KR101570783B1 - Transgenic silkworms producing antimicrobial peptide - Google Patents

Abstract

The present invention relates to a transgenic silkworm containing an antimicrobial peptide and a method for mass-producing a transgenic silkworm containing the antimicrobial peptide using the same, and more particularly, to a method for mass production of a transgenic silkworm containing an antimicrobial peptide, a marker gene- A transformant silkworm producing a silkworm containing an antimicrobial peptide can be produced by preparing a recombinant expression vector containing a gene construct in which the BmRelish1t gene is operably linked and transforming silkworm with the recombinant expression vector, It can be developed as a natural antibiotic and can be useful not only as a feed additive for livestock but also as a material for daily necessities such as cosmetics and toothpaste.

Description

Transgenic silkworms producing antimicrobial peptides containing antimicrobial peptides

The present invention relates to a transgenic silkworm containing an antimicrobial peptide and a method for mass-producing a transgenic silkworm containing the antimicrobial peptide using the transgenic silkworm.

The silkworm belongs to the taxonomic insecta, lepidoptera, Bombyxidae, Bombyx, and moth species. The silkworm is a complete transformational insect, which develops larvae hatching from eggs, becomes a pupa, becomes an adult (moth), gives birth to an egg, and finishes its life. The life of the silkworm is relatively short, on average 60 days, and it has an advantage as an experimental animal. Eggs that have been scattered are colored when left untreated, and hatched and hatch without coloring until the following spring. The number of hatchings per year is determined by the amount of dormant hormone produced in the ganglion below the esophagus by each gene action. In silkworm eggs, fertilized nuclei divide several times to form cleavage nuclei, surrounded by protoplasts around the nuclei, and they migrate toward the edges of eggs. For example, after 12 hours of spawning, ants silkworms form a syncytial blastoderm, and 20 hours later, it is possible to carry out pickling, one of the artificial incubation methods. At 30 hours after spawning, it becomes full yolk cells and hatches into ants silkworm after about 10 days.

Various studies have been conducted to produce silkworm transgenic silkworms due to their industrial value as well as research use. The development of transgenic silkworms was carried out by the Japanese Tamura et al. Transgenic transgenic vectors were constructed using the piggyBac gene derived from ni and microinjection into transgenic silkworm cultivars was succeeded in producing transgenic silkworms for the first time. Recently, transgenic silkworms producing various biopharmaceuticals have been reported. On the other hand, it has been reported that the transfection vector is injected into the silkworm eggs by the microinjection method used by Tamura et al. In 2000, and the injection efficiency can be improved by injecting the microinjection position in the middle part between the main part and the posterior part of the embryo have.

A transgenic animal is an animal in which an exogenous gene has been inserted into the genome of a host and a part of its trait has changed, and the foreign gene at that time is called a transgene. In the mid 1970's, we started to import foreign genes by using recombinant viruses in somatic cells or germ cells. In 1980, Gordon produced super-mice by microinjection.

Techniques for introducing a foreign gene include calcium phosphate, electroporation, DEAE-dextran, liposome, microinjection, and bombardment. Among these methods, the DEAE-dextran method and the electroporation method allow the cells to enter the cytoplasm directly through the open hole of the DNA, which may damage the DNA. The method using liposomes is widely used as a method of directly transferring DNA into a cell by fusing the DNA with a liposome, an artificial lipid vesicle, into the cell membrane. The microinjection method is a method of directly injecting DNA with a micro needle to such an extent that it does not damage the eggs by using a micro manipulator in the first embryo transfer embryo. Outpatient gene transfer technology at the practical stage will have infinite benefits to human beings if introduced foreign genes are related to growth rate control, tolerance in extreme environments, gene therapy.

In July 2011, it is urgently required to develop an effective antibiotic substitute to solve problems such as a decrease in productivity and an increase in the incidence of diseases, which may occur when the antibiotics used for promoting growth in livestock feeds are totally prohibited.

