KR100261044B1 - Nuecin cdna separated from bombyx mori - Google Patents

Abstract

PURPOSE: Provided is a cDNA of antimicrobial protein nuecin separated from Bombyx mori. And a recombinant antimicrobial protein nuecin is also provided which is effective on both gram positive and negative bacteria, so as to be applied to agricultural chemicals. CONSTITUTION: The cDNA of antimicrobial protein nuecin has the nucleotide sequence of figure (1) consisting of 852 bp and the amino acid sequence of figure (2) consisting of 214 amino acids. The expression vector pBacPAK8-nuecin shown in figure (3) contains the cDNA of antimicrobial protein nuecin. The recombinant virus vAc-nuecin is produced by transforming with the expression vector pBacPAK8-nuecin. The antimicrobial protein nuecin is produced by infecting Spodoptera frugiferda 9 with vAc-nuecin and collecting nuecin.

Description

CDNA of antibacterial protein gene silkworm isolated from Korean silkworm and its recombinant protein

1 shows the nucleotide sequence of silkworm _C DNA,

2 shows the imino acid sequence deduced from the silkworm _C DNA sequence,

3 shows a schematic diagram of a process for preparing a transfection vector for expressing recombinant baculovirus containing silkworm _C DNA,

4 is a result of Southern blot analysis of recombinant viral DNA to confirm that it contains recombinant baculovirus silkworm _C DNA,

5 shows protein electrophoresis analysis using 12.5% SDS-polyacrylamide gel of recombinant silkworm expressed in insect cell line (Sf21) by recombinant baculovirus.

The present invention relates to _C DNA of a new antibacterial protein gene silkworm isolated from Bombyx mori and recombinant protein produced using the insect baculovirus expression system.

Insects are a biological defense against invasion of microorganisms or foreign substances. Insects synthesize proteins from cellular immunity and fat and blood cells that remove invasive substances through phagocytosis, nodule formation, and encapsulation. And humoral immunity to secrete hemolymph to remove microorganisms (Boman & Hultmark, Annu. Rev. Microbiol., 41, 103-216, 1987: Dunn, Annu, Rev. Entomol., 31, 321-329 , 1986). Cecropin, lysozyme, and hemoline from wild silkworm moths and Hyalophora cecropia immunized with celiac injection of insect epidermal wounds or bacteria or lipopolysaccharides ) And proteins exhibiting antibacterial activity such as atacin were isolated. (Boman & Hultmark, Trends Biochem. Sci., 6, 306, 1981). Other proteins related to immunity have been reported, such as apidaecin, defensin, diptericin, and hypophancin. (Casteels et al., EMBO J., 8, 2387-2391, 1989: Lambert et al., Proc. Nat. Aacad. Sci. USA, 86, 262-266, 1989: Dimardq et al., Eur j. Biochem , 171, 17-22, 1988: Park et al., Korean J. Entomol., 24,191-198. 1994). Among these, atacin is a peptide having a molecular weight of about 20 to 30 kDa. Of the six forms reported so far, four have basic and two acidic isoelectric points (Dunn, Annu. Rev. Entomol., 31, 321- 329, 1986). Acidic atacin consists of 19 amino acid signal sequences and 28 amino acid sequences, and basic atacin consists of 17 amino acid signal sequences and 29 amino acid sequences, which form precursors Loatacin (preproattacin) is transported to the endoplasmic reticulum (Simon & Blobel, Cell, 65, 371-380, 1991), and the enzyme recognizes two basic residues (Arginine-Arginine) in the golgi body. Signal peptide is removed and converted to mature atacin (Pfeffer & Rothman, Annu Raev. Biochem., 56, 829-852, 1987), and the atacin N-terminal glutamine (pylutamine) It is converted into glutoglutamyl and secreted into hemolymph and exhibits biological activity (Gunne et al., Eur. J. Biochem., 187, 699-703, 1990). On the other hand, it is found that cecropin acts to destroy bacterial membranes, and sarcotoxin 11A, an attacin-like protein isolated from Sarcophaga peregrina (flesh fly), inhibits bacterial cell wall synthesis. (Ando & Natori, Biochmistry, 27, 1715-1721, 1988: Kanai & Natori, Mol. Cell., 10, 6114-6122, 1990). Atasin inhibits the biosynthesis of bacterial coat proteins (Carlsson et al., Infect. Lmmuno., 59, 3040-3045, 1991) and invades the outer membrane of E. coli to enhance the antibacterial activity of cecropin or lysozyme. However, its antibacterial activity is limited to Gram-negative bacteria (Engstrom et al., EMBO J., 3, 3347-3351, 1984). In addition, the atacin-like peptide Bmatt, isolated from silkworms, was reported to have a _C DNA size of 846 bp but no antibacterial activity spectrum was reported yet (Sugiyama, M. et al., Insect Biochem. Mol., 23, 385-). 392, 1995),

Attacin's application research has led to the introduction of attacin E gene to produce a disease-resistant transgenic plant that is resistant to the erwinia amylovora, an apple tree pathogen, and has succeeded in bioassay in plastic houses (Norelli et al., Euohytica, 77, 123-128, 1994).

