KR100267745B1 - Detection method of neutralizing antibody against porcine epidermic diarrhea virus - Google Patents

Abstract

PURPOSE: Provided is a spike gene and a method for detecting neutralizing antibodies of porcine epidemic diarrhea virus to prevent porcine epidemic diarrhea by using spike proteins which are mass-produced through genetic engineering. CONSTITUTION: The spike gene obtained from porcine epidemic diarrhea virus is located in the gene of recombinant baculovirus (KFCC 11014). And a method for detecting neutralizing antibodies of porcine epidemic diarrhea virus (PEDV) is characterized by the next step of: using a spike protein expressed in recombinant baculovirus (KFCC 11014) as an antigen in implementing indirect-ELISA. A method for manufacturing the recombinant baculovirus (KFCC 11014) is comprised of the following steps of: i) making expression vector pVL 1393 having a spike gene of PEDV and obtaining a medium lacking serum but including linear baculovirus, and mixture containing lipopectin; and ii) cotransfecting host cell Sf9 using the mixture.

Description

Detection of specific neutralizing antibodies of swine pandemic diarrheal virus

The present invention is to clone the spike gene of porcine epidemic diarrhea vrius (hereinafter referred to as PEDV) by using a recombinant recombinant protein expressed in the insect virus baculovirus (Korean spawn association strain accession number KFCC-11014) This is a rapid test for neutralizing antibodies against PEDV.

PEDV belongs to Coronauiridae and causes acute enterocolitis in pigs of all ages, especially in newborn piglets, with high mortality and high economic losses. The incidence of this disease has been reported mainly in Europe and Asia. Recently, PEDV has been widely reported in outdoor farms such as the transmissible gastroerteritis virus (TGEV), and the damage is more serious.

The antigenic association of PEDV with other coronaviruses was examined by neutralization, fluorescence antibody and immunoelectron microscopy, with slightly different cross-reactivity including nucleocap sid and membrane. Although reported by coronaviruses and Western blotting, cross-reactivity to spikes is unknown. Spikes are known to cause host cell receptor glycoprotein binding, cell fusion, cell mediated immunity and neutralizing antibodies. PEDV has been known to be difficult to cultivate in pig-derived cells in which TGEV is cultured, but can specifically grow in monkey kidney cells. In addition, the molecular biological characteristics of PEDV are not well known, but recent sequences of spikes and nucleocapsids, membranes, small membranes and ORF3 have been analyzed.

In Korea, the virus was isolated from swine diarrhea and carried out serological investigation, infection status and vaccine development. However, no diagnostic technology has been established. There is an urgent need for antibody diagnostics so that it can be used effectively.

Since the present invention is known to induce neutralizing antibodies of swine pandemic diarrheal virus and spike protein, porcine diarrhea by testing the swine pandemic diarrheal virus neutralizing antibody quickly and accurately using spiked protein (KFCC-11014) mass-produced by genetic recombination method The present invention has been completed so that it can be used as an antibody test that can be used for preventing and eradicating prematurely.

In other words, the present invention provides a method for detecting swine pandemic diarrheal virus neutralizing antibody by indirect sandwitch enzyme linked immunosorbent assay (IS-ELISA) using a swine epidemic diarrhea virus spike protein produced by genetic recombination. do.

Figure 1 is a photograph of the electrophoresis of the swine epidemic diarrhea virus spike gene amplified by the polymerase chain reaction method.

2 is a nucleotide sequence diagram of the swine epidemic diarrhea virus spike gene extracted from domestic diarrhea.

3 is a schematic diagram of a baculovirus expression vector cloned with the swine epidemic diarrhea virus spike gene.

Figure 4 is a photograph of the swine epidemic diarrhea virus spike protein expressed by the recombinant recombinant baculovirus using an anti-spike monoclonal antibody immunodetection method.

FIG. 5 is a photograph of porcine epidemic diarrhea virus spike protein expressed by a recombinant baculovirus using an anti-spike monoclonal antibody.

FIG. 6 is a photograph of porcine epidemic diarrhea virus spike protein expressed by genetically modified baculovirus using cell-immunochemical staining using anti-spiked monoclonal antibody.

