KR100267744B1 - Recombinant baculovirus and method of macelisa using the recombinant tgev spike protein for the detection of specific antibodies to tgev - Google Patents

Abstract

PURPOSE: Provided are a genetic recombinant baculovirus and a recombinant spike protein of transmissible gastroenteritis virus(TGEV) manufactured therefrom. And a monoclonal antibody capture enzyme linked immunosorbent assay for detecting the antibody of TGEV using the same protein is also provided, thereby the antibody of TGEV can be rapidly detected. CONSTITUTION: The recombinant spike protein of TGEV is produced by the steps of: isolating and identifying TGEV from pigs died of diarrhea; analyzing the nucleotide sequence of spike protein of TGEV; inserting the TGEV spike gene into the baculovirus gene to produce a recombinant baculovirus(KFCC-11015); infecting the recombinant baculovirus into SF9 cells to express TGEV spike protein. The antibody of TGEV is produced by inoculating the expressed recombinant TGEV spike protein into Guinea pigs. The monoclonal antibody capture enzyme linked immunosorbent assay using TGEV spike protein that is produced from the recombinant baculovirus(KFCC-11015) in Guinea pigs as an antigen is used in detecting the neutralizing antibody of TGEV.

Description

Enzyme-Immunosorbent Assay for Detection of Porcine Infectious Gastritis Virus Antibodies Using Genetically Recombinant Baculovirus and Recombinant Porcine Infectious Gastritis Virus Spike Protein

The present invention analyzes the nucleotide sequence of the spike gene of the isolated swine infectious gastroenteritis virus (NVRI 48) by cloning the spike glycoprotein gene and expresses the recombinant recombinant protein expressed in the insect virus baculovirus against swine infectious gastroenteritis virus. We wanted to find a way to quickly test for neutralizing antibodies.

Swine infectious gastroenteritis was first reported by Doyle and Hutching in 1946 and causes severe diarrhea, vomiting and high mortality in piglets less than two weeks old. The causative agent of swine infectious gastroenteritis is TGEV belonging to the coronavirus genus of Coronaviridae, a polymorphic virus with envelopes and club-shaped spikes of 60-220 nm in size. TGEV is an RNA virus with a positive-stranded genome of 23.6 kilobases, three major structural proteins, the nucleocapsid protein (N), the integral-membrane glycoprotein (M), and the spike glycoprotein. It consists of (glycoprotein (S)). Spike protein is about 20 nm in size and has a molecular weight of 220 kDa and is a highly glycosylated polypeptide that produces neutralizing antibodies that allow cell adhesion, cell membrane fusion and piglets to resist TGEV. It is known to have at least four major epitopes that lead to Therefore, several researchers have been trying to develop a vaccine to prevent swine infectious gastroenteritis by expressing the TGEV spike protein using a baculovirus, vaccinia virus or adenovirus expression system. The present invention was expressed in the baculovirus expression system using a domestically isolated pig TGEV spike protein that induces neutralizing antibodies, and by using the recombinant spike protein produced to test the pig TGEV neutralizing antibody quickly and accurately to prevent swine infectious gastroenteritis It was done for the purpose.

In other words, the present invention is to detect a neutralizing antibody to swine TGEV by developing a monoclonal antibody capture enzyme linked immunosorbent assay (MACELISA) using a pig TGEV spike protein produced by genetic recombination.

1 is a schematic diagram showing the spike gene region and primers of domestically isolated NVRI 48 weeks using enzyme polymerase chain reaction (PCR).

Figure 2 is a photograph of the electrophoresis of the NVRI spike gene amplification product using PCR.

3 is a schematic diagram of cloning for NVRI 48 spike gene sequencing.

4 is a subcloning schematic for NVRI 48 spike gene sequencing.

Figure 5 is the NVRI 48 week spike gene sequence.

Figure 6 is a schematic diagram of baculovirus expression vector cloned cloned NVRI 48-week spike gene.

Figure 7 is a photograph of the detection of TGEV spike protein expressed in recombinant baculovirus using a monoclonal antibody (5C8) that specifically binds to the TGEV spike protein by fluorescent antibody method.

Figure 8 is a photograph of the TGEV spike protein expressed in recombinant baculovirus detected by immunohistochemical staining using a monoclonal antibody (5C8) that specifically binds to the TGEV spike protein.

Figure 9 is a photograph of recombinant TGEV spike protein identification using immunoprecipitation (Immunoprecipitation).

