KR100270927B1 - An antiviral protein cap30 isolated from the chenopodium album var. - Google Patents

Abstract

PURPOSE: An antiviral protein CAP30 separated from Chenopodium album var. is provided, which can be used as antiviral bioagricultural drugs or human antiviral medicines. CONSTITUTION: The antiviral protein CAP30 having 30,000 dalton of molecular weight is isolated by the steps of: grinding the leaves of Chenopodium album var. and extracting it with a buffer solution A containing 50 mM of sodium chloride, 0.2 mM of PMSF, 20 mM of Tris-HCl, 1mM of EDTA, and 10 mM of beta-mercaptoethanol; filtering and centrifuging the extract and collecting the supernatant; adding 30 to 80% of ammonium sulfate and dialyzing it with the buffer solution A; passing the concentrate through the DE-52 cellulose column and subjecting it to linear gradient of buffer solution A to separate the active fragments; and purifying the active fragments by subjecting it to chromatography. The gene encoding the antiviral protein CAP30 is sequenced and its nucleotide sequence is represented by sequence ID No. 1.

Description

Antiviral Protein CAP30 Isolated from White Mage

The present invention relates to CAP30, which is an antiviral protein isolated from the leaves of the Korean native Magnolia (Chenopodium album var.).

Crops cultivated at home and abroad are widely damaged by plant viruses, resulting in severe agricultural product yield and quality deterioration.However, since viral diseases cannot be controlled using pesticides, resistant crops must be developed to reduce the damage. Can be.

In particular, recent advances in molecular biology have led to significant advances in understanding plant viruses and their replication mechanisms, and studies on the development of virus-resistant crops by introducing foreign genes into living organisms and regulating their expression. to be.

Genes that can be used for the development of such viral resistant crops include viral coat proteins, replicas, satellite RNA, movement proteins, antisense and lysine. Ribosomal inactivating protein.

Ribosome Inactivation Proteins (RIPs) are plant proteins widely distributed in the plant kingdom and are derived from various organs such as leaves, roots, and seeds, and their concentrations range from several μg to several hundred mg per 100 g of tissue depending on the type of plant. It is classified into two types, type 1 consisting of a single chain of about 30,000 Daltons and type 2 consisting of an A-chain and a galactose-binding lectin B-chain having ribosome inactivation activity. do.

These, RIPs can protect plants from hospital infections, and some RIPs may also inhibit plant virus infection.

Ribosome inactivation of RIPs is 5'-AGUACGA, a conserved sequence located 250-400 bases from the 3'-terminus of 23S / 25S / 28S rRNAs present in ribosomes from bacteria to mammals. ^* GAGGA-3 '(A ^* is a target adenin residues) through the cleavage of the N-glycosidic bond (ribose) between the ribose (ribose) and the specific adenine residues. The cleaved ribosomes cannot be attached to the EF-2 / GTP complex, so that protein synthesis is blocked at the translation stage, resulting in cell death, which eventually blocks cell-to-cell migration of the infected virus. Will become resistant.

As such a ribosome inactivating protein, various proteins such as PAP (Phytolacca Anti-viral Protein) isolated from Phytolacca americana L. are known.

However, the present inventors searched for another ribosome inactivating protein having excellent antiviral activity in addition to the ribosome inactivating protein to develop a virus resistant crop, and isolated from the leaves of domestic wild white tusks. The discovery that proteins can inactivate ribosomes has led to the completion of the present invention.

That is, it is an object of the present invention to provide CAP30, an antiviral protein derived from the leaves of Chenopodium album var.

1 is a photoelectrophoresis of the protein CAP30 isolated from the leaves of Korean native white Myeongju.

Figure 2 is a result of comparing the amino acid sequence homology of the active site between the CAP30 of the present invention and known ribosomal inactivation proteins.

<Explanation of drawing symbols>

cPAP: disadvantageous inactivating protein from Phytolacca americana

MAP: Disabling Inactivating Proteins from Miravilis jalapa

Abrin A: Disabling Inactivating Protein from Red Beans (Abrus precatorius)

Ricin A: Disabling Inactivating Protein from Castin (Ricinus communis)

Luffin-a: disadvantageous inactivating protein from Luffa cylindrica

Saporin 6: Disabling Inactivating Protein from Saponaria officinalis

Figure 3 is a photograph showing the results of assaying the ribosome inactivation capacity of plants treated with the protein CAP30 according to the present invention.