As related prior arts, Korean Patent No. 10-0267742 (filed on July 07, 2000, entitled "Fluorescent silkworms using recombinant baculovirus inserted with green fluorescent protein gene and preparation method thereof") and Korea Patent No. 10 -0323550 (registered on Jan. 24, 2002, titled: Transgenic silkworm transfection method and transgenic silkworm).

It is an object of the present invention to provide a recombinant expression vector comprising a gene construct operably linked to a silkworm-derived BmRelish1t gene, and an antimicrobial peptide transformed with the recombinant expression vector, in order to mass- The present invention provides a transgenic silkworm wherein the transgenic silkworm is expressed.

Another object of the present invention is to provide a method for mass-producing a transformed silkworm containing an antibacterial peptide using the transgenic silkworm according to the present invention.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

In order to achieve the above object, the recombinant expression vector of the present invention comprises a marker gene-regulated promoter, a marker gene, a nuactin-3 promoter, and a gene construct in which silkworm-derived BmRelish1t gene is operably linked.

The silkworm-derived BmRelish1t gene is a gene other than an extrinsic protein domain (ankyrin repeat domain) and a death-inducing protein domain (death domain).

The silkworm-derived promoter is a silkworm-derived actin-3 promoter.

The marker gene-regulated promoter is a 3xP3 promoter expressed in the eye and nervous system.

The marker gene is characterized by being a green fluorescent protein (EGFP) gene.

The gene construct is characterized by having the structure of FIG.

The expression vector is a piggyBac vector.

The transgenic silkworm to which the antimicrobial peptide of the present invention is expressed is characterized in that the recombinant expression vector is produced by transformation into silkworm (Bombyx mori) or silkworm.

The method for producing a transformed silkworm in which the antimicrobial peptide of the present invention is expressed comprises the steps of: 1) preparing the recombinant expression vector; 2) injecting the recombinant expression vector of step 1) into silkworm eggs to produce transformed silkworm eggs; And 3) hatching the transformed silkworm of step 2) to produce a transformed silkworm.

The transformation of step 2) is characterized by microinjection.

A method for producing a transformed silkworm wherein an antimicrobial peptide is expressed is characterized by further comprising the step of selecting a transformed silkworm by one of the following methods in the step 3): i) introducing a marker gene into an expression vector , A method of confirming the expression of the marker gene in a transgenic silkworm; Or ii) a method of confirming the expression of an antimicrobial peptide in a transgenic silkworm.

The method for mass production of the transformed silkworm containing the antimicrobial peptide of the present invention comprises the steps of: 1) preparing the recombinant expression vector of any one of claims 1 to 6; 2) transforming the recombinant expression vector of step 1) into silkworm or silkworm to produce a transformed silkworm; And 3) culturing the transformed silkworm of step 2) to obtain a transformed silkworm containing an antimicrobial peptide.

The transgenic silkworms of the present invention can be developed as natural antibiotics by producing transgenic silkworms containing antimicrobial peptides and can be usefully used as materials for household goods such as cosmetics and toothpastes as well as animal feed additives. In addition, the silkworm farmers can contribute to the improvement of income by breeding transgenic silkworms which produce high value-added natural antibiotics differentiated from common silkworms through the present invention.

1 is a diagram showing the structure of a transition vector (pG-3xP3-EGFP-BmA3-BmRelish1t).
Figure 2 is a diagram showing the fluorescence of EGFP in G2-transgenic silkworms.
A is egg, and F2 is a state in which fluorescence appears in the eye and nervous system of the fifth embryo.
(Arrows show the eye and nervous system)
B shows fluorescence in the eyes of the F2 3 insect in the larval case.
(Arrows show eyes)
FIG. 3 is a diagram showing the results of analysis of BmRelish1t gene using RT-PCR in transformed silkworms. FIG.
Lane M; 1 kb step DNA marker
Lane 1; PCR products of BmRelish1t
Fig. 4 is a diagram showing the antimicrobial activity of a transformed silkworm containing the BmRelish1t gene.
normal; Normal silkworm
BmRelish1t; Transgenic silkworm

Hereinafter, the present invention will be described in detail.