Figure 10 is a flow chart of porcine infectious gastroenteritis virus neutralizing antibody test using MACELISA.

Figure 11 is a determination of the appropriate dilution concentration of monoclonal antibody for MACELISA.

Figure 12 is a determination of the appropriate recombinant protein concentration for MACELISA.

Figure 13 is a determination of the appropriate serum dilution multiples for MACELISA.

14 is a titration conjugate dilution fold crystallinity for MACELISA.

Figure 15 is a chart comparing the detection efficiency between the current virus neutralization test and the developed MACELISA.

Hereinafter, the present invention will be described in detail.

Example 1

Viruses and cells

Domestically isolated TGEV (NVRI 48) strains were passaged in the Wine Testicle (ST) cell line, and the cell culture medium was 10% fetal calf serum (FCS, in alpha minimum essential medium (α-MEM, Gibco). Gemini)), and antibiotic-antimycotic (Gibco, 100 ×), were used in addition to the wild-type baculovirus Autographa californica nuclear polyhedrosis virus (Autographa californica nuclear polyhedrosis). virus (AcNPV: Pharmigen)) and recombinant virus were propagated and cultured in Spodoptera frugiperda (Sf9: Invitrogen) cell line, and the Sf9 cell line was in 10% fetal calf serum in Grace's media. Calf serum) and antibiotic-antimycotic (Gibco, 100 ×) were added to the medium at low temperature incubator at 28 ° C. The radioisotope was identified for identification. A methionine-deficient medium (methionine free Grace medium (Gibco, USA)) was used.

Example 2

Viral RNA Extraction and cDNA Synthesis

PCR primers were synthesized using a DNA / RNA synthesizer (Applied Biosystems) based on the Purdue and Miller strain spike gene sequences searched in the Gene bank to synthesize three sets. In the case of primers F1 and F2, restriction enzymes BamHI and EcoRI sites were introduced to facilitate cloning of amplification products (see FIG. 1). Total RNA of TGEV was inoculated with STRI cells incubated for 3 days and then harvested for cell degeneration when the cell degeneration effect began to occur. The RNA was extracted using Ultraspec II RNA isolation kit (BIOTECX). . Synthesis of cDNA was carried out by mixing about 5 ㎍ of total RNA with reverse primer, and boiled for 5 minutes, immediately put on ice, cooled for 5 minutes, and centrifuged at 10,000 rpm for 1 minute. first-strand cDNA contains 5 μg total RNA, 40 unit RNAsin (Promega), 50 mM Tris-HCl pH8.3, 3 mM MgCl ₂ , 75 mM KCl, 10 mM DTT, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dTTP, 0.4 mM dGTP and reverse primer were added to 50 μl of reaction solution and reacted at 50 ° C. for 2 minutes. Then, 4 units of reverse transcriptase (SUPERSCIPT II RNase H ^- Reverse Transcriptase, GIBCO BRL) were added and reacted at 50 ° C. for 50 minutes. . cDNA was denatured at 95 ° C for 5 minutes in Gene Amp PCR system 9600 (Perkin Elmer) using 3 primers synthesized and THERMALASE Tbr Kit (AMRESCO), followed by 50 ° C for 1 minute, 72 ° C for 2 minutes, and 92 ° C. After 29 cycles of 1 minute reaction, 50 ° C. for 1 minute and 72 ° C. for 5 minutes were amplified. (See FIG. 2)

Example 3

Cloning and DNA Sequencing of the NVRI 48-Week Spike Gene

DNA fragments amplified by PCR were 1,186bp, 1,362bp and 2,246bp, and the former two DNA fragments were immediately cloned (pGF2, pGF3) into pGEM-T vector (Promega) without restriction enzyme treatment. Treatment with KpnI and EcoRI was cloned (pTF2-K23) into the pTZ18R vector. The cloned three fragments were treated with appropriate restriction enzymes (1,186 bp: BamHI-XhoI, 1,362 bp: XhoI-KpnI, 2,246 bp: KpnI-EcoRI), and then cloned (4,458 bp) and cloned back into the pTZ18R vector (pTF5). -BE10) (see Figure 3). To facilitate sequencing of the entire NVRI48 spike gene, it was treated with restriction enzymes such as Sau3AI, HaeIII, and HindIII, divided into nine fragments, and each was subcloned into the pTZ18R vector (see Figure 4). The sequencing method was based on the dioxyoxy chain termination method. Sequenase version 2.0 DNA sequencing kit (USB, USA) and radioisotope [α- ³⁵ S] dATP were used. After the reaction was completed, the sample was electrophoresed on a 6% polyacrylamide gel containing 7M urea and exposed to X-ray film (Kodak, X-Omat AR) for 15-20 hours. Nucleotide sequences were read in both directions (see FIG. 5).