<Explanation of drawing symbols>

A) tobacco, b) tomato, c) white oyster

Figure 4 is a photograph showing the virus inhibitory effect of the antiviral proteins when inoculated cucumber cucumber virus on the leaves of the white Myeongju.

Figure 5 is a graph showing the inhibitory ability of in-flight protein synthesis by concentration of antiviral proteins.

In order to achieve the above object, the antiviral protein CAP30 of the present invention has a molecular weight of 30,000 Daltons, and its cDNA gene sequence is represented by SEQ ID NO: 1.

Hereinafter, the present invention will be described in more detail.

The antiviral protein according to the present invention, after drying and crushing the leaves of white mite, precipitated crude protein solution by leaching with ammonium sulfate, S-Sepharose colume, Blue-Sepharose column, Pure separation can be achieved by purification with FPLC Mono-S column and Sprose 12 HR 10/30 column. The molecular weight of this isolated and purified protein is 30,000 Daltons and is antiviral vitality.

Therefore, the CAP30 of the present invention can be used as a biopesticide for virus control, and genes isolated from the CAP30 cDNA library are inserted into a vector allowing expression of a foreign gene in a plant to prepare an antiviral protein gene expression vector. By transforming and culturing the vector into plant cells, such as pepper, tobacco, tomato, lily, carnation, garlic, and the like, the vector can be used to produce plants that produce antiviral proteins. In addition, by producing a transgenic plant that produces and expresses an antiviral protein, plants resistant to various types of viruses can be developed.

Hereinafter, the present invention will be described in more detail.

Example 1 Isolation of Antiviral Proteins

(1) Extraction and Purification of Proteins

White Mengju (Chenopodium album var.) From Hwaseong-gun, Gyeonggi-do was collected wildly, and the leaves were separated and stored in a freezer at -80 ° C. 1 kg of white amber leaf was frozen in liquid nitrogen and pulverized, and then ground in a waring blender. Buffer A (20 mM Tris-HCl, 1 mM EDTA, 10 mM β-mercaptoethanol) with 50 mM sodium chloride and 0.2 mM PMSF was added. , pH 7.5). Since the entire experimental process was carried out in a low temperature room 4 ℃. The extract was first filtered with miracloth and centrifuged at 15,000 × g for 30 minutes to obtain a crude protein extract. The crude extract was again precipitated with 30-80% ammonium sulfate and dialyzed in Buffer A. The concentrate was injected into a DE-52 cellulose column (5 × 20 cm) continuously connected to an S-Sepharose colume (5 × 20 cm) previously equilibrated with Buffer A, and then eluted with Buffer A. The adsorbed protein was then eluted with a linear gradient with buffer A containing 0-0.5 M sodium chloride. At this time, while continuously monitoring the concentration of the protein in the eluate, in-flight protein synthesis inhibition rate measured by the in-flight protein synthesis inhibition rate measurement method described in Example 4 to select the active fraction eluted protein with the highest in-flight protein synthesis inhibition Precipitate the protein with 80% Ammonium sulfate in the active fraction, and then concentrate the dialysis solution in Buffer A on a Blue Sepharose column (2 × 25 cm), and then 20 mM Tris-HCl buffer containing 0.1 M sodium chloride. The active fraction was eluted with (pH 8.6), and then the active fraction was eluted with a linear gradient in FPLC system (Pharmecia) with buffer A containing 0-0.5 M sodium chloride in a Mono-S column (1 ml). The reaction mixture was concentrated and finally separated by a Sprose 12 HR 10/30 column (25 mL). The isolated protein was named "CAP30".

