TWI399382B - Mutant ibdv vp2 protein and use thereof - Google Patents

Description

Mutant chicken infectious bursal disease virus VP2 protein and use thereof

The present invention relates to a mutant chicken infectious bursal disease virus (IBDV) VP2 protein produced by site-directed mutagenesis, which has better yield and immunogenicity than the wild type. The invention further relates to the use of such mutant VP2 proteins for IBDV testing and for the manufacture of vaccines.

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease (IBD) in chickens. The epidemic began in Gumboro, 1962, and is also known as Gumboro. It is a disease of Gan Paulo (Nick et al., J. Virol. 18:227-234, 1976). The infectious bursal sac belongs to the genus Avirerinavirus of the Birnaviridae virus family in the virological classification. It has no enveloping envelope in the capsid of the virus, and the virus form is a right-handed icosahedron of T=13. (icosahedral symmetry), particle size of about 61-65 nm (Ozel and Gelderblom, Semin Cell Biol 5:83-93, 1985), the monolayer outer sheath structure consists of the outer VP2 protein and the VP3 protein located on the inner side. In addition, there are a small amount of protein VP1, VP4 and non-structural protein VP5 on the inner side (Mundt et al., J. Virol. 71: 5647-5651, 1997). There are two double-stranded RNA fragments inside (Kibenge et al., J. Gen. Virol. 69:1757-1775, 1988). The virus is mainly divided into two serotypes, of which serum type I mainly infects young chickens; serum type II is mainly Infected turkeys are not pathogenic (Wyeth and Cullen, Vet Rec 102: 362-363, 1978; Okoye and Uzoukwu, Avian Dis 25: 1034-1038, 1981).

When a young chicken is infected with infectious bursal disease virus, the virus destroys the lymphocytes in the chicken's bursal tissue, causing damage to the group and reducing the immunity of the chicken (Fahey et al., J. Gen.Virol . 70: 1473-1481, 1989), so other infectious diseases, such as Newcastle disease (ND) and Marek's disease (MD), cause serious economic losses in the chicken industry. (Wyeth and Cullen, 1978, as mentioned above). After being infected with infectious bursal disease virus, chickens will only have symptoms such as squatting, lack of energy, loose feathers, poor color, poor appetite, weight loss, etc., and their viral proliferation is mainly in the Fahrenheit sac, so the virus-infected Fahrenheit often There will be inflammation, swelling and even bleeding. In the current method for controlling infectious bursal disease virus, in addition to vaccinating strains to immunize chickens, it is also possible to immunize hens by dead or acclimated live virus strains, and then The antibodies produced protect chickens, but live vaccines may still destroy Fahrenheit cells and reduce immune responses, which may have adverse effects (Macreadie et al., Vaccine 8: 549-552, 1990).

The genome of infectious bursal disease virus contains two double-stranded RNA fragments, which are large fragment A (segment A) and small fragment B (segment B), wherein fragment A contains two open reading frames (open reading frame, ORF), the larger ORF A1 (about 3.4 kb) will translate polypotein with a molecular weight of about 110 kDa, which will result in independent pVP2 protein (52 kDa) and VP3 protein (32 kDa). VP4 protein (28 kDa) (Hudson et al, Nucleic Acids Res 14: 5001-5012, 1986). The small fragment, called segment B, is 2.9 kb in length and can translate a protein product VP1 of approximately 90 kDa. VP1 is a double-stranded RNA-dependent RNA polymerase (Morgan et al., Virology 163: 240-242, 1988), which binds to the VP3 protein to form a VP1-VP3 complex, which assists in the addition of VP1 protein to the assembled viral particles during viral replication. The process plays an important role (Maraver et al., J. Virol. 77: 2459-2468, 2003).

pVP2 is a precursor of VP2. After proper protein cleavage, it becomes a VP2 with a molecular weight of about 42 kDa, which is a mature VP2 (VP2-441) protein. The VP2 protein content accounts for about 51% of the whole virus composition. It is one of the major structural proteins of infectious bursal disease virus and is also the main host protective antigen, which can induce the host to produce a virus-negative antibody to protect chickens (Becht et al., J. Gen. Virol. 69) : 631-640, 1988; Azad et al, Vaccine 9: 715-722, 1991; and Wang et al, Biotechnol Bioeng 67: 104-111, 2000). According to the results of the past few years, the neutralization antibody epitopes of IBDV can be divided into two types, the first one is structurally dependent; the second is non-structural dependent, the first one is structurally dependent. The study also used a single antibody and phage display to find the conformational epitope associated with the amino acid position 206-350 in the highly variable region of the VP2 protein (Cui et al. J Virol Methods 114: 109-12, 2003; Heine et al, J. Gen. Virol. 72: 1835-43, 1991; Yamaguchi et al, Arch Virol 141:1493-507, 1996). The second non-structural-dependent type is also found in VP2. The two sequences are amino acid positions 197-209 and 329-337, respectively. They are linear epitopes with neutralizing ability, and there is no special stereo. A spatial configuration in which 197-207 is at the N-terminus of the highly variable region and overlaps 4-6 amino acids with a structurally dependent epitope formed by amino acids 206-305 (Wang et al., Viral Immunol 18: 549-57, 2005).