Figure 7 is a photograph confirming the expression of the recombinant spike protein using Western blotting (Western blotting).

8 is a diagram showing the expression level of recombinant protein by culture days.

Figure 9 is a schematic diagram of the neutralizing antibody detection method of swine epidemic diarrhea using indirect sandwitch enzyme linked immunos or bent assay (IS-ELISA).

Figure 10 is a figure that determines the concentration of monoclonal antibody for the detection of neutralizing antibodies.

Figure 11 is a figure that determines the optimal antigen concentration for the detection of neutralizing antibodies.

Figure 12 is a figure that determines the optimal serum dilution concentration for antibody detection.

FIG. 13 shows the antibody detection method of swine pandemic diarrheal virus using indirect sandwitch enzyme linked immunosorbent assay (IS-ELISA) and the virus neutralization test using conventional tissue culture. This is a table examining the concordance rate.

The present invention is isolated from the swine epidemic diarrhea virus and cells, viral FNA extraction and cDNA preparation

Amplification of Spike Gene by Polymerase Chain Reaction, Cloning and Sequence Analysis of PEDV Spike Gene, Preparation of PEDV Spike Gene Expression Vector and Recombinant Baculovirus, Identification of Recombinant Protein by Immunization, Indirect Fluorescent Antibody, Cell Immunostaining Expression of recombinant protein, Western blot analysis, production condition of expression protein, enzyme immunoassay using recombinant protein, determination of optimal anti-spike monoclonal antibody adsorption, determination of optimal antigen concentration for antibody detection It consists of steps. It will be described in detail in the following examples, which are not intended to limit the scope of the invention.

Example 1 Isolation of Viruses and Cells

1 g of pig diarrhea infected with swine pandemic diarrhea virus was suspended in 10 ml in a minimal medium (alpha-minimal essential medium: α-MEM, Gibco), and centrifuged at 3,000 rpm for 20 minutes. Used as. The wild type baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV: Pharmigen) and the recombinant virus are Spodoptera frugiperda (Sf9: Invitrogen)) were grown and cultured in the cell line, and the Sf9 cell line was added with 10% fetal calf serum and antibiotic-antimycoic solution (Gibco, 100 ×) to Grace's media. One medium was incubated in a low temperature thermostat at 28 ℃.

Example 2 Viral RNA Extraction and cDNA Preparation

RNA extraction was performed using an Ultraspec ^™ II RNA isolation system (BIOTECX Laboratories, Inc, USA). 0.5 ml of fecal supernatant was added to a sterile RNAse free-eppendorf tube, and 1.0 ml of Ultraspec ^™ RNA reagent was mixed and shaken for 2 minutes. 200 μl of chloroform was added and centrifuged at 4 ° C. and 12,000 rpm. 1.0 ml of the supernatant was collected, transferred to a new tube, 500 µl of isopropanol and 40 µl of RNA Tack ^™ resin were mixed, and allowed to stand at room temperature for 5 minutes. After centrifugation at 4 ° C. and 12,000 rpm for 2 minutes, 1 ml of 70% ethyl alcohol was added and washed twice by centrifugation at 10,000 rpm for 30 seconds. The precipitated RNA-Resin was vacuum dried in a vacuum dryer for 5 minutes, and then 50 μl of sterile distilled water was added thereto. After reacting at room temperature for 5 minutes, the mixture was centrifuged for 2 minutes, and the supernatant was harvested to extract RNA. cDNA was prepared by using reverse transcriptase (Superscrip II) between Gibco / BRL for 90 minutes at 42 ° C to prepare cDNA.

