KR101423260B1 - Expression of Tomato yellow leaf curl virus(TYLCV)-C4 gene in Escherichia coli and production of polyclonal antibody against the recombinant TYLCV-C4 protein - Google Patents

Abstract

The present invention firstly synthesizes the C4 gene of TYLCV ( Tomato yellow leaf curl virus ) and induces overexpression in Escherichia coli to produce TYLCV-C4 recombinant protein To the process. 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 TYLCV-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 Tomato yellow leaf curl virus (TYLCV) -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 TYLCV (Tomato yellow leaf curl virus) in Escherichia coli, producing recombinant C4 protein, isolating and purifying it, ) In order 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. Tomato yellow leaf curl virus (TYLCV) is a bee predominant virus with a monopartite genome consisting of single-stranded DNA. It is mediated by Bismuthia tabaci and is widely distributed worldwide . TYLCV has been mutated in a short period of time and many variants have been reported internationally. In Korea, the virus has been spreading nationwide since 2008, causing damage to tomato plant farms and various genetic isolates have been frequently reported [Kim et al., 2011, Virus Genes 42: 117-127].

Gemini viruses infect host plants and express the C4 gene encoded in the genome to synthesize the 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).

For TYLCV V2 protein it is silent after the transfer; this role is known as an inhibitor of the (post transcriptional gene silencing PTGS) but [. Zrachya et al, 2007, Virology 358: 159-165], chopping the other species of the parent virus Tomato leaf curl virus (ToLCV) -C4 protein 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 AC4 protein of this segmental genomic bee virus, African cassava mosaic virus (ACMV) and Sri Lankan cassava mosaic virus (SLCMV), also acts as a PTGS inhibitor by binding to miRNA and siRNA, ≪ / RTI > [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 TYLCV, 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.

To accomplish the above object, in the present invention, the TYLCV-C4 gene was amplified by PCR, cloned into a pET28a vector, and then transformed into E. 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 TYLCV-C4. For this, TYLCV-C4-6X His fusion protein was eluted by Ni-NTA resin chromatography under denaturing conditions.

The TYLCV-C4 recombinant protein obtained by the above method was injected into an experimental animal rat three times to collect an antiserum, and purified immunoglobulin G (IgG) .

In this project, viruses are detected in plants infected with TYLCV and provided with a production process of antibodies commonly used for interaction studies, which can be useful for the development of virus diagnostic kits and virus-host interaction studies. 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 the TYLCV-C4 gene by PCR, electrophoresis in order to confirm its size, agarose gel staining with ethidium bromide,
3 is a pET-28a vector DNA digested with restriction enzymes BamHI and HindIII.
Figure 4 shows the amplified target gene C4 (lane 2) digested with restriction enzymes BamHI and HindIII,
FIG. 5 is a graph showing the relationship between the recombinant vector pET-TYLCV-C4
Figure 6 shows that the target gene is present in the recombinant vector pET-TYLCV-C4 through restriction enzyme cleavage
Figure 7 confirms whether the target gene is present in the recombinant vector pET-TYLCV-C4 through PCR synthesis
FIG. 8 shows that TYLCV-C4 protein expression is induced by treating IPTG with various concentrations of IPTG at 37 ° C for 4 hours (lanes 1-5: water soluble fraction of E. coli lysate, lanes 6-7: E. coli lysate Of the insoluble fraction)
FIG. 9 shows the purification of TYLCV-C4 recombinant protein by Ni-NTA resin chromatography under denaturing conditions
FIG. 10 shows the cross-reactivity of various concentrations of HYVV-C4 and TYLCV-C4 recombinant proteins produced by Western blot analysis after producing rat antiserum against TYLCV-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,
Figure 11 shows SDS-PAGE of IgG separated and purified from antiserum against TYLCV-C4 recombinant protein. Lanes 1-6: 10, 5, 1, 0.5, 0.25, 0.1 g rat TYLCV-C4 antiserum
FIG. 12 shows cross-reactivity observed by SDS-PAGE / Western blotting of IgG separated and purified from mouse antiserum against TYLCV-C4 recombinant protein at various concentrations as a primary antibody. M: protein ladder, lanes 1-4: TYLCV-C4 recombinant protein (10, 5, 1, 0.5 g)

{Example}

1. Virus and virus genomic DNA

The virus used in the present invention is a TYLCV- [Bus] (GQ141873) isolate isolated from Busan. PGEM T-Easy [Kim et al., 2011, Virus Genes 42: 117-127] containing the entire DNA of the genome was used as the template for the PCR reaction described below.

2. PCR Synthesis

 (1) TYLCV-C4 protein gene-specific primer

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

  - forward primer: 5'-GGATTCATGGGGAACCACATCTCCATGTG-3 '

- reverse primer: 5'-AAGCTTATATATTGAGGGCCTCGGATTTACTG-3 '

 (2) PCR reaction

PCR was performed using the above-described TYLCV-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) Ex Taq 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. TYLCV-C4 gene pET28a vector cloning

 (1) pET28a vector

The TYLCV-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) described in 3- (1) above and the TYLCV-C4 DNA (Fig. 4, lane 2) synthesized in 2- (2) were cut with the same two restriction enzymes BamHI and HindIII After that, 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-TYLCV-C4 (FIG.