Insect antibacterial peptides can be used not only for the creation of disease-resistant transgenic plants, but also as biological pesticides, and their simple structure makes them highly valuable as new biomaterials that can replace existing synthetic antibiotics.

Therefore, the present invention isolated new antibacterial peptide silkworm _C DNA from Bombyx mori and produced recombinant protein by using insect baculovirus expression vector system and antibacterial activity against gram negative bacteria as well as gram positive bacteria. The purpose of the present invention is to provide such antibacterial peptides for the production of disease-resistant transgenic plants, biopesticides and new biological materials.

Hereinafter, the present invention will be described in detail.

[summary]

The present invention has been found to be related to the amino acid sequence deduced from the new anti-bacterial gene silkworm God _C DNA and the DNA separated from the _C jipnue a new gene on June 29, 1997 was registered as GenBank Registration No. AF005384.

In the present invention, standard single characters are used to represent nucleotide sequences and amino acid sequences, and the meanings of these abbreviations are standard biochemistry textbooks and molecular biology experiments, such as Leninginger, Principles of Biochemistry, Worth Publishers Inc., ANew Uo. , pp96, 789, 1984: Sambrook, J. et al., Molecular Cloning, A Laboratory MANUALS, cold Harbor Laboratory Press, NY, PP174-184, 1989).

[submission]

It is deposited in the Biotechnology Research (KRIBB) (KRIBB) under the regulations published under the Budapest Treaty and Treaty on the International Recognition of Microbial Deposits for the purpose of the Baculovirus vAc-nuecin Patent Procedure. The expression transfer vector sample may be available or available to industrial property authorities and the individual who is legally authorized to receive them under the provisions of Article 4 of the Enforcement Decree of the Patent Act relating to the Treaty.

The KRIBB accession numbers and deposit dates for the deposits are as follows:

[Summary of invention]

The present invention also relates to a recombinant baculovirus expression transfer vector and a recombinant baculovirus transformed thereby, including the _C DNA. As an example, the transition vector pBacPAK8-nuecin and the recombinant baculovirus vAc transformed thereby are provided. -nuecin.

In the present invention, by infecting the transformed recombinant baculovirus vAc-nuencin to the insect cell line (Spodoptera frugiperda 9), the recombinant protein silkworm is expressed and mature silkworms secreted into the silkworm form of maturation as a culture medium in the insect cell line. have.

In the present invention, the antibacterial activity of the recombinant protein silkworm expressed by the _C DNA is Gram-negative bacteria E. coli, Erwinia chrysantuemi, Xanthomonas campestris pv. oryzae, Xanthomonas campertris pv. Citri and gram positive bacteria Bacillus thuringiensis, Bacillus megaterium, Bacillus thuringiensis kurstaki, Bacillus subtilis and Staphylococcus aureus were tested. The invention is illustrated in more detail by the following examples.

[Description of the Invention]

Example 1

[ _C DNA Cloning and Nucleotide Sequence and Amino Acid Sequence Analysis of Silkworm]

After 8 hours of injection of E. coli K12 (4 × 10 ⁶ cells / larva) in logarithmic phase into the larval cavity of the larvae, an immuno-induced silkworm _C DNA-rich bank was produced using silkworms. Selection of increasing immune-induced silkworm genes was performed by differential screening (Hoog et al., Nuclein Acids Res. 19, 6123-6127, 1991). In order to analyze the sequencing of the differentially selected _C DNA clones, a deletion set was prepared using the Exo / Mung bean nuclease deletion kit (pronega), and the sequencing analysis was completed with the dideoxy chain termination. (dideoxy chain termination) method (Sangenase version 2.0 kit, manufactured by United States Biochmical) by the method (Sanger et al., Proc. Natl. Acad. Sci USA, 74, 5463-5467, 1997). It was. Denature the 0.5-1.0 μg single stranded DNA with 1.5 μl T3 primer (Stratagene, 5′-AATTAACCCTCACTAAAGGG-3 ′, 20-mer, 3.125 ng / μl) and 2.0 μl 5X sequencing enzyme buffer at 70 ° C. for 2 minutes. Afterwards, the temperature was slowly lowered to below 35 ° C. so that the primer and template DNA could be combined. And 2.0 μl dGTP (or 7-deaza-dGTP, dITP) labeling mixture with 1.0 μl 0.1MDTT (dithiothreitol), 0.5 μl [α- ³⁵ S] dATP (10 mCi / ml) or [α- ³² P] dATP ( 10 mCi / ml) and 2.8 units (U) of sequencing enzyme were added and reacted at room temperature. The reaction product carried out by the above method was dispensed in 3.5 ㎕ each of the ddGTP, ddAPT, ddTTP, ddCTP containing a still mixture, and terminated for 5 minutes at 37 ℃. Then, 4.0 μl of stop buffer was added to the gel for sequencing to determine the nucleotide sequence, and the results are shown in FIG. 1.