Example 3 (Amplification of Spike Gene by Polymerase Chain Reaction)

Spike gene, a structural protein of swine pandemic diarrheal virus, has been reported in CV777 and Br1 / 87 strains. Based on these results, primers were synthesized to amplify the spike gene by polymerase chain reaction (PCR). 5'-GCG GGA TCC ATG AGG TCT TTA ATT TAC TTC-3 'anti-sense primer (SR3) 5'-AAT ACC CTC ACC TTT AAA GCC ATA GAT AGT-3 'and sense pimer (SF3) for amplifying 1813-3702 bp 5'-CGG AAT TCG CGT TCG GTA GTG GTG TTA AGT-3' and anti-sense primer (SR6) 5'-CTG GTC GCT AGT CAG ATT GAC ATA GGT GA-3 ', and the sense pimer (SF5) to amplify 3233-4370 bp was 5'-CTC TAC GTG AGC CTG GCT TAG TCT TGT T-3, anti-sense primer ( SR7) was synthesized as 5'-CGC GGA TCC TGC CAA CAT AAT ATA ATT GCG CCT-3 'and used for amplification. The reaction conditions were treated first at 95 ℃ for 5 minutes, the reaction was 50 cycles 1 minute, 72 ℃ 2 minutes, 92 ℃ 1 minute in 29 cycles, and finally 1 cycle 50 ℃ 1 minutes, 72 ℃ 5 minutes.

Example 4 (PEDV spike gene cloning and sequencing)

Each of the 1977, 1890 and 838 bp genes was amplified, and each gene was cloned into pGEMT, a cloning vector capable of cloning the amplified gene by polymerase chain reaction, and pSL1190, a general cloning vector, as shown in FIG. 1. . The genes were linked to the complete spike genes, and sequence analysis was performed to confirm that the complete spike genes were inserted correctly. The analysis was performed using a Dye terminater kit using ABI's Taq polymerase (polym erase) and fluorescent labeled Dideoxynucleotides (Applied Biosy stems International Prism system). 7.0) was used to analyze the sequence. As a result, it was confirmed that the spike gene was inserted to purify (see FIG. 2).

[Example 5] (Preparation of PEDV spike gene expression vector and recombinant baculovirus)

The cloned spike gene was inserted into the BamHI site of pVL1393, a baculovirus expression vector, and a recombinant virus was prepared using lipofectin as follows. That is, using a baculovirus expressi on system of Pharmingen (USA), 100 μl of serum-free Grace's medium containing 5 μg of expression vector pVL1393-spike plasmid DNA and 0.5 μg of baculovirus linearized DNA were used. Cotransfection mixture was prepared by mixing with lipofectin (0.1mg / ml, Gibco), and allowed to stand at room temperature for 20 minutes, and then added to Sf9 cells (3x10 ⁶ cells) prepared in 60mm Petri dish for 6-12 hours at 28 ° C. Remove the solution, add 2-3 ml of fresh Grace's medium containing 5% FCS, and incubate at 27 ° C for 4-6 days, harvesting when cytopathic effect (CPE) is observed. Was prepared (see also 3).

Example 6 (Isolation of Proteolytic Protein by Immuno-Drip Method)

In order to confirm the expression of the recombinant protein, an immunodropping method was used. Infected Sf9 cells were harvested by centrifugation, followed by sonication with saline (0.85% NaCl) in the same amount as the culture supernatant and centrifuged at 12,000 × g for 5 min. Dilution was carried out using a negative pressure on nitroce lulose paper. After using the anti-spike monoclonal antibody for 1 hour at room temperature and washing, the anti-mouse IgG HRP (KPL was reacted for 40 minutes and washed with diaminobenzidine (DAB, Pierce)) was confirmed by color development (see FIG. 4).

Example 7 (Indirect Fluorescent Antibody Test)

Recombinant virus was immobilized with 80% cold acetone at 3 days after inoculation into Sf 9 cells and reacted for 40 minutes using PEDV-spike monoclonal antibody as a primary antibody in a humid chamber and anti-mouse as a secondary antibody. -FITC was used. Under fluorescence microscopy, specific reaction was observed only in infected cells expressing the spike protein, thereby confirming the expression of the spike protein (see FIG. 5).

