JPH07159408A - Antigen for identifying aujeszky's desease virus-infected antibody - Google Patents

Abstract

PURPOSE:To make it possible to measure an antigen for identifying an Aujeszky' s desease virus-infected antibody with higher specificity in reaction with a gX antibody and with sufficiently high reliability by an ELISA method and further, the providing of the antigen for identification at a low cost with higher production efficiency by a simple purification method. CONSTITUTION:An antigen for identifying an Aujeszky's deseases virus-infected antibody is made up of a fused protein of a membrane glycoprotein of swine herpes 1 virus and glutathione S transferases. In the production of the antigen, a gene which has the membrane glycoprotein gX of swine herpes 1 virus and the glutathione S transferases coded is integrated with a plasmid vector to develop the fused protein which is refined by glutathione column chromatography. The fused protein thus refired is applied for an ELISA method as an antigen for identification to detect the Aujeszky's desease virus-infected antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antigen for identifying a virus infection antibody of Aujeszky's disease which infects domestic animals such as pigs, a method for producing the same and a method for detecting an Aujeszky's disease virus infection antibody.

[0002]

2. Description of the Related Art Aujeszky's disease is an infectious disease caused by the infection of porcine herpes 1 virus (hereinafter abbreviated as ADV) belonging to the alphaherpesvirus subfamily of the herpesviridae. , Cows, sheep, cats, dogs, and other mammals. When infected with such ADV, young pigs show serious symptoms and may die. Adult respiratory pigs often present with transient respiratory symptoms and mild fever symptoms. In addition, when the virus latently infects the pig body and becomes a new source of infection or infects pregnant pigs, the death rate is high.
There is also a miscarriage, which causes great damage to the livestock industry.

At present, in Japan, as a vaccine for the purpose of preventing this infectious disease, a viral membrane glycoprotein that is not considered to be involved in the growth or infectivity of the virus, g
A virus deficient in genes encoding I antigen, gIII antigen and gX antigen is adopted as a strain for producing a live vaccine. This is because pigs infected with Aujeszky's disease need to be separated from naturally infected pigs and vaccinated pigs to detect only naturally infected pigs in accordance with the Japanese epidemics policy of eliminating and cleaning the pigs.

[0004] As a method for identifying an antibody caused by natural infection or an antibody caused by vaccination, a method of quantitatively detecting an antibody against the defective antigen by an ELISA method is generally used.

The gX antigen used in the above-mentioned detection method when a virus deficient in the gene encoding the gX antigen is employed as a vaccine-producing strain is centrifuged from the virus culture supernatant and concentrated by salting out. And then extract,
It was purified by gel filtration, ion exchange column, affinity column chromatography and the like.

[0006] Here, as a method for producing a large amount of gX antigen necessary for the ELISA method, it is conceivable to adopt a genetic engineering method as follows. That is, it is a method in which a gene encoding gX among the structural genes of Aujeszky's disease virus is cloned and inserted into a virus, plasmid or phage vector gene to express gX.

[0007]

However, gX obtained by adopting the genetic engineering method as described above is contaminated with a contaminating protein as a bacterial component in the expression with a bacterial plasmid, and gX is expressed with a viral vector. In the case of No. 3, nonspecific reaction was observed due to contamination with contaminating protein as a component of the cells to be grown, and if it was left as it was, the highly reliable measurement could not be carried out by the ELISA method, and the above-mentioned purification should be carried out.

[0008] Further, even when the conventional identification antigen gX extracted from the virus culture supernatant is used, contaminating proteins are contaminated, and as a matter of course, nonspecific reaction is not observed, and complicated purification is required. is there.

As described above, any of the conventional identification antigens gX requires a complicated purification step, resulting in a high production cost and inefficient production.

Therefore, the present invention solves the above-mentioned problems, and the antigen for identifying an Aujeszky's disease virus-infected antibody has a high specificity in the reaction with the gX antibody without complicated purification and is reliable by the ELISA method. It is an object of the present invention to make it possible to perform sufficiently high measurement, that is, to improve the production efficiency of this identifying antigen by a simple purification method and to provide it at low cost.