Example 4

NVRI 48 Week Spike Gene Baculovirus Expression Vector Construction

pTF4-BK1 (see FIG. 3) was used as template DNA and PCR amplified with forward primer (5'-CGGGATCC ATGAAAAAACTATTTGTGGTTT-3 ') and reverse primer (5'-TCCATCAGTTACGCCG AACG-3'). The amplified product and pTF4-BK1 were ligated after BamHI and XhoI to prepare pTF6-A. pVL1393 containing pTF6-A and a universal stop sequence (5'-TAATT AATTAA-3 ') were ligated after BamHI and EcoRI to prepare pF8A. 5'-CCATCACCATCACCATCACTAAG-3 'and 3'-CATGGGTAGTGGTAGTGGTAGTGATTCTTAA-5' were synthesized to insert 6 histidines at the COOH end to facilitate purification of the synthesized protein, and the two strands were combined to make 2-strand DNA. DNA containing 6X histidine sequence and pTF6-A were treated with KpnI and EcoRI and ligated to prepare pTF6-AH. pTF6-AH and pVL1393 were ligated with BamHI and EcoRI to prepare pF6AH (see FIG. 6).

Example 5

Cotransfection and Recombinant Baculovirus Preparation

Cortfection was carried out using a baculovirus expression system from Pharmingen (USA), and a serum-free gray medium containing 5 μg of expression vectors (pF8A, pF6AH) plasmid DNA and 0.5 μg of baculovirus linearized DNA 100 A cotransfection mixture was prepared by mixing μL and the same amount of lipofectin (lipofectin (0.1 mg / ml, Gibco)), and allowed to stand at room temperature for 15 minutes, and then prepared Sf9 cells (1.5 × 10) in 35 mm Petri dishes. ⁶ cells), incubated for 5 hours at 28 ℃, remove the supernatant, add 5 ml of fresh Grace medium containing 10% FCS, incubated for 4-6 days at 27 ℃ harvested when CPE is observed and recombinant baculo Virus was written.

Example 6

Confirmation of Recombinant TGEV Spike Protein Expression

After infecting the recombinant virus with Sf9 cells, the infected cells were fixed with 100% cold acetone for 10 minutes and monoclonal antibody (5C8) that specifically binds to the TGEV spike protein. Washed and reacted with anti-mouse FITC (KPL, German) and observed by fluorescence microscopy (see Figure 7). In addition, the infected cells were fixed with 80% cold acetone for 10 minutes at 20 ° C, and then reacted with monoclonal antibody (5C8) for 40 minutes at room temperature. It was confirmed by color development with diaminobenzidine (DAB, Pierce) (see Figure 8). Indirect fluorescence antibody test and immunohistochemical staining showed specific expression only in recombinant virus-infected cells, indicating the expression of recombinant TGEV spike protein. The recombinant viruses thus identified were named pF8A-Bac and pF6AH-Bac.

Example 7

Protein Identification Using Immunoprecipitation

Normal Sf9 cells and wild type baculovirus as a negative control, and extracts of Sf9 cells infected with recombinant baculovirus were immunoprecipitated with monoclonal antibody and subjected to SDS-PAGE followed by autoradiography. First, Sf9 cells were cultured to 3 × 10 ⁶ cells in a 25 cm 2 flask, and the inoculum was removed after 1.5 hours of inoculation with recombinant and wild baculovirus 5-10 PFU (plaque forming unit). Subsequently, 5 ml of 5% FCS-containing Grace medium was added and cultured for 42 hours at 28 ° C., followed by 1 hour starvation in methionine deficient Grace medium (Gibco BRL), followed by ³⁵ S-methionine (Amersham, UK) at ⁵⁰ μCi / ml. It was labeled by incubating for 22 hours. The labeled Sf9 cells and the supernatant were harvested separately and the cells were washed once with PBS, followed by the addition of 200 μl of lysis buffer (0.5% TritonX-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) and on ice. After standing for 10 minutes, the mixture was centrifuged at 12,000 × g for 10 minutes, and the supernatant was stored at −20 ° C. and disclosed as an immunoprecipitation sample. Immunoprecipitation was performed using the immunoprecipitation kit (Behringer Mannheim: BM), and protein A agarose was added to 100 μl of cell lysate for 15 hours at 4 ° C., followed by centrifugation at 12,000 rpm for 30 seconds. After removing the supernatant. 3 μl (3 μl) of anti-TGEV spike protein monoclonal antibody was added to the supernatant and shaken at 4 ° C. for 1 hour. Add 50 μl of protein A (BM) agarose, shake at 15 ° C. for at least 15 hours, centrifuge at 12,000 × g for 20 minutes, remove the supernatant, and wash each with 1 wash buffer according to the manufacturer's method. Washed once. Add 100 μl of the sample buffer solution (10% -mercaptoethanol, 10% SDS, 25% Glycerol, 10 mM Tris-Hcl pH 6.8) to the sample, boil for 5 minutes, centrifuge for 5 minutes at 12000 × g, and then use Lammli's method. After electrophoresis on 7.5% polyacrylamide gel, autoradiography was performed. The molecular weight of the recombinant TGEV spike protein labeled with ³⁵ S-methionine was found to be about 100 kDa (see FIG. 9).