(2) Confirmation of purity of separated protein

The degree of pure separation of the protein is usually confirmed by SDS electrophoresis using polyacrylamide gel. Also, if the separated protein is analyzed by protein amino acid sequencing, it can be determined whether it is a complex protein or a pure protein.

a) SDS electrophoresis

CAP30, the protein isolated in Example 1, was subjected to electrophoresis on 12.5% SDS-polyacrylamide gel, and the results as shown in FIG. On the other hand, electrophoresis of the standard protein together to compare the moving distance ratio according to the molecular weight size can be measured molecular weight, the standard protein used in the molecular weight measurement bovine serum albumin 66,200 daltons, egg albumin 45,000 daltons, carbonic anhydrous dehydrogenase (carbonic anhydrase) 31,000 daltons, trypsin inhibitor 21,500 daltons, lysozyme 14,400 daltons. From Figure 1, it can be seen that the CAP30 isolated from the leaves of white tuna is a pure protein having a molecular weight of 30,000 Daltons.

b) N-terminal amino acid sequencing of the protein

4 μg of purified protein CAP30 was electrophoresed on SDS-PAGE, followed by electroblotting on PVDF membranes at room temperature, 50 V, and 170 mA for 30 minutes. As the blotting buffer, 10 mM 3-cyclohexylamino-1-propanesulfonic acid dissolved in 10% methanol was used. Blotted bands were identified by Coomassie blue staining and then cut. Determination of the N-terminal amino acid sequence was analyzed in liquid phase conditions with a protein sequence analyzer 476A (applied Biosystems) against phenylthiohydantoin derivatives. The analyzed N-terminal amino acid sequence is shown in Table 1.

Residue number amino acid Residue number amino acid One Ala 15 Thr 2 Asp 16 Tyr 3 Ile 17 Asn 4 Thr 18 Thr 5 Phe 19 Phe 6 Lys 20 Met 7 Leu 21 Gln 8 Glu 22 Val 9 Pro 23 Ile 10 Lys 24 Arg 11 Pro 25 Asn 12 Thr 26 Gln 13 Gln 27 Ala 14 Asn

Example 2. Isolation of the Gene of CAP30

In order to isolate the gene fragment of CAP30, a degenerated probe for gene selection was constructed using the N-terminal amino acid sequence of the CAP30 protein. ') And Che-2 (5'-TTTCGAACGACTATAGTGAAAATTCGAACTCGGTTTCGGT TGAGTCTTGTGAATGTTGTGACGTCC-3') were synthesized, and then subjected to annealing and klennow treatment and inserted into pCR ^TM 2.1 cloning vector (Invitrogen), and E. coli TG1 (ATCC 13329). ) And the correct insertion was confirmed by sequencing.

After amplifying the prepared carriers, cleavage was carried out with Hind III and PstI, and only fragments were separated, labeled with ³² P-dATP isotopes using a random primed DNA labeling kit (BM 1004760), and used as a CAP30 gene selection probe. It was.

To prepare the cDNA library, approximately 2 mg of total RNA was isolated from 8 g of the leaves of the same white tusk, which was used for protein isolation, and then mRNA was isolated using the QuickPrep mRNA purification Kit (Pharmacia). It was. The cDNA library was prepared using a ZAP-cDNA Gigapack II Gold Cloning Kit (Stratagene 200402) and the efficiency was very good as 1.5 × 10 ⁷ pfu / μl. Seven positive phages were selected by screening about 500,000 phages using the CAP30 gene selection probe prepared above. Selected positive phage fragments were reinserted into pBluscript II SK + (Stratagene), and then reacted using a sequencing kit (Sequencing Kit Version 2.0: Biochemical, USA), followed by FB-SEQ 3545 (Fisher Scientific) DNA Sequencing system. The base sequence was determined in. As a result of analyzing the nucleotide sequence of the CAP30 cDNA fragment, as shown in SEQ ID NO: 1, the total was 1,137 bp, and there was an opening reading frame (ORF) that recognized the 840 hexane sequence (279 amino acids). Since the 26 amino acid sequences of the N-terminal region used as the probe exist identically, it was confirmed that the CAP30 protein gene was selected. In addition, there were signal peptides (underlined in SEQ ID NO: 1) at the N-terminus, and as shown in FIG. 2, homology between the entire nucleotide sequences of known ribosomal inactivating proteins (RIPs) was low. However, since the amino acid sequence of AIQMVAEAARFKYI (the reverse phase in SEQ ID NO: 1), which is a site of very high homology, is an active site of RIPs, it can be assumed that CAP30 of the present invention has an ability to inactivate ribosomes.