[0011]

In order to solve the above problems, in the present invention, a fusion protein of the membrane glycoprotein gX of porcine herpes 1 virus and glutathione S transferase is used for identifying an Aujeszky's disease virus-infected antibody. It constituted the antigen.

The above-mentioned antigen for identification is obtained by incorporating a gene encoding a membrane glycoprotein gX of porcine herpes 1 virus and glutathione S transferase into a plasmid vector to express a fusion protein, and purifying the obtained fusion protein by glutathione column chromatography. It can be manufactured by

Further, a gene encoding the membrane glycoprotein gX of porcine herpes 1 virus and glutathione S transferase was inserted into a plasmid vector to express a fusion protein, and the obtained fusion protein was purified by glutathione column chromatography and purified. Using the fusion protein thus prepared as an identification antigen, an Aujeszky's disease virus-infected antibody was detected by an ELISA method.

To obtain a membrane glycoprotein gX (hereinafter abbreviated as gX) which is a raw material of the fusion protein of the present invention, first, A
A gene encoding gX is cloned from a cDNA library prepared by inoculating sensitive cells with DV and extracting and purifying the collected infected cells by an immunoscreening method using gX immune rabbit serum.

The ADV used in the present invention is not particularly limited to one isolated from a specific strain, and other known wild strains such as wild strains isolated from infected mammals such as pigs, etc.
Iwate stock can also be used.

Glutathione S transferase and gX
Fusion protein, for example, a gene encoding glutathione S transferase production is incorporated into a bacterial plasmid vector, and a recombinant plasmid in which the gX gene is ligated downstream thereof is constructed, which is further transformed into Escherichia coli (Escherichia coli) or the like. Can be infected with and expressed. In this case, gX is fused to the C-terminus of glutathione S transferase.

The fusion protein expressed in this manner is used as a glutathione column (affinity) as follows.
Purified by chromatography. That is, glutathione S-transferase reacts with its substrate, glutathione, in a highly specific manner, and thus is adsorbed on a gel filtration column to which a substrate, glutathione is added. Next, wash the column with a buffer solution at or near the pH optimum for the enzyme, wash away the non-adsorbed components, and then use the buffer solution to which glutathione has been added to flush out the adsorbed fusion protein and purify it. can do.

The fusion protein thus obtained was used as an identifying antigen to detect an antibody against gX in the serum by the ELISA method. , It is possible to accurately distinguish a naturally infected individual from serum (including an antibody by the membrane glycoprotein gX).

[0019]

【Example】

(Cloning of gX gene) ADV Iwate strain infection RK-
Based on mRNA extracted and purified from 13 cells according to a conventional method, M-MuLV was prepared using XhoI linker primer.
CDNA was synthesized by RT RNA-dependent DNA polymerase, DNA-polymerase I, and inserted into the phage vector λZAP. This is the phagemid vector (p
Bluescript) library and subjected to colony screening with E. coli.

For colony screening, LB
/ Allow colonies to form on a nitrocellulose filter placed on an ampicillin plate and culture overnight, then IPT
Protein expression was induced by G.

Then, a replica filter was prepared from the above-mentioned filter, treated with chloroform and lysozyme, reacted with anti-gX antibody, and colored by the system of POD and DAB to obtain a positive clone.

(Incorporation of gX Gene into E. coli Expression Vector) The step of incorporating the gX gene into an E. coli expression vector will be described below with reference to FIG.

As schematically shown in the step on the right side of FIG. 1, plasmid DNA was extracted from the positive clone (pBluescript-gX), Eco RI, Kpn.
The cDNA was cut out with I and the ends were blunted with T4 DNA polymerase. After separating it by agarose gel electrophoresis, a pBamHI linker (12mer) was ligated and inserted into the BamHI site of an E. coli expression vector (AMRAD: pGEX-2T).

