KR20020015919A - A combined-antigen ELISA using the ORF5- and ORF7recombinant proteins of porcine reproductive and respiratory syndrome virus - Google Patents

Abstract

PURPOSE: Provided is an improved combined-antigen of ELISA(enzyme-linked immunosorbent assay) using ORF5- and ORF7- recombination protein to detect an antibody for porcine reproductive and respiratory syndrome virus(PRRSV) from the serum of an infected animal, thereby improving antibody detection rate and accuracy. CONSTITUTION: The ELISA comprises the steps of: expressing ORF5 and ORF7 recombination gene using E. coli expression system to obtain a recombinant protein; analyzing the reactivity of antigen and antibody of the recombination protein by Western blotting method; determining optimal concentration of the separated pure and refined recombination protein as ELISA antigen; and developing and testing the combined-antigen of ELISA.

Description

A combined-antigen ELISA using the ORF5- and ORF7 recombinant proteins of porcine reproductive and respiratory syndrome virus}

Enzyme-linked immunosorbant assay (ELISA) detects antigens or antibodies from infected animals by coordinating proteins, such as antigens or antibodies, on the plastic surface of the microplate, using the interaction of hydrophobic moieties with nonpolar plastic surfaces. It's a diagnostic. In addition, the antigen or antibody adsorbed to each well of the microplate is very small (see Winston, SE et al., Current Protocols in Molecular Bioloar, Wiley and Sons, inc., New York, p, 11.28-11.2). .9 (1989), the ELISA method using them is very economical, has high specificity and sensitivity, and has the advantage of inspecting a large number of specimens in a short time.

However, in addition to the sensitivity and specificity of the ELISA method, additional considerations generally required for the ELISA method for diagnosing animal diseases include: 1) an inexpensive, economical, and short-term inspection of large quantities of specimens; and 2) a significant period of time after the animal has been infected. The diagnosis should be possible after the diagnosis.

So far, the methods of detecting antibodies from pigs infected with swine genital respiratory virus have been indirect fluorescence antibody test (Indirect fluorescence antibody test), Immunoproxidase monolayer assay (IPMA), etc. ELISA using virus antigens from Denac, H. et al. Journal of Virological Methods 65: 169 (1997) and porcine alveolar lung macrophage (PAM) (Albina, E) et al. Ann.Rech.Vet. 23: 167 (1992), etc., but indirect fluorescent antibody tests of these antibody detection methods generally have high specificity and low sensitivity and specificity compared to ELISA. In addition, the experimental results may be subjective because the results are visually read using a fluorescence microscope.The ELISA method reported by Albina (1992) et al. Since swine genital respiratory virus was cultured in lung macrophage and used as ELISA antigen, macrophage cell components and other calf serum and other components in cell culture medium exist in the antigen. It is a disadvantage that accurate diagnosis is difficult due to mass production of pulse positives.The ELISA method using a nucleocapside protein (ORF7) recombinant protein, reported by Denac H. et al., Uses ORF7 recombinant protein as a single antigen. Antibodies to proteins can be detected, but antibodies to other viral proteins, such as ORF5, cannot be detected, and antibodies against ORF7 proteins can be detected early (1 to 2 weeks after infection) compared to other proteins of the swine genital respiratory virus. Antibodies are produced quickly and become infected 10 weeks after infection. For this to be a considerable time after infection, because this property almost disappears after which an in vivo was found to be diagnosed by the detection of antibodies to the ORF7 protein impossible [see: Fig. 12]. Antibodies to the ORF5 protein are rarely detected in the body of infected pigs during the early stages of infection but have been reported to detect antibodies from three weeks to almost one year after infection.

Considering that the production time and the extinction period of the antibodies against ORF5 and ORF7 proteins are different under the background described above, this study was performed by mixing ORF5 and ORF7 recombinant proteins as ELISA antigens. Technical challenges are: 1) higher sensitivity (detection rate) when using ORF5 or ORF7 single antigens by using complex antigens; 2) The use of complex antigens not only enables early diagnosis of the disease, but also develops an improved ELISA method that can be diagnosed even after a long time after infection.

(1) is an expression and purification of the ORF5 gene recombinant protein in E. coli M15 arrow indicates the isolated purified ORF5 protein

Figure 2 is a Western blot testing the reactivity of the isolated and purified ORF5 gene recombinant protein with the antibody

(3) shows the expression and purification of the ORF7 gene recombinant protein in Escherichia coli M15, and the arrow shows the purified ORF7 protein.

Figure 4 is a Western blot testing the reactivity of the isolated purified ORF7 protein with the antibody

Figure (5) is a graph measuring the titration concentration as ELISA antigen of ORF5 recombinant protein

(6) is a graph measuring the titration concentration as ELISA antigen of ORF7 recombinant protein

Figure 7 is a graph showing the optimal antibody dilution ratio for the optimal concentration of ORF5 recombinant protein (100 ng / well) antigen.

