KR101423256B1 - Expression of Honeysuckle yellow vein virus(HYVV)-C4 gene in Escherichia coli and production of polyclonal antibody against the recombinant TYLCV-C4 protein - Google Patents

Abstract

The present invention primarily relates to a process for producing HYVV-C4 recombinant protein by synthesizing C4 gene of Honeysuckle yellow vein virus (HYVV) and inducing it to overexpress in Escherichia coli . Secondly, it is separated and purified, then inoculated into an experimental animal with an immunogen, and then provided for the purpose of producing a polyclonal antibody. Since the HYVV-C4 protein is known to play a variety of roles in viral-host interactions, production and production of antibodies to the C4 protein can be widely used in biological, molecular, and immunological studies. Therefore, production of polyclonal antibodies to C4 purified recombinant proteins through this process can be useful for virus-host interaction studies and virus diagnostic kits.

Description

(Expression of Honeysuckle yellow vein virus (HYVV) -C4 gene in Escherichia coli and production of polyclonal antibody against the recombinant TYLCV-C4 protein}

The present invention relates to a method for inducing the expression of C4 gene of HYVV (Honeysuckle yellow vein virus) in Escherichia coli, producing recombinant C4 protein, isolating and purifying it, and then immunizing it with an immunogen And to provide a process for producing a polyclonal antibody after inoculation into an experimental animal.

In recent years, the onset of viruses classified as Geminiviridae family and Begomovirus genus in tomato plant houses in various regions in Korea has been rapidly spreading, resulting in serious economic loss to domestic tomato farms have. Among them, HYVV and TYLCV (Tomato yellow leaf curl virus) are beemo viruses with a monopartite genome consisting of single-stranded DNA, and tobacco is widely distributed throughout the world as mediated by Rui (Bemisia tabaci) . In Korea, HYVV and TYLCV have been spreading nationwide since 2008, causing damage to tomato plant farms and various genetic isolates have been reported in a short time [Wang et al., 2011, Eur. J. Plant Pathol. 129: 361-370; Kim et al., 2011, Virus Genes 42: 117-127].

The two viruses listed above have a narrow host range and are quite redundant, especially the symptoms that appear in tomatoes are very similar. To date, no two viruses have been reported in the same package or in the same plant at the same time.

HYVV was first reported in Japan from honeydew (Honeysuckle, scientific name: Lonicera japonica) [Osaki et al., 1979, Ann. Phytopathol. Soc. Jpn. 45: 62-69). After that, the nucleotide sequences of HYVV- [UK1] (NC005807) and HYVV- [UK2] (AJ543429) were reported in the UK and in Japan, the nucleotide sequences of HYVV- [Masuda] (AB236325) and HYVV- [Fukui] (AB236321) The sequence has been reported [Ueda et al., 2008, Archives of Virology 153, 417-426]. In Korea, HYVV- [Jeju] (FJ434943, 2763 nucleotides) and HYVV- [DJ] (HQ189431, 2764 nucleotides) were reported from tomato [Wang et al., 2011] and β satellite (GQ495268 HYVV- [Kr] (GQ477135, 2763 nucleotides) in which 1227 nucleotides are present [Lee et al., 2011, Arch. Virol. 156: 785-791. HYVV- [UK2] and HYVV- [DJ] have 89.4 and 92.8% homology with Japanese HYVV- [Masuda] respectively, and HYVV- [Kr] And the like.

Begomoviruses infect host plants and express the C4 gene encoded in the genome to synthesize C4 protein. The C4 protein of the bee monkey virus with the monopartite genome is known to bind not only viral DNA but also a variety of single strand and double strand DNA and RNA in a non-specific manner [Teng et al., 2010, PLoS ONE 5 (6): e11280; Amin et al., 2011, Mol. plant Microbe Interact. 24: 973-983).

In addition, Tomato leaf curl virus (ToLCV) -C4 protein, another beemo virus, is known to act as an inhibitor of PTGS and is also known to interact with host shaggy-like kinase (SISK) [Dogra et al , 2009, Plant Mol. Biol. 71: 2538]. On the other hand, the segmented genome chopping parent virus, African cassava mosaic virus (ACMV) and Sri Lankan cassava The AC4 protein of the mosaic virus (SLCMV) is also known to act as a PTGS inhibitor by binding to miRNAs and siRNAs as well as C4 of the monoclonal genomic bee viral virus [Vanitharani et al., 2004, J. Gen. Virol. 89: 2063-2074; Chellappan et al., 2005, Proc. Natl. Acad. Sci. USA 102: 1038110386].