The present invention first uses a point mutation to replace His-249 and His-253 on VP2-441 subviral particles, and explores its effect on the assembly and immunogenicity of VP2 subviral particles. The present invention discloses that point mutations of His249 and His253 do not affect the formation of secondary virus particles, and can enhance the immunogenicity of the original VP2 subviral particles, and have a 100% protective effect on chickens, It can be effectively applied to the diagnosis and vaccine control of infectious bursal disease virus.

The objects and advantages of the invention will be set forth in part or in the description.

It is an object of the present invention to provide a chicken infectious bursal disease virus (IBDV) VP2 protein variant in which the histidine acid at position 249 and/or 253 of the mature VP2 (VP2-441) protein amino acid is replaced Other amino acids, wherein the VP2 protein variant retains the ability to assemble into Subviral Particles (SVP). In one embodiment, the histidine at position 249 and/or 253 of the amino acid is replaced with alanine.

Another object of the present invention is to provide a diagnostic reagent for detecting the presence of chicken infectious bursal disease virus (IBDV) antibody, which comprises chicken infectious bursal disease virus (IBDV) VP2 protein variant H249A, H253A or H249. 253A, and detection buffer and secondary antibody (anti-chicken serum antibody).

According to still another object of the present invention, a vaccine for protecting a chicken against infectious bursal disease virus (IBDV) comprising an amino acid of a mature chicken infectious bursal disease virus VP2 (VP2-441) protein The histidine acid at position 249 and/or 253 is replaced with a VP2 protein variant of the other amino acid, and a veterinary acceptable adjuvant or excipient. In a specific aspect, the VP2 The protein system is selected from one of H249A, H253A and H249.253A.

Other features of the invention will become apparent from the following detailed description of the embodiments.

The invention will be described in detail with particular embodiments. These specific examples are provided by way of illustration of the invention and are not intended to limit the invention. The present invention is intended to embrace these and other modifications and variations within the scope and spirit of the invention.

Example Example 1. Expression of point mutation protein of VP2-441 in an insect baculovirus system

The Escherichia coli expressing the DNA encoding the VP2-441 site-directed mutagen protein was constructed in our laboratory to express the plastids TOPO-VP2-441 H249A, TOPO-VP2-441 H253A and TOPO-VP2-441 H249.253A (these utilize Constructing a part of VP2 gene in pCR from Taiwan traditional strain p3009 T7/CT-TOPO The plastid TOPO-1323 is used as a template for PCR, and a forward and reverse primer with partial sequence overlap is designed, and the nucleotide sequence at the position of the amino acid to be mutated is replaced with a side chain having no functional group and no positive and negative charges. Alanine, which does not affect protein folding, is substituted for histidine (Histidine) at amino acid positions 249 and 253, and baculovirus transfer plastid pBlueBac4 (purchased from Invitrogen) to limit enzyme NheI After cutting with EcoRI , the target gene and the baculovirus transfer plastid pBlueBac4 backbone were purified by TAE agarose gel electrophoresis, and purified by PCR clean-up kit (Genemark Technology Co., Ltd., Taiwan). The cells were recovered and added to T4 DNA ligase (Promega) or the like, and subjected to ligation at 16 °C. Co-transfection of Sf - 9 with baculovirus DNA, homologous recombination to obtain recombinant baculovirus, and various mutated recombinant baculovirus H249A by virus plaque assay , H253A and H249.253A. Then, the recombinant virus was infected with Hi5 cells with higher expression amount by MOI 10, and when the cell survival rate was 65-75%, the cell pellet after infection was collected, and the soluble protein was centrifuged to obtain 12.5% SDS-polymerization. Acrylamine gel electrophoresis was then stained with Coomassie brilliant blue to determine whether the recombinant protein was correctly expressed. The results showed that the protein of each point mutation was indeed and unmutated with VP2-441 protein on a 12.5% SDS-polyacrylamide gel. Consistent (as shown in Figure 1), the yield of mutant protein was roughly estimated to be more than two times higher than that of the unmutated VP2-441 protein.