Example 8 Expression Confirmation of Recombinant Protein by Cell Immunostaining

Infected Sf9 cells and normal Sf9 cells were fixed with 80% cold ace one on day 3 of inoculation, reacted with anti-PEDV-spike monoclonal antibody at room temperature and washed three times, followed by anti-mouse IgG-HRP (Kpl) for 1 hour. The reaction was carried out three times with PBST and developed. As a coloring agent, 10 μl of hydrogen peroxide (H ₂ O ₂ ) was added to 10 ml of diaminobenzidine (DAB, 0.9 mg / 100 ml) (see FIG. 6).

Example 9 (Western blot analysis)

After 4 days of inoculation of the recombinant virus, the infected cells were sonicated, sonicated, centrifuged at 3,000 rpm for 20 minutes, suspended in SDS-PAGE sample buffer, and treated at 95 ° C for 5 minutes, and then subjected to SDS-PAGE. After SDS-PAGE was transferred to nitrocellulose paper. A blocking solution (blocking solution: 5% lactalbumin hydrolysate and skim milk in PBS) was added and blocked for 1 hour at room temperature. After reacting with anti-PEDV spike monoclonal antibody and washing with PBST (PBS-0.05% Tween20) three times, anti-nouse IgG-HRP (Kpl) was reacted for 1 hour, washed three times and diaminobenzidine ( 10 μl of H ₂ O ₂ was added to 10 ml of DAB, 0.9 mg / 100 ml). The molecular weight of the expressed spike protein was found to be about 150 kd. (See Figure 7.)

Example 10 (Investigation of Production Conditions of Expression Protein)

In order to measure the amount of expression, the indirect ELISA method was used. After monolayer culture of Sf9 cells on 6 well TC microplate, the selected recombinant virus was inoculated, and the recombinant protein expressed in culture supernatant and infected cells was applied to 96 well polysorb ELISA plate every 24 hours in a buffer buffer (BuPH ^TM Carbonate Bicarbonate buffer Packs, Pierce, USA) was diluted twice by step and 100ul was added and coated at 4 ℃ 18 hours. The supernatant was discarded and washed four times with PBST (PBS-0.05% Tween20) and then blocked with 200 μl of blocking solution (5% lactalbumin hydrolysate and skim milk in PBS) for 1 hour. Porcine epidemic diarrhea virus specific monoclonal antibody was diluted 1,000-fold in PBST and reacted at 37 ° C for 1 hour. After washing four times with PBST, anti-mouse-HRP (kpl) was diluted 1,000-fold in PBST and reacted at 37 ° C for 1 hour. After washing 4 times with PBST, 100 μl of OPD (o-pheylenediamine dehydrochloride, 5mg / 12.5ml in citrate phosphate buffer) was added, and after 15 minutes in the dark, 2.5MH ₂ SO ₄ was added to stop the reaction. Readings were measured for absorbance at 490 nm by ELISA reader. The determination was positive when the positive serum absorbance / negative serum absorbance ratio was 2 or more (see FIG. 8).

Example 11 (Immunosorbent Assay Using Recombinant Protein: IS-ELISA)

The purified anti-spike monoclonal antibody was diluted to an optimal concentration using an adsorption coating buffer (BuPH ^TM Carbonate Bicarbonate Buffer Packs, Pierce, USA), and 100 μl of the plate was immunized on an immunoassay plate. Time to adsorb. Discard the adsorbed antibody from the plate and wash once with ELISA 1st washing solution (Tris-base 2.42g, NaCl 22.22g, Merthiolate 0.1g, Tween 20 0.5ml / liter, pH7.3) and then block buffer (5% gelatin). , 100% of 1% bovine serum albumain) is reacted at 37 ℃ for 2 hours. The recombinant spike protein is diluted in PBS with an optimal dilution concentration and dispensed in 100 μ portions, followed by reaction at 37 ° C. for 2 hours, and then washed three times with the primary wash solution. Dispense 100 μl of blocking buffer (5% gelatin, 1% bovine serum albumain) and react at 37 ° C for 1 hour. The prepared porcine serum is diluted in a separate dilution plate with an optimal dilution drainage as the primary washing solution. Discard the blocking solution, discard the blocking solution, add 100 µl of diluted serum and react at 37 ° C for 1 hour. After the reaction, the mixture is washed three times with a second washing solution (Tris-base 2.42g, NaCl 29.22g, Merthiolate 0.1g, Tween 20 1.0ml / liter, pH 7.7). At this time, each wash is performed for 5 minutes. The anti-swine IgG peroxidase conjugate is diluted to the optimal dilution concentration in the primary wash solution, and 100 μl each is added, followed by reaction at 37 ° C. for 1 hour. After the reaction, the solution is washed three times or more, and the remaining solution is removed. To develop color, ABTS is used as a coloring agent. After color development, absorbance is measured at 405nm using ELISA reader. Determination of serum antibody titers was performed by selecting five standard positive and negative serums, respectively, as a control group, and diluting two-fold, followed by ELISA. The inverse of the dilution factor in which the positive serum absorbance / negative serum absorbance ratio falls below 2 is determined by the antibody value. The procedure for porcine epidemic diarrhea virus neutralizing antibody test using ELISA is shown in FIG.