As described above, the C4 protein of beemo virus is known to play various roles in virus-host interaction. Therefore, biological, molecular, and immunological studies of C4 proteins are needed.

In order to solve the above-mentioned necessity, it is absolutely necessary to produce a C4 recombinant protein of HYVV, a monosaccharide bee viral virus, and a method of producing an antibody thereto. Hereinafter, the solution of the present invention will be described in more detail.

In order to achieve the above object, in the present invention, the HYVV-C4 gene was amplified by PCR, cloned into a pET28a vector, and then transformed into Escherichia coli. E. coli colony transformants were selected and cultured. Optimum conditions were determined by adding various concentrations of IPTG to express the recombinant proteins. The recombinant proteins were separated and purified to prepare antibodies against HYVV-C4. For this, HYVV-C4-6X His fusion protein was eluted by Ni-NTA resin chromatography under denaturing conditions. The HYVV-C4 recombinant protein obtained by the above method was injected into an animal rat three times to collect an antiserum, and purified and purified immunoglobulin G (IgG) .

This task can be used to develop a virus diagnostic kit and to study virus-host interaction by providing the production process of antibody that is commonly used to search for viruses in plants infected with HYVV and to study interaction. Hereinafter, specific methods of the present invention will be described in detail with reference to examples. However, the scope of the present invention is not limited to the following examples.

Fig. 1 (left) is a map of the pET-28a vector used to produce the target protein by inserting the target gene DNA (left). In Fig. 1 (right), agarose gel stained with ethidium bromide was subjected to electrophoresis to confirm the size of the pET-28a vector.
FIG. 2 shows the results of amplification of HYVV-C4 gene by PCR, electrophoresis in order to confirm its size, agarose gel stained with ethidium bromide,
Fig. 3 shows the amplified target gene C4 (lane 2) cleaved with the same restriction enzyme as pET-28a vector DNA (lane1) digested with restriction enzymes EcoRI and SalI.
Fig. 4 shows the results of the transformation of the recombinant vector pET-HYVV-C4
FIG. 5 shows that the target gene is present in the recombinant vector pET-HYVV-C4 through restriction enzyme cleavage
Figure 6 confirms whether the target gene is present in the recombinant vector pET-HYVV-C4 through PCR synthesis
FIG. 7 shows the results of treatment of HYVV-C4 protein with various concentrations of IPTG, followed by incubation at 37 DEG C for 4 hours (lanes 1-5: soluble fraction of E. coli lysate, lanes 6-7: E. coli lysate Of the insoluble fraction)
FIG. 8 shows the separation and purification of HYVV-C4 recombinant protein by Ni-NTA resin chromatography under denaturing conditions
Figure 9 shows the cross-reactivity of HYVV-C4 and TYLCV-C4 recombinant proteins at various concentrations after Western blot analysis after producing rat antiserum against HYVV-C4 recombinant protein. M: protein ladder, lanes 1-4 HYVV-C4 recombinant protein (10, 5, 1, 0.5 g), lanes 5-8 TYLCV-C4 recombinant protein (10,
Fig. 10 shows SDS-PAGE of IgG separated and purified from antiserum against HYVV-C4 recombinant protein. Lanes 1: Normal rat serum, lanes 2-5: rat HYVV-C4 antiserum
FIG. 11 shows cross-reactivity observed by SDS-PAGE / Western blotting of various concentrations of HYVV-C4 recombinant protein using IgG separated and purified from mouse antiserum as a primary antibody. M: protein ladder, lanes 1-4: HYVV-C4 recombinant protein (10, 5, 1, 0.5 g)

{Example}

1. Virus and virus genomic DNA

The virus used in the present invention is HYVV- [DJ] (HQ189431) isolate isolated from Dangjin, Chungnam Province. PGEM T-Easy containing the entire DNA of the genome [Wang et al., 2011, Eur. J. Plant Pathol. 129: 361-370] was used as a template for the PCR reaction described below.

2. PCR Synthesis

 (1) HYVV-C4 protein gene-specific primer

For PCR amplification, a specific base sequence of the following HYVV-C4 protein gene was used as a primer.