The present invention provides a recombinant expression vector comprising a marker gene-regulated promoter, a marker gene, a silkworm-derived promoter, and a gene construct operably linked to the silkworm-derived BmRelish1t gene.

In one embodiment of the present invention, a gene encoding a silkworm-derived BmRelish1t gene, which is introduced to produce an antimicrobial peptide by immunization induction in silkworm, is a gene other than an ankyrin repeat domain and a death domain, .

In the recombinant expression vector, it is preferable that the silkworm-derived promoter uses nuhectin 3 (BmA3) for the expression control of the amplified BmRelish1t gene.

In the recombinant expression vector, the marker gene-regulated promoter that regulates expression of the marker gene is preferably a 3xP3 promoter, but not limited thereto, and any promoter capable of expressing the marker gene can be used.

In the recombinant expression vector, all of the genes that express the fluorescent protein can be used as the marker gene for selection of transformants, and it is more preferable to use EGFP (green fluorescent protein) gene, but the present invention is not limited thereto.

In one embodiment of the present invention, a fluorescent protein (EGFP) was used as a marker gene for the selection of transformants, and a 3xP3 promoter specifically expressed in the eye and nervous system was used to regulate the expression of the marker gene.

In the recombinant expression vector, the gene construct preferably has a construct comprising the structure of FIG. 1, but is not limited thereto.

In the recombinant expression vector, the expression vector into which the gene construct is introduced is preferably a piggyBac vector, but is not limited thereto.

The present invention also provides a transformed silkworm in which an antimicrobial peptide is expressed by transforming the recombinant expression vector according to the present invention into silkworm (Bombyx mori) or silkworm eggs.

In addition,

1) preparing the recombinant expression vector according to the present invention;

2) transforming the recombinant expression vector of step 1) into silkworm eggs to produce transformed silkworm eggs; And

3) hatching the transformed silkworm in step 2) to produce a transformed silkworm, wherein the transformed silkworm is expressed.

In the above method, the transformation in step 2) is preferably performed using microinjection, but not limited thereto, and all known transformation methods can be used.

In the above method, in the step 3), it may further include selecting a transformed silkworm by one of the following methods.

i) a method of inserting a marker gene into an expression vector and then confirming the expression of the marker gene in the transformed silkworm; or,

ii) A method of confirming the expression of an antimicrobial peptide in a transgenic silkworm.

In one embodiment of the present invention, silkworm eggs were transformed with the recombinant expression vector using a known microinjection method, and some of them were hatched with larvae, and some adult moths were mated with each other to generate F1 generations Of silkworm eggs were obtained. Then, the transgenic plants were selected by observing the expression of the marker gene in the eye or nerve tissue in the early embryo, larva, pupa, or adult after spawning of the F1 generations. Then, finally, only these were crossed to obtain F2 generation transformants.

In addition,

1) preparing the recombinant expression vector according to the present invention;

2) transforming the recombinant expression vector of step 1) into silkworm or silkworm to produce a transformed silkworm; And

3) culturing the transformed silkworm of step 2) to obtain a transformed silkworm containing an antimicrobial peptide, wherein the transformed silkworm is cultivated in the step 2).

The transgenic silkworm of the present invention can produce transgenic silkworms containing a large amount of antimicrobial peptides, and thus can be usefully used as a material for daily necessities such as natural antibiotics, animal feed additives, cosmetics, and toothpastes.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Production of Transformation Transformation Vector

In order to produce transgenic silkworms expressing the BmRelish1t gene in silkworms, the nuactin 3 promoter and the BmRelish1t gene were introduced into piggyBac vector.

First, in order to obtain the silkworm actin 3 promoter, the following primers were used for PCR amplification. The sense primer used was 5'-GGCGCGCCGCGCGTTACCATATATGGTG-3 'including the translation initiation codon ATG and Asc I restriction enzyme recognition sequence and the antisense primer was amplified by PCR using 5'-GCTAGCCTTGAATTAGTCTGCAAGAAA' containing Nhe I restriction enzyme Amplified and cloned into pGEM-T Easy Vector System (Promega, Madison Wis.).