Then, it is purified by sucrose gradient and purified by Sucrose gradient centrifugation. The buoyant density is about 1.19 g/cm ³ , and the buoyant density of each point-mutated protein is the same after purification analysis in the above manner. The protein does not change its physical properties due to point mutations. Further, when observed under a transmission electron microscope (Transmission Electron Microscopy, JEOL JEM 1200EX-2, Japan), secondary virus particles having a particle diameter of about 23 to 25 nm were observed (see Fig. 2 for the results).

These results indicate that the VP2 recombinant protein after point mutation is identical to the physical property analysis of the unmutated wild-type VP2-441 protein regardless of the molecular weight of the protein, physical properties and microscopic observation. It was also demonstrated that the point mutation of either H249 or H253 did not affect the ability of the VP2 subviral particles expressed by the insect baculovirus to assemble themselves.

Example 2. Adsorption analysis of VP2 recombinant protein variants and monoclonal antibodies

In order to confirm the mutation of the two positions of the amino acid H249 and H253 of the VP2 protein, the immunogenicity of the subviral particles was changed, and the monoclonal antibody No. 1 (Mab SVP-1) was used. And neutralizing monoclonal antibody No. 4 (Mab SVP-4) (monoclonal anti-VP2 antibody is an antigen of IBDV VP2-441 expressed by the insect baculovirus expression system as an antigen, and is mediated by Zhuoshuixi Biotechnology The company (Taiwan, Taoyuan) screened for hybridoma cells to obtain several different monoclonal antibodies, and in this experiment, Mab SVP-1, Mab SVP-4) and recombinant protein were used for enzymes. Linkage immunoassay (ELISA). The purified and quantified recombinant protein was added to a 96-well plate (EIA/RIA strip plate, Costar), 0.5 nM was added to each well, and 200 μl of carbonate-bicarbonate buffer (1.56 mM Na ₂ CO ₃ , 1.36 was added. mM NaHCO ₃ , pH 9.6), was subjected to a protein adsorption reaction at 4 ° C for 16 hours. Thereafter, blocking was performed with 5% skim milk at 37 ° C for 1 hour, followed by washing 3 times with PBS-T (0.1% Tween 20 in PBS), and then 100 μl of primary antibody was added to each well (diluted multiples from 4000x~128000x, primary antibody was monoclonal antibody No.1 and No.4 respectively. After reacting for 2 hours at 4°C, it was also washed 3 times with PBS-T, and then added 200 μl of secondary antibody (dilution factor 5000x, secondary antibody) Goat anti-mouse Conjugated Horseradish Peroxidase, Jackson) After 2 hours of reaction at 4 ° C, it was also washed 3 times with PBS-T, then 100 μl of ABTS was added per well. (ABTS Peroxidase Substrate, KPL, USA) After 10 minutes of color reaction, the absorbance at OD 405 nm was read by an enzyme immunoassay analyzer (Dynex Technologies, Chantilly, Virginia, USA).

The results shown in Fig. 3 show that the signals recognized by the amino acid H249 or H253 mutated to alanine (Ala) via the monoclonal antibodies No. 1 and No. 4 are higher than the unmutated VP2-441. The part of the monoclonal antibody No. 1 was about 2 times higher, and the part of the single antibody No. 4 was about 1.4 times higher. As a result of this experiment, the binding of a neutralizing monoclonal antibody or a non-neutralizing monoclonal antibody to a recombinant protein of H249 or H253 point mutation is shown. The force is higher than the unmutated VP2-441, so the recombinant protein deduced to be H249 or H253 point mutation has the ability to induce neutralizing antibodies in chickens more than wild type VP2-441, and has a more sensitive detection of anti-IBDV. The ability of the VP2 antibody can be effectively used to detect the presence of anti-IBDV antibodies in chicken serum.

Example 3. Immunogenicity analysis of recombinant subviral particles

To further confirm that the HVR and H253 point mutations of the secondary virus particles can induce protective neutralizing antibodies in chickens, and are more able to induce the ability of chickens to produce neutralizing antibodies than unmutated VP2-441 virus particles. Quantitative VP2-441, VP2-441-H249A, VP2-441-H253A, VP2-441-H249.253A were used as immunizing antigens, and PBS was used as a negative control group, and two weeks old were absent. Specific pathogen free (SPF) chicks, each of which was given 10 grams of VP2 antigen. After four weeks of immunization, the recombinant protein was induced to induce specific antibody production against VP2 (VP2). -specific antibody), the evaluation method is to determine the specific antibody producing identifiable VP2 virus particles in the blood of the immunized chicken by AC-ELISA (antigen capture ELISA). The purified secondary virus particles (VP2-441 SVP) were adsorbed on a 96-well plate, 0.1 μg per well, and allowed to act at 4 ° C for 8 hours, and then added with 5% skim milk at 60 ° C for 60 minutes. The PBS-T buffer solution was washed 5 times, and the serially diluted serum was added thereto, and reacted at 4 ° C for 4 hours, followed by washing 5 times with PBS-T. HRP-conjugated goat anti-chicken IgG (H+L) diluted 5000-fold was added, and reacted at 4 ° C for 4 hours, followed by washing 5 times with PBS-T. Finally add 100 μl of ABTS to each hole (ABTS Peroxidase Substrate, KPL, USA) After 10 minutes of color reaction, the absorbance at OD 405 nm was read by an enzyme immunoassay analyzer (Dynex Technologies, Chantilly, Virginia, USA). The valence of VP2-specific IgG in serum is determined by the absorption of the serum samples of each serial dilution, and the absorbance at 405 nm is taken to be the closest to the background absorbance and 0.1 unit higher than the background absorbance. The reciprocal of the serum dilution factor is the VP2-specific IgG titer of the serum. The background group is not adsorbing secondary virus particles.