  - forward primer: 5'-GAATTCATGGGAGCCCTCATCTCCAC-3 '

  - reverse primer: 5'-GTCGACATATAGACAACGCCGATGACTTTGATC-3 '

 (2) PCR reaction

PCR was performed using the above-described HYVV- [DJ] genomic DNA as a template. 50 μl of the PCR reaction solution contained 5 μl of 10 × buffer (Dakara Bio, Inc., Japan), 10 μl of DNA (final concentration: 10 ng), 4 μl each of 2.5 mM dNTPs, 1 μl of forward and reverse primers -9㎕ 25mM MgCl ₂ (final concentration: 1.5-4.5mM), 0.5㎕ (1U) ExTaq It was composed of polymerase. The PCR reaction was carried out by denaturation of the DNA at 95 ° C for 2 minutes, followed by 35 cycles of continuous reaction (denaturation at 94 ° C for 1 minute, annealing at 56 ° C for 2 minutes, amplification at 72 ° C for 3 minutes) Lt; RTI ID = 0.0 > 72 C < / RTI >

As a result of the experiment described above, MgCl ₂ Regardless of the concentration, the target band, the C4 gene, was synthesized and the size of the gene DNA band was 294 bp (Fig. 1).

3. HYVV-C4 gene pET28a vector cloning

 (1) pET28a vector

The HYVV-C4 gene DNA amplified by the PCR reaction described in 2- (2) above was purchased from Novagen (Germany) for cloning into the pET28a vector. The pET28a vector diagram and vector size are shown in FIG.

 (2) Cloning

For the target gene cloning, the pET28a vector (Fig. 3, lane 1) described in 3- (1) above and the HYVV-C4 DNA (Fig. 3, lane 2) synthesized in 2- (2) were ligated with the same two restriction enzymes EcoRI and SalI , 1.5 μl of C4 DNA, 2 μl of pET28a vector, 1 μl of 10 × buffer, 5 μl of distilled water and 0.5 μl of T4 DNA ligase (Fermentas, Canada) were mixed and ligation was performed at 16 ° C. Escherichia coli was plated on Luria-Bertani (LB) solid medium containing kanamycin (30 μg / ml), and 5 μl of the mixture was used for transformation of E. coli (XL1-Blue) The transformed colonies were selected and cultured at 37 ° C inoculated into LB (+ kanamycin) liquid medium. The supernatant was removed by low-speed centrifugation, and the precipitate was harvested. The plasmid was separated according to the manufacturer's instructions using a High Speed Plasmid Kit (Geneaid, Taiwan) and named pET-HYVV-C4 (FIG.

To examine whether the target gene C4 was properly inserted into the recombinant plasmid (pET-HYVV-C4) obtained as described above, pET-HYVV-C4 was treated with restriction enzymes EcoRI and SalI, (Fig. 5). PCR was performed using pET-HYVV-C4 DNA as a template and the primer indicated in 2- (1) above, and C4 DNA was synthesized (Fig. 6). In addition, the nucleotide sequence of pET-HYVV-C4 was determined, and it was confirmed that the target gene was properly inserted into the vector and there was no problem in the codon frame.

4. Recombinant protein expression

 (1) induction and analysis of IPTG expression

The HYVV-C4 coli genes containing a pET-HYVV-C4 to induce the expression in E. coli on LB (+ kanamycin) broth OD _600nm And then IPTG (Isopropyl-β-D-thio-galactoside) was added at a concentration of 0.6. For the expression of proteins regulated by Lac operon, IPTG was added to 0.1-5 mM (final concentration) medium instead of lactose and incubated at 37 ° C for 4 hours before optimal conditions were determined. Samples were subjected to low-speed centrifugation and the precipitated E. coli was harvested and dissolved in a lysis buffer, followed by sonication three times (30 sec / one time and one minute). The degraded samples were harvested as supernatant and sediment fractions after low-speed centrifugation. The insoluble fraction was dissolved in 1X PBS (phosphate-buffered saline, pH 7.4), and analyzed by SDS-PAGE. The total amount of protein in the supernatant was determined as a soluble fraction and the precipitate as an insoluble fraction. Respectively.