The completed plasmid was named 'pGEMT-BmA3'. Next, pGEMT-BmA3 was treated with restriction enzymes Asc I and Nhe I to prepare fragments. These fragments were cloned into a pG3xP3-EGFP vector, a piggybac transfer vector restricted with Asc I and Nhe I, and named pG3xP3-EGFP-BmA3.

BmRelish1t gene was obtained by PCR amplification using the following primers.

(BmRelish1t genes: caagaagagaaggatgaatgcctcatgttctacttcccctgcttcgtctgggtcgttgaagagtatcagcgatctgcccgcccttgtgactctcaataacaatttcaacgatgaaaacaacattgaaatgccgaaaactgagctgccggccgccgcggccgccgccccctctctgggactgtgcgacctcgccgacgcgctcatgggctccgagagcgccgtggacctacactcggtcgccgggaacgcgatgtggagcccgagtgcgcccgtcgagacgatgagcgtcgagcagttcccgaatctgcaactgaattcgacggagttcgaaaagataataacgaacgcccttccgccggaggaaaggaacgacttcacggaatatgccctaagttcgtacgaatcgttcgatgacatcgacgatgatccaaatggatggaagttcatacgtacgttacagatgatgcaaaccgactctgcacgcagtaggcagcagacaccgcagactagtaaacccgcactgaaaactgttgaaccccaagagagctcagacaggaccgagctgaatgtaccgtctgatgcgaaagatgcaaacgaatatagcgcttattacaacgcccaagacggcctcgaagtgaaacagctactaagagaactgTAA)

sense primer used 5'-AAAATGTCTACAACTGCCAGTG-3 'containing the translation initiation codon ATG and Nhe I restriction enzyme recognition sequence and the antisense primer used 5'-TTATAATACATAAGTCAATGGAT-3' containing Afl II restriction enzyme Respectively.

The thus amplified BmRelish1t cDNA was cloned into pGEM-T easy vector (Promega Co.) to construct pGEMT-BmRelish1t. The BmRelish1t ORF fragment between NheI / AflII of pGEMT-BmRelish1t was inserted into the NheI / AflII site of the piggyBac vector (PG-3xP3-EGFP-BmA3-BmRelish1t) (Fig. 1).

≪ Example 2 > Production and selection of silkworm transformants

<2-1> Preparation and breeding of silkworm

Bombyx mori, a silkworm kept at the National Institute of Agricultural Science and Technology (RDA), was used for the transformation of the silkworm (sleep 123 × sleep 124) Relative humidity, 70% - 90%).

<2-2> Production of silkworm transformant

The production of silkworm transformants was carried out with reference to the microinjection method used by Tamura et al. In 2000 (Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas (2000) Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nat Biotechnol 18, 81-84)

Specifically, the concentration ratio of the transition vector 'pG-3xP3-EGFP-BmA3-BmRelish1t' prepared in Example 1 and the helper plasmid pHA3PIG was 1: 1 (200 ng / And diluted to a concentration of 0.2 μg / μl in a buffer solution for microinjection (5 mM KCl, 0.5 mM Phosphate buffer, pH 7.0). The microinjection of the silkworm early blastocyst was injected into the middle part of the middle between the main and posterior parts of the embryo. The procedure was as follows. First, a small hole is drilled in the railing of the silkworm with a tungsten needle, the tip of the microcapillary containing the DNA solution is inserted into the hole, and the DNA solution is injected into the egg using the air pressure of the microinjector Respectively. At this time, the amount of DNA solution injected into each embryo was 10-15 nl, and the hole in the railing was blocked with a cyanocrylate adhesive. A total of 1800 silkworm eggs were microinjected. After microinjection, silkworm eggs were placed in a moistened patridish and protected until incubated at 25 ° C.