To examine whether the target gene C4 was properly inserted into the recombinant plasmid (pET-TYLCV-C4) obtained as described above, pET-TYLCV-C4 was treated with restriction enzymes BamHI and HindIII, (Fig. 6). PCR was carried out using pET-TYLCV-C4 DNA as a template and the primer indicated in 2- (1) above. As a result, C4 DNA was synthesized (Fig. 7). In addition, the nucleotide sequence of pET-TYLCV-C4 was determined, and it was confirmed that the target gene was properly inserted into the vector and there was no problem with the codon frame.

4. Recombinant protein expression

 (1) induction and analysis of IPTG expression

To induce expression of TYLCV-C4 gene in E. coli, Escherichia coli containing pET-TYLCV-C4 was added to LB (+ kanamycin) liquid medium at 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 TYLCV-C4-6X His fusion protein

Purification of TYLCV-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 TYLCV-C4 recombinant protein was overexpressed regardless of the IPTG concentration added in the insoluble fraction, and the molecular weight was observed to be about 16.0 kDa (FIG. 8). Therefore, the TYLCV-C4 recombinant protein (FIG. 9) 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 TYLCV-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) Antigen detection of recombinant TYLCV-C4 protein: Western blot

The TYLCV-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 TYLCV-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-described method, the TYLCV-C4 protein was more strongly reacted with the heterologous HYVV-C4 recombinant protein (control) than the homologous TYLCV-C4 protein. The limit of TYLCV-C4 antiserum search was possible up to 5 μg of antigen (Figure 10)

 (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-mentioned method (Fig. 11). As a result, IgG was more strongly and clearly reacted with the antigen than the antiserum by the homologous TYLCV-C4 recombinant protein. The limit of TYLCV-C4 antiserum search was possible up to 0.5 μg of antigen (FIG. 12).

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

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<110> Paichai University Industry-Academic Cooperation Foundation <120> Expression of Tomato yellow leaf curl virus (TYLCV) -C4 gene in          Escherichia coli and production of polyclonal antibody against          the recombinant TYLCV-C4 protein <130> 13P130860 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 294 <212> DNA <213> Tomato yello leaf curl virus (TYLCV) <400> 1 ttaatatatt gagggcctcg gatttactgc ctgaattgag tgcttcggca tatgcgtcgt 60 tggcagattg ctgacctcct ctagctgatc tgccatcgat ttggaaaact ccaaaatcaa 120 tgaagtctcc gtctttctcc acgtaggtct tgacatctgt tgagctctta gctgcctgaa 180 tgttcggatg gaaatgtgct gacctgtttg gggataccag gtcgaagaac cgttggttct 240 tacattggta tttgccttcg aattggataa gcacatggag atgtggttcc ccat 294 <210> 2 <211> 97 <212> PRT <213> Tomato yello leaf curl virus (TYLCV) <400> 2 Met Gly Asn His Ile Ser Met Cys Leu Ser Asn Ser Lys Ala Asn Thr   1 5 10 15 Asn Val Arg Thr Asn Gly Ser Ser Thr Trp Tyr Pro Gln Thr Gly Gln              20 25 30 His Ile Ser Ile Arg Thr Phe Arg Gln Leu Arg Ala Gln Gln Met Ser          35 40 45 Arg Pro Thr Trp Arg Lys Thr Glu Thr Ser Leu Ile Leu Glu Phe Ser      50 55 60 Lys Ser Met Ala Asp Gln Leu Glu Glu Val Ser Asn Leu Pro Thr Thr  65 70 75 80 His Met Pro Lys His Ser Ile Gln Ala Val Asn Pro Arg Pro Ser Ile                  85 90 95 Tyr    

Claims (5)

AGCTTATATATTGAGGGCCTCGGATTTACTG-3 'as a forward primer and 5'-GGATTCATGGGGAACCACATCTCCATGTG-3' as a reverse primer using the pGEM T-Easy vector containing the whole genome of TYLCV- [bus] (GQ141873) Amplifying the TYLCV-C4 gene DNA having the nucleotide sequence of SEQ ID NO: 1 by performing a reaction;
Treating the amplified TYLCV-C4 DNA with restriction enzymes BamH1 and HindIII, and introducing the amplified TYLCV-C4 DNA into a pET28a vector to prepare a plasmid pET-TYLCV-C4;
Transforming E. coli with pET-TYLCV-C4;
Culturing the transformed E. coli in a medium containing 0.1-5 mM IPTG at 35-38 DEG C for 3-5 hours;
Wherein the TYLCV-C4 protein has the amino acid sequence of SEQ ID NO: 2. Producing a TYLCV-C4 protein according to claim 1;
Purifying the produced TYLCV-C4 protein using chromatography;
Injecting the purified TYLCV-C4 protein into a mouse to produce antiserum;
Separating and purifying the polyclonal antibody in the produced antiserum;
&Lt; RTI ID = 0.0 &gt; TYLCV-C4. &Lt; / RTI &gt; delete delete delete

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