- lysis buffer: 10mM imidazole, 300mM NaCl, 50mM NaH 2 PO 4, pH8.0

 (2) Purification of HYVV-C4-6X His fusion protein

Purification of HYVV-C4-6X His fusion protein was eluted using Ni-NTA resin (Qiagen, USA) chromatography under denaturing conditions (8M Urea).

  The elution buffer was 6 M guanidine hydrochloride + 0.2 M actic acid

 (3) SDS-PAGE analysis: Molecular weight and overexpression

After each step of the separation and purification process as well as the fraction thus extracted was completed, a sample was secured and the denaturation sample was heated for 3-5 minutes by adding a denaturing buffer [Laemmli, 1970, Nature 227: 680-685] Was loaded on the prepared gel and subjected to 15% SDS-PAGE electrophoresis. After the electrophoresis was completed, the gel was stained with Coomassie blue solution (methanol: glacial acetic acid: water, 5: 1: 5, 0.05% Coommasie brilliant blue R-250 dissolved in v / v / v mixture) Respectively.

As a result of SDS-PAGE analysis as described above, the HYVV-C4 recombinant protein was overexpressed regardless of the IPTG concentration added in the insoluble fraction, and the molecular weight was observed to be about 16.3 kDa (FIG. 7). Thus, HYVV-C4 recombinant protein (FIG. 8) isolated and purified through multi-step washing was prepared as an antigen for injection into experimental animals after quantification.

5. Production of antiserum against recombinant protein and polyclonal antibody purification

 (1) Production of antiserum

  - Antigen: isolated purified HYVV-C4 recombinant protein

  - Animals: Sprague-Dawley rats (6 weeks old) male

  - 3 injections (every 2 weeks) and blood collection (1 week after injection)

    Primary injection: Recombinant protein (100 μg concentration) + complete adjuvant → Blood collection

    Secondary injection: Recombinant protein (concentration 50 μg) + complete adjuvant → Blood collection

    Third injection: Recombinant protein (concentration 50 μg) → blood sampling

 (2) Antigenic screening of recombinant HYVV-C4 protein for antibodies: Western blot

The HYVV-C4 recombinant protein was diluted with 1 × PBS buffer (10 μg, 5 μg, 1 μg, 0.5 μg), and the denaturation buffer was added. After heating for 3-5 minutes, the denatured sample was loaded on the prepared gel and analyzed by SDS-PAGE Electrophoresis was performed. After the electrophoresis was completed, the gel was transferred to nitrocellulose membrane (NCM) by electro-transfer method for immunological analysis without staining. In order to inhibit nonspecific reactions, NCM was immersed in a blocking solution of 5% nonfat milk dissolved in 1 × PBS, treated for 1 hour on a stirrer, and then washed with 1 × PBS buffer containing 0.03% Tween-20 And washed. Primary antibody was prepared by diluting antiserum or IgG against HYVV-C4 recombinant protein in 1X PBS (1: 1,000, V / V). The secondary antibody was a goat anti-rat alkaline phosphatase conjugate (Sigma Co.). , USA) was diluted (1: 30,000, v / v) before use. The primary and secondary antibodies were reacted for 1 hour each and washed three times after each step. Finally, to observe the antigen-antibody reaction visually, NCM was treated with color development buffer containing nitroblue tetrazoilium (NBT) / 5-bromo-4-chloro-3-indolylphosphate (BCIP).

- color development buffer: 100 mM Tris, 100mM NaCl, 5mM MgCl 2, pH 9.5

As a result of searching for the recombinant protein by the above-mentioned method, it reacted more strongly with the homologous HYVV-C4 recombinant protein as compared with heterogeneous TYLCV-C4 recombinant protein (control). The limits of the HYVV-C4 antiserum search were up to 1 μg of HYVV-C4 recombinant protein antigen (Figure 9)

 (3) Purification of polyclonal antibody IgG

In order to isolate and purify TYLCV-C4-IgG from the antiserum, Montage Antibody Purification Kit and Prosep-G media were purchased from Millipore and tried as follows according to the manufacturer's instructions. First, the antiserum was equilibrated with 10 ml of binding buffer A using PROSEP-G media, and impurities in the antiserum were removed using a 0.22 μm Sterflip-GP apparatus. The filtered antiserum was diluted (1: 1, v / v) with attachment buffer A, loaded on a spin column, and centrifuged at 150 g for 20 min. In order to remove the detached impurities from the spin column, 10 ml of binding buffer A was added, followed by centrifugation at 200 g for 2 minutes. The attached IgG was eluted with 10 ml of elution buffer B2, And transferred to a new spin column containing 1.3 ml of neutralization buffer C to keep it. After centrifugation at 500 g for 5 min, the supernatant containing IgG was collected and desalted and concentrated using an Amicon Ultra-15 centrifugal filter device.