<2-3> Screening of silkworm transformants

Selection of silkworm transformants was performed by fluorescence microscopy. Specifically, the silkworms were selected for each generation and period by using a LEICA MZ16FA microscope (Leica, USA) and a Microscope MZ FLIII Flourescence Filter EGFP fluorescence filter (Leica, USA). According to the previously reported documents, the 3xP3 promoter is known to act in the eyes, neural tissues and larval eyes of the early stage of silkworm, and the transformants were selected based on these characteristics.

As a result, green fluorescence was observed in the eyes and nervous tissues of the silkworms from the third day after spawning, and a total of two transformants (G1) having EGFP positive could be selected (Table 1).

In addition, the larvae of the green fluorescent eye were selected from the transformant (G1) (Fig. 2), and finally the transformants of F2 generation were selected by crossing only these genes.

Transformation ratio of construct DNA to Baekokjang embryo Injected embryo A hatched embryo G1 amount EGFP positive
G1 amount
600 grains 80 grains 18 grains Two

White birch was used as a host species.

Vector plasmid pG-3xP3-EGFP-BmA3-BmRelish1t (200 ng / ul) and helper plasmid (200 ng / ul) were used for injection.

<Example 3> Expression analysis of BmRelish1t gene in silkworm transformant

The expression of BmRelish1t gene in silkworm transformants was analyzed by RT-PCR.

Specifically, 2 ug of total RNA was isolated from transgenic silkworms of the second generation (F2) 5th instar larvae using TRI REAGENT (Molecular Research Center), and a High-Capacity cDNA Reverse Transcription kit Applied Biosystems). PCR was carried out using 5'-GCGCGTTACCATATATGGTG-3 'and 5'-CTTGAATTAGTCTGCAAGAAA-3' gene specific primers.

PCR proceeded as follows. First, it was performed once at 95 ° C for 5 minutes, then repeated 30 times at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 50 seconds, and was performed once at 72 ° C for 5 minutes. The amplified PCR products were confirmed by electrophoresis on 1% agarose gel.

As a result, gene specific primers capable of amplifying BmRelish1t cDNA were used. As a result, an expected 1,686 bp band of BmRelish1t cDNA was confirmed (FIG. 3).

<Experimental Example 1> Antimicrobial activity of antimicrobial peptides produced by BmRelish1t gene in silkworm transformants

In order to investigate the activity of antimicrobial peptides produced by the BmRelish1t gene in silkworm transformants, we used the Radial diffusion assay (RDA).

Bacteria cultured on sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type agarose and 0.03% TSB (4 × 10 ⁶ colony forming units / ml) and mix. After mixing in the culture dish, make a 3 mm diameter hole and add 5 μl of 1 mg / ml peptide. Peptide is allowed to diffuse for 3 hours at 37 ° C, then pour overlay gel (6% TSB, 1% agarose) and incubate at 37 ° C again. The antimicrobial activity of each peptide appeared after 18 hours, and it was confirmed by the size of the clear zone that the bacteria could not grow.

As a result, it was confirmed that the transformed silkworms expressing BmRelish1t gene exhibited antibacterial activity as compared with normal silkworms (FIG. 4).

Thus, it was confirmed that silkworm transformants producing BmRelish1t combined protein in silkworm were produced.