As shown in FIG. 1, after selection of the Nuecin gene with increased expression after immuno-induction, the total nucleotide sequence was determined, and this _C DNA had a total size of 852 bp and was initiated at the 35th base at the 679 position. It has an open reading frame and it is confirmed that there is a provisional transcription termination signal 'AATAAA' at the 812th position. The base sequence shown in FIG. 1 was found to be a new antibacterial protein gene by database search. In Fig. 1, the vertically arranged numbers at the left ends indicate the order of bases.

The amino acid sequence was deduced by the base sequence of the determined _C DNA, and the results are shown in FIG.

As shown in FIG. 2, 214 amino acids were deduced by the base sequence of silkworm _C DNA. Also, in FIG. 2, the newly listed numbers at the left ends indicate the order of amino acids.

Example 2

[Recombinant baculovirus production comprising silkworm _C DNA]

5′-primer (5′-CGGGATCCATGTCCAAGAG-3 ′, 19-mer) containing the translation start codon of the silkworm gene to produce a recombinant protein of silkworm _C DNA having the nucleotide sequence determined in Example 1 above A 3′-primer containing a signal (5′-CGGAATTCTTAGAATTCG-3 ′, 22-mer) was synthesized to amplify the DNA fragment containing the coding region, and then inserted into a pGEMT vector (promega), and the recombinant plasmid Was named pGEMT-nuecin. The BamHl / EcoRl DNA fragment of PGEMT-nuencin was inserted at the BamHl / EcoRl position of pBacPAK8 (Clontech) to prepare a recombinant baculovirus transfer vector pBacPAK8-nuecin. The schematic is shown in FIG.

In order to produce a recombinant virus into which the silkworm gene was introduced, the recombinant transfer vector (pBacPAK8-nuecin) shown in FIG. 3 was mixed, and the same amount of lipofectin (lipofectin, manufactured by Gibco) was added and reacted at room temperature for 15 minutes. The mixture was inoculated into a 35 mm 2 cell culture dish containing 1.0 × 10 ⁶ Sf9 cell lines, transfected at 27 ° C. for 5 minutes, replaced with fresh culture solution, and cultured at 27 ° C. for 5 days.

The selection of recombinant virus (vAc-nuecin) was performed by plaque assay (Summers & Smith, Texas Agriculture Experiment Station, Billetin No. 1555, 1987). Incubate the culture medium of insect cells with 10 ^-4 to 10 ^-8 marginal dilution and inoculate 1.5 × 10 ⁶ cells, and then cryolytic agarose (FMC) containing X-gal at a final concentration of 120 μl / ml. Product) and recombinant viruses of plaques that were not stained with ex-gal were selected while incubating at 27 ° C. The selected recombinant virus was reinoculated and propagated. Confirmation of whether the recombinant virus contained silkworm _C DNA was performed by Southern blot analysis by separating the DNA of the recombinant virus.

The cell cultures inoculated with the recombinant virus were centrifuged at 2,000 rpm for 10 minutes and the supernatant was further centrifuged at 30,000 rpm for 1 hour to collect a precipitate. Then, SDS 1%, proteinase K was added to a concentration of 0.5 mg / ml (manufactured by Sigma) and treated at 37 ° C for at least 4 hours. Then, the same amount of phenol / chloroform-isoamyl alcohol (phenol / chloroform-isoamylalcohol, 24: 1) was added and mixed, and the supernatant was collected by centrifugation at 15,000 rpm for 5 minutes. To this, 2 times of cold ethanol was added, the DNA was precipitated for 15 minutes at 15,000 rpm, and the recombinant viral DNA separated by 70% ethanol washing was treated with various restriction enzymes and subjected to agarose gel electrophoresis. 0.5M NaOH, 1.5M NaCl) was immersed for 15 minutes and neutralized with neutralizing solution (1M Tris-HCL, pH7.4, 1.5M NaCl). The neutralized gel was transferred to nylon membrane with 20 × SSC solution (3M NaCl, 0.3M Sodium citrate), and the DNA was fixed with UV crosslinker. The treated membrane was reacted with a biotin-labeled silkworm _C DNA with a probe, and the result was confirmed by Southern Light Chemiluminescent Detection System (manufactured by TROPIX).