(8) is a graph showing the optimal serum (antibody) dilution factor for ORF7 recombinant protein antigen

(9) is a graph showing the optimal serum (antibody) dilution factor for ORF5 and ORF7 mixed recombinant protein

(10) is a graph showing the results of comparing the ORF5 and ORF7 single antigen ELISA and complex antigen ELISA

Hereinafter, the present invention will be described in more detail.

RNA was extracted from the pure purified porcine respiratory donor virus and amplified the ORF5 and ORF7 genes by reverse transcription-polymerase chain reaction, and cloned the gene into pGEX2T plasmid vector and pQE30plasmid vector, respectively. After transformation, the recombinant proteins for ORF5 and ORF7 genes were produced and purified as shown in Figs. (1) and (3). In order to investigate the reactivity of ORF5 and ORF7 recombinant proteins against porcine genital respiratory antiviral antibodies, Western blot was performed with artificially infected virus-positive pig serum, as shown in Figs. (2) and (4). Similarly, it was confirmed that ORF5 and ORF7 recombinant proteins specifically reacted with viral antibodies, so that ORF5 and ORF7 recombinant proteins were suitable as ELISA antigens. As a result of measuring the optimum amount as ELISA antigens for the ORF5 and ORF7 recombinant proteins, as shown in Figs. (5) and (6), 100ng / well was found to be the optimal ELISA antigen amount, respectively. Therefore, the amount of antigen per well of the complex antigen ELISA was set to 200 ng / well. The adsorbing (coating) of the antigen to the microplate was diluted to a concentration of 200ng / 50μl in a carbonate buffer (pH 9.0), 50μL injected into each well, and then allowed to stand for 1 day at 4 ℃. As a result of measuring the optimal serum dilution concentration for ELISA, as shown in FIGS. 7, 8 and 9, the optimal serum dilution factor for ORF5, ORF7 and complex antigen was 1: 200.

Swine genital respiratory virus antibodies were detected in serum collected at weekly intervals from pigs infected with ORF5, ORF7 and complex antigen ELISA. As shown in FIG. 10, ORF5 ELISA was contacted with infected pigs. Antibodies were detected from 6 weeks and showed high antibody titers up to 11 weeks of contact infection. In ORF7 ELISA, antibody detection was possible from 5 weeks after contact with infected pigs, but antibody titer rapidly decreased and showed low titer around 11 weeks. The complex antigen ELISA showed high antibody titers from 5 weeks after contact to 11 weeks, indicating that the complex antigen ELISA was superior to the initial antibody detection time and the detectable time period compared to the single antigen ELISA.

Example 1

In this example, blood was collected at intervals of one week after inoculation from a pig infected with contact, and ORF5 ELISA, ORF7 ELISA, and complex antigen ELISA were compared for the initial antibody detection time, antibody detection period, and the like. ELISA tests were performed according to known methods. As shown in FIG. 10, the ORF7 ELISA was able to detect antibodies one week earlier than ORF5, whereas the antibody rapidly disappeared, preventing the detection of the antibody later in infection. Although possible, high antibodies were detected up to 11 weeks. Therefore, in the combination antigen ELISA containing the two antigens, the results were obtained with the advantages of both ORF7 ELISA and ORF5 ELISA.

Example 2

In this example, comparative tests were performed on 772 pig serum collected from ORF5 ELISA, ORF7 ELISA and complex antigen ELISA using a single recombinant protein antigen in an outdoor farm. Indirect fluorescent antibody test is the most commonly used swine genital respiratory virus diagnosis method in Korea and abroad. The ELISA test and indirect fluorescent antibody method were carried out as generally known methods. In comparison with the results of this comparative test, the antibody detection rates of indirect fluorescent antibody method, ORF5 ELISA, ORF7 ELISA and complex antigen ELISA were 59.0% (465/722), 65.0% (502/772), 60.2% (466 / 772) and 75.5% (583/772), but the highest antibody detection rate of the combined antigen ELISA was ORF5 or ORF7 ELISA and the indirect fluorescent antibody method.

As described and demonstrated in detail above, the present invention increased the antibody detection rate of ELISA by using purely isolated virus protein produced by genetic recombination technology as ELISA antigen, and the complex antigen ELISA method was used to infect disease compared to ELISA using single antigen. Diagnosis was possible at the initial stage by antibody detection, and it was proved that diagnosis by antibody detection was possible even after a long time after viral infection. In addition, by refining and recombining the recombinant protein of a specific viral gene in the E. coli expression system, it is possible to increase the purity of the antigen and lower the production cost of the antigen. As a result, the present invention is inexpensive and rapid and accurate pig genital respiration. Can provide diagnostics

Claims (2)

Improved Antigen ELISA Method Using a Porcine Genital Respiratory Virus ORF5 Recombinant Protein and ORF7 Recombinant Protein as an ELISA Antigen An improved complex antigen ELISA method using all ORF recombinant proteins of porcine respiratory virus in claim 1