Example 3 Inactivation Capacity Assay of Plant Ribosomes Treated with CAP30

In order to assay the ribosomal inactivation ability of CAP30 in tobacco, white tuna and tomato, the efficiency of depurination of ribosomal RNA of tobacco, white tuna and tomato leaf was investigated. That is, after separating the ribosome of tobacco, white tuna and tomato leaves, and treated with CAP30, it was observed whether the cleaved fragment is formed.

After separating the ribosomes of tobacco, white malt, and tomato leaves, each of the ribosomes (15 μg) was treated with CAP30 (1 μg), followed by RAM solution (25 mM HEPES-KOH, 2 mM MgOAC, 2 mM ATP-KOH, 0.1). Mg / ml tRNA) at 30 ° C. for 30 minutes. Then, the ribosomal RNA was isolated, annealed and reacted on ice for 30 minutes, followed by electrophoresis on formamide gels, and Northern blot with a probe to confirm cleavage. The result was shown in FIG.

In general, since the nucleotide sequence of the ribosome rRNA is known to be highly homologous between plants, tomato 25S to recognize fragments separated by annealing treatment upon inactivation in order to examine the presence or absence of ribosome inactivation with or without CAP30 treatment. Primers were prepared for the C-terminus of the ribosomal ribohexane RNA gene (EMBL # LE25SSRIB). The base sequences of the prepared primers were P-1 (5'-TGACGAGAGGAACCGTTG-3 ') and P-2 (5'-ATCTCAGTGGATCGTGGC-3'), respectively, and 335bp (3356) was obtained by PCR synthesis of the genome DNA of tomato. PCR product of -3021) was obtained and used as a probe of the above experiment.

3, in the case of tobacco and tomato ribosomes, ribosome RNA was cleaved by anneal only when CAP30 was treated, but when not treated, the ribosome inactivation ability of CAP30 was confirmed. However, in the case of the ribosome of the white gingival leaves, the cleavage fragments were formed not only in the CAP30 treatment but also in the non-treatment, because it was affected by the CAP30 itself. Therefore, it can be seen that CAP30 is a protein having a strong ribosomal inactivation ability based on the above experiment.

Example 4 Antiviral Vitality Against Plant Viruses

For the antiviral activity, two methods were used: a bioassay in which the virus was directly inoculated into the plant and assayed in the laboratory using the biochemical properties of the antiviral protein.

a) activity assay against cucumber mosaic virus

The antiviral protein (CAP30) isolated from the white M. oleander leaves was made to the concentration shown in Table 2 below, and the tobacco leaves infected with cucumber mosaic virus (CMV) were extracted at a ratio equal to a predetermined amount of the extract extracted at the ratio of 1 g / 5 ml buffer solution. The mixture was smeared on the leaf surface of tobacco (Nicotiana tabacum cv. Samsun). In order to compare the activity with known antiviral proteins, pokeweed antiviral protein (PAP), which was distributed by Professor Irvin of Souyhwest Texas State University, USA, was also treated at the concentrations shown in Table 2 below. The control group was treated with only CMV and non-inoculated treatment (100 mM Tris-HCl, pH 7.5). Viral symptoms were examined at 8, 11 and 15 days after inoculation, respectively. The results are shown in Table 2.

Protein concentration (ng / ml) Sickness 8 days after inoculation 11 days after inoculation 15 days after inoculation PAP 1,000 - - - PAP 500 - ++ +++ PAP 250 ++ +++ +++ CAP 250 - - - CAP 100 - - - CAP 50 - - ++ CAP 25 - + +++ CAP 12.5 + ++ +++ CMV +++ +++ +++ Buffer Treatment - - -

As can be seen in Table 2, the CAP30 at 100ng / ㎖ or more, PAP did not show viral infection symptoms until 15 days after inoculation at a concentration of 1,000ng / ㎖.

b) activity assay for tobacco mosaic virus

Instead of the cucumber mosaic virus of (a), the virus symptom is different from that of tobacco mosaic virus (Tobacco mosaic virus (TMV); TMV U1 strain isolated from the Pathology Department and the Virus Laboratory of Rural Development Administration). The assay was carried out and the results are shown in FIG. 4 and Table 3. In Table 3, PAP-S is a US pore antiviral protein expressed only in seeds, and PRIP-2 is a protein isolated from amber.