As shown in FIG. 4, about 4.3 kb of recombinant viral DNA treated with restriction enzyme EcoRl (lane 2), about 2 kb (lane 3) treated with BamHl, and about 0.6 kb treated with BamHl / EcoRl. Bands were combined with the silkworm _C DNA probe of kb (lane 4). However, since the band was not found in the DNA of the wild-type virus (BacPAK B6) using the control, the recombinant virus was found to contain the silkworm gene.

Example 3

Expression of Recombinant Silkworm Protein in Insect Cell Line by Recombinant Baculovirus Infected with the insect cell line (Spodoptera frugiferda 9), the recombinant virus with the silkworm gene introduced in Example 2 was identified and confirmed the intracellular expression of the recombinant silkworm protein. In order to check the intracellular expression of recombinant silkworm protein by performing recombinant protein electrophoresis (Laemmli, Nature, 227, 680-685, 1970) by recovering the cells at an insect cell line infected with a recombinant virus every three days. . Recombinant virus-infected cells were collected, centrifuged at 2,000 rpm for 10 minutes, and the precipitated cells were washed twice with PBS (phosphate buffered saline) solution, and twice the protein sample solution (0.0625M Tris-HCI, pH6.8). , 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.001% brbomophenol blue) were mixed. After heating at 100 ° C. for 5 minutes, electrophoresis was performed on 12.5% SDS-polyacrylamide gel and stained with Coomassie brilliant blue. The results are shown in FIG.

As shown in Figure 5, a silkworm band of about 23 kDa was not observed in uninfected normal cell lines (lane 2) and wild line virus infected cell lines (lane 3), but in recombinant cell infected cell lines 1 Day (lane 4) showed high expression levels on the 2nd day (lane 5) and 3rd day (lane 6).

Example 4

[Antibacterial Activity Assay of Recombinant Silkworms]

In order to assay the antibacterial activity of the recombinant silkworm produced in Example 3, 40 ml (0.5 × 10 ⁶ cells / ml, inoculation concentration 1 MOl) 4 days after infection by recombinant virus (vAc-nuecin) Was concentrated and assayed by an inhibition zone assay (Lambert et al., Proc. Natl. Acad. Sci. USA, 86, 262-266, 1989). ⁵ ml of agar medium (1% nutrient agar, pH 7.2) containing logarithmic bacteria (2 × 10 ⁵ / ml) was coagulated in a sterile culture dish (87 × 15 mm). A filter cell (Whatmann 3MM) having a diameter of 6.1 mm was placed therein, and concentrated cell culture solution (1.2 μL silkworm) was added dropwise to each filter paper, and cultured at 37 ° C., and then the size of the growth zone formed was measured. Four gram negative bacteria (E. coli, Erwinia chrysantuemi, Xanthomonas camperstris pv. Oryzae, Xanthomonas campestris pv. Citri) and five Gram-positive bacteria (Bacillus thuringiensis, Bacillus megaterium, Bacillus thuringiensis kurstaki) , Bacillus subtilis, Stphylococcus aureus) were used.

The results are shown in Table 1.

TABLE 1

* Antibacterial activity was determined by the size of growth inhibitory ring as follows. ++, growth inhibitory ring of 10-15 mm; +++, growth inhibition ring of 15-20 mm; Growth inhibition ring of 20 mm or more; -Indicates no effect.

As shown in Table 1, the antibacterial activity spectrum of recombinant silkworm showed high antibacterial activity in the gram-negative bacteria of Escherichia coli and Erwinia chrysantuemi, but the aerobic bacteria Xanthomonas campestris pv. In citri, the activity was relatively low, and Xanthomonas campestris pv. No activity against oryzae was observed. Among the Gram-positive bacteria, Bacillus subtilis, which is widely distributed in soil and gourd, is a cause of decay such as rice. In addition, Staphylococcus auresus, which produces enterotoxin to induce food poisoning, and Bacillus thuringiensis without pathogenicity, did not show activity.

From the above analysis, recombinant silkworm has antibacterial activity to Gram-positive bacteria as well as Gram-positive bacteria, so that silkworm _C DNA isolated from houseworms is functional and active only to Bmatt and Gram-negative bacteria isolated from houseworms so far published. It was confirmed that it is a new gene different from atacin.

Claims (6)

_C DNA encoding a protein isolated from silkworm, characterized by having the following amino acid sequence

The _C DNA according to claim 1, which has the following nucleotide sequence

Recombinant baculovirus expression transfer vector comprising the _C DNA of claim 2. A recombinant baculovirus comprising the recombinant baculovirus expression transfer vector of claim 3. Recombinant silkworm protein produced by the recombinant baculovirus of claim 4. An antimicrobial pesticide comprising the recombinant silkworm protein of claim 5 as an active ingredient.

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