  - Adhesion buffer A: 1.5 M glycine-NaOH, 3 M NaCl, 0.1% sodium azide, pH 9.0

  Elution buffer B2: 0.2 M glycine-HCl, 0.1% sodium azide, pH 2.5

- Neutralizing buffer C: 1 M Tris-HCl, 0.1% sodium azide, pH 9.0

IgG was isolated from the antiserum by the above-described method (FIG. 10). As a result, homologous HYVV-C4 recombinant protein was detected and IgG was more strongly and vigorously reacted with the antigen than antiserum. The limit of the TYLCV-C4 antiserum search was possible up to 0.5 μg of antigen (FIG. 11).

The production of polyclonal antibodies to the HYVV-C4 recombinant proteins devised from the above results can be applied to virus-host interaction studies and to identify viruses from HYVV-infected plants.

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<110> Paichai University Industry-Academic Cooperation Foundation <120> Expression of Honeysuckle yellow vein virus (HYVV) -C4 gene in          Escherichia coli and production of polyclonal antibody against          the recombinant TYLCV-C4 protein <130> 13P130861 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 294 <212> DNA <213> Honeysuckle yellow vein virus (HYVV) <400> 1 tcaatataga caacgccgat gactttgatc ctgagttgat tgcctcggca tatgcgtcgt 60 tggcagactg gcaacctcct ctagctgatc ttccatcgac ttggaaaact ccagaatcaa 120 tgacgtctcc gtctttttcc atgtaggtct taacgtccga cgagctttta gctccctgaa 180 tgttcggatg gaaatgtgct gacctggatg gggatacgag atcgaagaat cgttgatttg 240 tgcatctgta ttttccctcg aattggatga ggatgtggag atgagggctc ccat 294 <210> 2 <211> 97 <212> PRT <213> Honeysuclke yellow vein virus (HYVV) <400> 2 Met Gly Ala Leu Ile Ser Thr Ser Ser Ser Ser Ser Glu Asn Thr   1 5 10 15 Asp Ala Gln Ile Asn Asp Ser Ser Ile Ser Tyr Pro His His Gly Gln              20 25 30 His Ile Ser Ile Arg Thr Phe Arg Glu Leu Lys Ala Arg Arg Thr Leu          35 40 45 Arg Pro Thr Trp Lys Lys Thr Glu Thr Ser Leu Ile Leu Glu Phe Ser      50 55 60 Lys Ser Met Glu Asp Gln Leu Glu Glu Val Ala Ser Leu Pro Thr Thr  65 70 75 80 His Met Pro Arg Gln Ser Thr Gln Asp Gln Ser His Arg Arg Cys Leu                  85 90 95 Tyr    

Claims (5)

PCR was performed using 5'-GAATTCATGGGAGCCCTCATCTCCAC-3 'as a forward primer and 5'-GTCGACATATAGACAACGCCGATGACTTTGATC-3' as a reverse primer, using the pGEM T-Easy vector containing the entire genome of HYVV- [DJ] Having the nucleotide sequence of SEQ ID NO: 1 Amplifying HYVV-C4 DNA;
Preparing a plasmid pET-HYVV-C4 by treating the amplified HYVV-C4 DNA with a restriction enzyme of EcoRI and SalI and then introducing it into a pET28a vector;
Transforming E. coli with pET-HYVV-C4;
Culturing the transformed E. coli at 35-38 DEG C for 3-5 hours in a medium containing 0.1-5 mM IPTG;
Having the amino acid sequence of SEQ ID NO: 2 Production method of HYVV-C4 protein. Producing a HYVV-C4 protein according to claim 1;
Purifying the produced HYVV-C4 protein using chromatography;
Injecting the purified HYVV-C4 protein into a mouse to produce antiserum;
Separating and purifying the polyclonal antibody in the produced antiserum;
Lt; RTI ID = 0.0 &gt; HYVV-C4. &Lt; / RTI &gt; delete delete delete

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