 145 150 155 160 Ala His Cys Ala Glu Ile Asn Glu Leu Tyr Val Val Arg Ile Gly Asp                 165 170 175 Leu Asn Leu Val Arg Asn Asp Asp Gly Ala His Pro Val Gln Ile Glu             180 185 190 Ile Glu Ser Lys Ile Ile His Pro Asp Tyr Ile Ser Gly Val Thr Lys         195 200 205 His Asp Ile Ala Ile Leu Lys Leu Val Glu Glu Val Pro Phe Ser Glu     210 215 220 Tyr Val Tyr Pro Ile Cys Leu Pro Val Glu Asp Asn Leu Arg Asn Asn 225 230 235 240 Asn Phe Glu Arg Tyr Tyr Pro Phe Val Ala Gly Trp Gly Ser Leu Ala                 245 250 255 His His Gly Pro Gly Ser Asp Asp Leu Met Glu Val Gln Val Pro Val             260 265 270 Ile Ser Asn Thr Glu Cys Lys Asn Ser Tyr Ala Arg Phe Ala Ala Ala         275 280 285 His Val Thr Asp Thr Val Leu Cys Ala Gly Tyr Thr Gln Gly Gly Lys     290 295 300 Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Leu Pro Lys Lys 305 310 315 320 Phe Thr Phe Tyr Gln Ile Gly Val Val Ser Tyr Gly His Lys Cys Ala                 325 330 335 Ala Ala Gly Tyr Pro Gly Val Tyr Thr Arg Val Thr Ser Tyr Leu Asp             340 345 350 Asp Phe Ile Leu Pro Ala Met Gln         355 360 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PAP forward primer 1 <400> 4 atgacgggct ccaagatgct gttc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PAP reverse primer 1 <400> 5 tacagctggc ttaccaccga ccac 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PAP forward primer 2 <400> 6 gtggtcggtg gtaagccagc tgta 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PAP reverse primer 2 <400> 7 ttattgcatc gctgggagaa taaa 24 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> BmPPO-1 forward primer <400> 8 ggatccatgc tgacgccaag aacaac 26 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BmPPO-1 reverse primer <400> 9 ctcgagcccc tgctggccgc gctggcg 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BmPPO-2s forward primer <400> 10 ggatccatgg ctgacgtttt tgaaagc 27 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> BmPPO-2s reverse primer <400> 11 gcggccgcaa ctgacatggg agggttccg 29  

Claims (3)

CDNA of SEQ ID NO: 1 encoding the prophenoloxidase active protease of bumblebee venom. Genomic DNA of SEQ ID NO: 2 encoding the prophenoloxidase active protease of bumblebee venom A polypeptide encoded by the cDNA sequence of claim 1.

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