Here, the commercially available Escherichia coli expression vector (AMRAD: pGEX-2T) shown on the left side of FIG.
It is produced by the method described in Japanese Patent Publication No. 1-503441, and the glutathione S-transferase derived from Schistosoma japonicum is incorporated into a bacterial plasmid.

(Expression, collection and purification of fusion protein) gX
Escherichia coli expression vector incorporating the gene
After transforming into E. coli and culturing the E. coli at 37 ° C. until the OD ₆₀₀ reaches 0.6 to 1.0, IPTG
Is added to 0.1 mM, and further at 37 ° C. for 3 to
Cultured for 5 hours.

This culture solution was centrifuged at 3000 rpm for 10 minutes, and the resulting precipitate was diluted with 1% Triton X.
After suspending in phosphate buffered saline (PBS) containing -100, sonication was carried out, and 3000 rpm, 1 again.
Centrifugation was carried out under the condition of 0 minutes. The centrifugation supernatant was applied to a glutathione Sepharose 4B column (Pharmacia: Glutathione Sepharose 4B), non-adsorbed components were washed away with PBS, and then adsorbed with 0.1 M TrisHCl buffer (pH 8.0) containing 15 mM glutathione. The components were eluted to obtain a fusion protein of gX and glutathione S transferase (hereinafter abbreviated as gX-GST).

The obtained purified fusion protein fractions were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting, and the results are shown in FIG.

As is clear from the results shown in FIG. 2, the gX fusion protein was mainly a fusion protein of 80 Kd, and all four types were detected. These were derived from all nucleotide sequences (origs).
in) was a expressed protein, and the partially read protein had a small molecular weight. These several fusion proteins
It had the ability to detect gX antibodies.

(Detection of Antibody in Serum of Infected Pig by ELISA) 0.5 μ of gX-GST purified as described above was used.
It was prepared with 0.05 M carbonate buffer so as to be g / 50 μl / well and used as a solid phase. Add 300 μl / well of phosphate buffered saline containing 1% bovine serum albumin,
Incubation was at 2 ° C. for 2 hours. Next, 0.05% T
Phosphate buffered saline containing Tween 20 (T-PBS)
After washing the plate with 100 μl of diluted pig serum
In addition, the mixture was incubated at 25 ° C for 1 hour. T-PBS
After washing with 100 μl, 100 μl of peroxidase-labeled anti-porcine IgG diluted with the same buffer was added and incubated at 25 ° C. for 1 hour. After the reaction, the plate was washed with T-PBS, 100 μl of the substrate (ABTS) solution was added, and the mixture was incubated at 25 ° C. for 1 hour. And this reaction is the same amount of 0.1N
After stopping by adding NaOH, the absorbance (OD value) at 492 nm was measured using a microplate auto reader. In the above detection experiment, 46 pigs naturally infected with ADV and ADV vaccine (without gX as a marker)
Twenty-nine inoculated pigs were used as samples, and the results are shown in FIG.

As is clear from the results of FIG. 3, the neutralizing antibody titer and the ELISA O. D. There is a correlation with the value,
It is possible to detect an antibody in the serum of infected pigs, and it is understood that this fusion protein of gX and glutathione S transferase can be used as an antigen for identifying an Aujeszky's disease virus-infected antibody.

(Confirmation Experiment for Accuracy of Antibody Detection by ELISA of Vaccinated Pig and Serum of ADV-Infected Pig) Confirmation Experiment for Vaccinated Pig: Three 3-week-old pigs were used, and the vaccine was ear-rooted twice at 3-week intervals. It was inoculated intramuscularly. Serum to be tested is, before vaccination, 3 weeks after the first inoculation, 2, 6, 8, 11, 14, 1 after the second inoculation.
Neutralizing antibody titer was measured and EL was measured using serum after 7 weeks.
Infection discrimination by ISA was evaluated and these results are shown in Table 1.
It was shown to.