Example 8

Enzyme Immunoassay Using Recombinant TGEV Spike Protein (pF6AH-Bac) (MACELISA)

After concentration with ammonia sulfate, dialysis purified monoclonal antibody (5C8) was diluted to an optimal concentration for use as a coating buffer, and 100 μl of an immunoassay plate (Nunc) was adsorbed at 37 ° C. for 15 hours. After the adsorption-absorbed antibody was discarded from the plate, washed once with ELISA washing solution, 100 μl of blocking buffer was dispensed, and then reacted at 37 ° C. for 3 hours, and then washed three times with washing solution. The prepared porcine serum is diluted with the optimal dilution factor with serum dilution buffer solution. After the blocking reaction is completed, the blocking solution is discarded, washed three times with the washing solution, and 100 μl of diluted serum is added and reacted at room temperature for 1 hour. After the reaction, wash with washing solution three times or more. At this time, each wash is performed for 5 minutes. Anti-swine IgG peroxidase conjugate (KPL) was diluted in optimal dilution concentration in serum dilution buffer and added to 100 μl and reacted at room temperature 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. Once color development is completed, absorbance is measured at 405 nm using an ELISA reader. The antibody titer of serum was positively determined more than two times by the ratio of test serum to negative serum. Porcine TGEV neutralizing antibody assay using ELISA is the same as the 10th.

Example 9

Determination of titer of monoclonal antibody dilution for MACELISA

The dilution concentration of the optimal monoclonal antibody was 1: 4000 or 0.3 μg / ml (see FIG. 11).

Example 10

Determination of Proper Recombinant Protein Concentration for MACELISA

The dilution concentration of the appropriate recombinant protein was 1: 100 or 5 μg / ml (total amount of protein measured) (see FIG. 12).

Example 11

Determination of Proper Serum Dilution Concentration for MACELISA

The optimal serum dilution factor for antibody detection was 1/40, and the result is shown in FIG. 13.

Example 12

Determination of titrant conjugate dilution concentration for MACELISA

The proper dilution for detecting neutralizing antibodies was 1: 1,000 (see FIG. 14).

Example 13

Porcine Infectious Gastritis Virus Using Enzyme Immune Antibody (MACCELISA)

Investigation of detection agreement between the neutralizing antibody detection method and the virus neutralization test method using the existing tissue culture (see Fig. 15).

As a result of investigating the antibody detection correlation between the virus neutralization test method and the MACELISA method of the present invention under the conditions of the monoclonal antibody, antigen and serum determined above, the correlation was as high as 0.91, with specificity of 91.9% and sensitivity of 91.6%. It is demonstrated that the MACELISA method can detect the neutralizing antibodies to Swine infectious gastroenteritis virus and the results are shown in FIG. 15. In view of the above results, the MACELISA method of the present invention is evaluated as a method capable of quickly and accurately testing pig infectious gastroenteritis virus intermediate antibody.

Claims (2)

Recombinant baculovirus KFCC-11015 transformed with pF8A or pF6AH of FIG. 6 containing the Spike gene of FIG. 5 isolated from porcine infectious gastroenteritis virus (TGEV). Pig infectious gastroenteritis virus neutralizing antibody serum test using enzyme immunoassay, porcine infectious gastroenteritis using enzyme immunoassay characterized in that the antigen is the TGEV spike protein produced by the recombinant baculovirus KFCC-11015 of claim 1 Neutralizing antibody serum test method of virus.