Example 12 Determination of Optimal Anti-Spike Monoclonal Antibody Adsorption Concentration

The optimal monoclonal antibody concentration for the detection of neutralizing antibody against swine pandemic diarrheal virus was the highest reactivity at (1/1000, 10µg / ml).

Example 13 Determination of Optimal Antigen Concentration for Antibody Detection against Recombinant Spike Protein

The optimal antigen concentration for detecting the swine pandemic diarrhea virus neutralizing antibody was 1/10, that is, the best reactivity was 5 ㎍ / ml and the result was 11 degrees.

Example 14 Determination of Optimal Serum Dilution Concentration for Antibody Detection Against Recombinant Spike Protein

The proper serum dilution factor for antibody detection was 1/50 and the result was 12 degrees.

Test example (investigation of antibody detection agreement rate with IS-ELISA and virus neutralization test)

The antibody detection correlation between the existing monoclonal pandemic diarrheal virus neutralization test and the IS-ELISA method of the present invention under the conditions of monoclonal antibody, antigen, and serum determined above was found to be about 0.706, with specificity of 83.3% and sensitivity. 83.3% showed that ELISA can detect the swine pandemic diarrheal virus neutralizing antibody. In view of the above results, the IS-ELISA method of the present invention is evaluated as a method capable of quickly and accurately testing the swine epidemic diarrhea virus intermediate antibody.

Swine epidemic diarrhea causes vomiting and watery diarrhea regardless of the age of the pig. The cause is swine panic diarrhea virus. Spike, a structural protein, causes neutralizing antibodies. Therefore, the spike gene is inserted into insect virus baculovirus. The specific spiked protein was developed to detect specific neutralizing antibodies of the swine epidemic diarrheal virus.

This method enabled the detection of neutralizing antibodies to swine pandemic diarrheal disease within 4 hours, and can be useful for the examination of immunity level and the status of infection after vaccination for effective prevention of swine diarrheal disease.

Claims (4)

Swine pandemic diarrheal disease (PEDV) spike gene isolated from pig diarrhea and contained in the genetically engineered baculovirus (KFCC 11014) gene, the gene sequence of the pig is characterized by the following structure Spike gene for detecting specific neutralizing antibodies of virus (PEDV). Recombinant baculovirus (KFCC 11014) transformed using the baculovirus expression vector pVL 1393 of FIG. 3 containing the swine epidemic diarrheal virus (PEDV) spike gene of claim 1. Obtaining a mixture of a baculovirus expression vector pVL 1393 containing the swine pandemic diarrheal virus (PEDV) spike gene of claim 1 and a serum-free medium containing a linear baculovirus and lipofectin, wherein the mixture is a host cell. Method for producing a recombinant baculovirus (KFCC 11014), characterized in that it comprises the step of cotransfection in addition to Sf9 (Cotransfection). In a method for detecting specific neutralizing antibodies of swine pandemic diarrheal virus by using an enzyme-linked immune antibody method, an indirect enzyme-linked immune antibody method (lndirect-) using a spike protein expressed in a recombinant baculovirus (KFCC 11014) as an antigen. A method for detecting specific neutralizing antibodies of swine pandemic diarrhea virus using ELISA).