For the evaluation of ELISA, the test serum was diluted 100-fold, and the color of POD-labeled anti-porcine IgG antibody and ABTS (substrate) was used to determine the reactivity of ADV field-negative pig serum based on the reactivity. When the difference in OD value between the positive antigen and the negative antigen was less than 0.3, it was defined as negative, and when it was more than that, it was defined as positive.

The vaccine used in the above experiment was carried out by using the cell line PK-15 derived from ADV-infected pig kidney as Tr.
The supernatant solubilized with iton X-100 and ultracentrifuged at 100,000 × g was applied to an anion exchanger and a heparint yopearl column, washed with PBS, and then washed with 2M NaC.
It is eluted with PBS (pH 7.4) containing 1 g, dialyzed against PBS, inactivated with formalin and mixed with an oil adjuvant (Sepic: ISA70) to give gII.
A vaccine containing I 60% or more and not containing gX was used.

[0034]

[Table 1]

As is clear from the results shown in Table 1, a favorable increase in neutralizing antibody titer was observed in all pigs, which was the highest. The serum 14 weeks after the second vaccination of No. 1 showed a 700-fold or more value, but the E against the gX antigen was increased.
No antibody was detected by LISA at any time.

Confirmation experiment on ADV-infected pigs: 3 pigs were used, each having an ADV of 10 ^6.0 TCID50
Yamagata S81 strain was administered intranasally. As the test serum,
Neutralizing antibody titers were measured using sera before infection, 2 weeks after infection, 1, 2, 3, 4, 5 and 6 months after infection, and ELI
The infection distinguishability by SA was evaluated in exactly the same manner as described above, and these results are shown in Table 2.

[0037]

[Table 2]

As is clear from the results shown in Table 2, an increase in neutralizing antibody titer was observed 2 weeks after infection in all pigs, and the value was almost maintained even 6 months later. Also in the ELISA against the gX antigen, antibodies were detected in all sera up to 6 months after infection.

[0039]

As described above, according to the present invention, since the antigen for identifying an Aujeszky's disease virus-infected antibody is composed of a fusion protein of the membrane glycoprotein gX and glutathione S transferase, the specificity of the reaction with the gX antibody is high. Highly ELISA
It becomes a discriminating antigen that can be measured with sufficiently high reliability by the method, and this fusion protein is expressed by incorporating a membrane glycoprotein gX and a predetermined enzyme into a plasmid vector, and the obtained fusion protein is analyzed by glutathione column chromatography. There is an advantage that it can be produced by a relatively simple method of purification, the production efficiency of this product can be improved, and the product can be provided at low cost.

[Brief description of drawings]

FIG. 1 is a schematic diagram illustrating a method for producing an antigen for identifying an Aujeszky's disease virus-infected antibody.

FIG. 2 is an immunoblotting image of the purified fusion protein fraction against anti-gX antibody.

FIG. 3 ELISA O. D. Chart showing the relationship between values and neutralizing antibody titers

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12P 21/02 C12R 1:19)

Claims (3)

[Claims] 1. A membrane glycoprotein gX of porcine herpes 1 virus
An antigen for identifying an Aujeszky's disease virus-infecting antibody, which comprises a fusion protein of: and a glutathione S transferase. 2. Membrane glycoprotein gX of porcine herpes 1 virus
A method for producing an antigen for identifying an Aujeszky's disease virus infectious antibody, which comprises incorporating a gene encoding a glutathione S transferase into a plasmid vector to express a fusion protein, and purifying the obtained fusion protein by glutathione column chromatography. 3. Membrane glycoprotein gX of porcine herpes 1 virus
The fusion protein was expressed by incorporating the gene encoding the enzyme and glutathione S transferase into a plasmid vector, the obtained fusion protein was purified by glutathione column chromatography, and the purified fusion protein was used as an identifying antigen for ELISA. A method for detecting an Aujeszky's disease virus-infected antibody, which comprises detecting a key disease virus-infected antibody.