As can be seen from Table 3, at the protein concentration of 0.5 µg / ml or less, CAP30 treatment showed better virus infection inhibition effect than PAP treatment.

Protein concentration (ng / ml) ^♪ number of local lesions CAP 30 PAP PAP-S PRIP-2 2.0 0 1 ± 1 1 ± 1 NT 1.0 1 ± 1 15 ± 7 4 ± 2 NT 0.5 4 ± 2 20 ± 8 6 ± 4 NT 0.25 33 ± 12 27 ± 7 19 ± 6 42 ± 23 0.125 47 ± 14 56 ± 17 58 ± 13 56 ± 16 0.0625 NT NT NT 58 ± 17 TMV alone 64 ± 9 ♪: Local lesions of 6 white leaves. NT: No local lesions.

c) Determination of in-flight protein synthesis inhibition

In vitro protein synthesis inhibition was measured by an in vitro translation system (Promega) using rabbit reticulocyte lysate. That is, 3.5 μl of ribonuclease free H ₂ O in a eppendorf tube (1.5 mL), 0.5 μl of RNasin ribonuclease inhibior (40 unit / μl), 1 mM amino acid mixture ((- ) -Leucine) 0.5 [mu] l, [ ¹⁴ C] leucine leucine; 2.5 μl of 50 μCi / ml) and 1.0 μl of Bromine mosaic virus mRNA (0.5 μg / μl) were added and mixed. Then, 3 μl of each protein fraction and 17.5 μl of rabbit reticulocyte lysate were added thereto. The reaction was allowed to proceed for 60 minutes at. After the reaction was stopped by adding 10 µl of 1.5 M potassium hydroxide, 8 µl of the reaction solution was adsorbed onto the glass filter. Radioactive amino acid residues included in the synthesized protein were removed by washing. The washing was performed in 10 minutes in a cooled 10% trichloroacetic acid (TCA) solution, 5 minutes in a 5% TCA solution bathed in boiling water, twice in a 5% TCA solution, twice in an ethanol wash. After the glass filter was washed and dried, the cpm was measured in a Liquid Scintilation Counter (Beckman LS5801) to measure the protein synthesis inhibition rate, and the results are shown in FIG. 5. As can be seen in Figure 5, 50% inhibition of in-flight protein synthesis was 14pM for PAP-S, 9.2pM for CAP30, 23pM for PAP, and 29pM for PRIP-2.

Therefore, it can be seen that the CAP30 according to the present invention has a superior inhibitory effect on the virus than the protein reported to show strong antiviral vitality.

As described above, since CAP30 of the present invention is a protein having inactivation of ribosome and antiviral vitality, it can be used as a biopesticide for virus control, and is combined with an antibody that recognizes a virus that infects humans. Can be used as a sex agent. In addition, the cDNA gene of CAP30 can be used as a viral resistance gene for disease-resistant crop development. When the gene is introduced and expressed in a plant, it can reduce the damage caused by the development of plant viruses in the cultivation of major crops.

Sequence List

SEQ IN NO: 1

Sequence length: 840 bp (hexane) 279aa (protein)

Sequence type: hexane and amino acid

Strandedness: 2 strands (hexane)

Topology: linear (hexane), unknown (protein)

Molecule type: cDNA for rRNA, protein

Hypothetical: No

Anti-sense: no

Original source

(A) Organism: Chenopodium album var.

(B) Strain: Chenopodium album var.

(C) Individual isolate clone name: CAP30

(D) Organelle: leaf

Immediate source

(A) Library name: Chenopodium album L. Library

(B) Clone name: CAP30

Feature of Sequence

(A) Characteristic symbol (Name / Key): CAP30

(B) Identification method: E

Claims (1)

An antiviral protein CAP30 isolated from the leaves of native Korean Magnolia vulgaris (Chenopodium album var.), Molecular weight 30,000 Daltons, and the nucleotide sequence of the gene represented by SEQ ID NO: 1.