Thus, the transgenic silkworm can be developed as a natural antibiotic by producing a transgenic silkworm containing an antimicrobial peptide. Thus, the transformed silkworm can be usefully used as a material for household goods such as cosmetics and toothpastes as well as animal feed additives. In addition, the silkworm farmers can contribute to the improvement of income by breeding transgenic silkworms which produce high value-added natural antibiotics differentiated from common silkworms through the present invention.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Transgenic silkworms producing antimicrobial peptide <130> p2013-0093 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> sense primer included ATG and Asc I restriction enzyme <400> 1 ggcgcgccgc gcgttaccat atatggtg 28 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> antisense primer included Nhe I restriction enzyme <400> 2 gctagccttg aattagtctg caagaaa 27 <210> 3 <211> 661 <212> DNA <213> BmRelish1t gene <400> 3 caagaagaga aggatgaatg cctcatgttc tacttcccct gcttcgtctg ggtcgttgaa 60 gagtatcagc gatctgcccg cccttgtgac tctcaataac aatttcaacg atgaaaacaa 120 cattgaaatg ccgaaaactg agctgccggc cgccgcggcc gccgccccct ctctgggact 180 gtgcgacctc gccgacgcgc tcatgggctc cgagagcgcc gtggacctac actcggtcgc 240 cgggaacgcg atgtggagcc cgagtgcgcc cgtcgagacg atgagcgtcg agcagttccc 300 gaatctgcaa ctgaattcga cggagttcga aaagataata acgaacgccc ttccgccgga 360 ggaaaggaac gacttcacgg aatatgccct aagttcgtac gaatcgttcg atgacatcga 420 cgatgatcca aatggatgga agttcatacg tacgttacag atgatgcaaa ccgactctgc 480 acgcagtagg cagcagacac cgcagactag taaacccgca ctgaaaactg ttgaacccca 540 agagagctca gacaggaccg agctgaatgt accgtctgat gcgaaagatg caaacgaata 600 tagcgcttat tacaacgccc aagacggcct cgaagtgaaa cagctactaa gagaactgta 660 a 661 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer included ATG and Nhe I restriction enzyme <400> 4 aaaatgtcta caactgccag tg 22 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> antisense primer included Afl II restriction enzyme <400> 5 ttataataca taagtcaatg gat 23 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> gene specific primer <400> 6 gcgcgttacc atatatggtg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> gene specific primer <400> 7 cttgaattag tctgcaagaa a 21

Claims (12)

A marker gene-regulated promoter, a marker gene, an actin-3 (BmA3) promoter derived from a silkworm, and a gene construct operably linked to a silkworm-derived BmRelish1t gene,
The recombinant expression vector of Fig. 1, wherein the silkworm-derived BmRelish1t gene comprises the nucleotide sequence of SEQ ID NO: 3.
delete delete The method according to claim 1,
Wherein the marker gene-regulated promoter is a 3xP3 promoter that is expressed in the eye and nervous system.
Recombinant expression vector.
The method according to claim 1,
Wherein the marker gene is a green fluorescent protein (EGFP) gene.
Recombinant expression vector.
delete The method according to claim 1,
Wherein the expression vector is a piggyBac vector.
Recombinant expression vector.
A recombinant expression vector according to any one of claims 1, 4, 5, or 7,
Transgenic silkworms.
1) preparing a recombinant expression vector according to any one of claims 1, 4, 5, or 7;
2) injecting the recombinant expression vector of step 1) into silkworm eggs to produce transformed silkworm eggs; And
3) hatching the transformed silkworm in step 2) to produce a transgenic silkworm in which the antimicrobial peptide is expressed.
A method for producing a transformed silkworm in which an antimicrobial peptide is expressed.
10. The method of claim 9,
Characterized in that the transformation of step (2) uses microinjection.
A method for producing a transformed silkworm in which an antimicrobial peptide is expressed.
10. The method of claim 9,
The method for producing a silkworm silkworm according to any one of claims 1 to 3, wherein the step (3) further comprises the step of selecting a transformed silkworm by one of the following methods:
i) a method of inserting a marker gene into an expression vector and then confirming the expression of the marker gene in the transformed silkworm; or
ii) A method of confirming the expression of an antimicrobial peptide in a transgenic silkworm.
1) preparing a recombinant expression vector according to any one of claims 1, 4, 5, or 7;
2) transforming the recombinant expression vector of step 1) into silkworm or silkworm to produce a transformed silkworm; And
3) culturing the transformed silkworm of step 2) to obtain a transformed silkworm containing an antimicrobial peptide.
Method for mass production of transgenic silkworms containing antimicrobial peptides.

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