According to the AC-ELISA results of Figure 4, the average titer of VP2 antibody in the blood of pre-immune-free young chickens was 6, virgin virgin VP2-441, VP2-441-H249A, VP2-441- The average titers of VP2 antibodies from H253A and VP2-441-H249.253A from the first week to the fourth week after immunization gradually increased with the increase of the number of immunization weeks, and the fourth period after immunization Zhou's serum can measure the average price of immune VP2-441 is 3200, the average price of VP2-441-H249A is 2987, the average price of VP2-441-H253A is 10240, and the average of VP2-441-H249.253A The price is 3840. The average force of VP2 antibody was applied to PBS buffer solution. The price is 90. From the results of AC-ELISA, it was found that the VP2-specific antibody valence in the serum was similar to that of the unmutated VP2-441 in the chickens immunized with the sub-viral particles other than VP2-441-H253A. The force price of VP2-441-H253A was 3.2 times higher than that of VP2-441; it was 3.4 times higher than VP2-441-H249A; it was 2.7 times higher than VP2-441-H249.253A.

To further determine the protective effect of specific antibodies to VP2 subviral particles on chickens, this study used a neutralizing antibody assay to determine the neutralizing antibody titer in the blood of immunized chickens. Serum collected weekly after immunization of chickens was not activated for 30 minutes prior to 56 °C. The growth medium was added to a 96-well tissue culture dish, 25 μl was added to each well, and 25 μl of the non-activated serum was mixed with GM medium for 2-fold serial dilution, and serial dilutions were made to 4096-fold dilution. Then, 50 μl of 200TCID ₅₀ (tissue cell infection dose) of IBDV virus solution (P3009 strain) was added to each well, and it was allowed to act for 1 hour at 37 ° C to effect serum and IBDV, and finally 50 μl was added to each well ( 2 x 10 ⁴ cell/well) chicken embryo fibroblasts were cultured at 39 ° C for 72 hours, during which time a continuous observation of cytopathic effects occurred. 72 hours after infection, the supernatant was removed and stained with crystal violet. If the cells survived, they can be stained with the dye. By this method, the virus-neutralized serum antibody is neutralized in the serum to maintain the highest serum survival. The dilution factor is the neutralizing antibody titer. The results are shown in Figure 5.

The results showed that in the second week to the fourth week after immunization, the serum obtained by applying VP2-441, VP2-441-H249A, VP2-441-H253A and VP2-441-H249.253A antigens was neutralized. There is a tendency to gradually increase over time. In contrast, in the control group administered with PBS, the neutralizing antibody had a titer of less than 2. From the above results, it is speculated that the increase in the VP2 anisotropy antibody should be correlated with the neutralizing antibody titer. And in the fourth week, the neutralizing antibody titer of VP2-441-H249A was about 1792, VP2-441H253A was about 3072, VP2-441 was about 1536, and VP2-441-H249.253A was about 2048, of which VP2-441H253A was more than VP2- 441 is about 2 times higher. From the above two analysis results, the VP2-specific antibody and the neutralizing antibody produced by the chicken immunized with VP2-441-H253A were higher than the VP2-441.

Four weeks after immunization, we conducted a chicken challenge test. Each chicken was orally infected with a dose of 100 LD ₅₀ vvIBDV virus. The symptoms and mortality of the chickens were observed every day. After 3 days of infection, the chickens were analyzed. To assess the clinical symptoms of the chicken after challenge and the proliferation of the virus in the Fahrenheit capsule (see Figure 6 for the results). First, four groups of chickens were administered VP2-441, VP2-441-H249A, VP2-441-H253A and VP2-441-H249.253A. After the challenge, there was no infection and no chicken died. No virus was detected (as shown in Figure 6) and the protection was 100%. However, in the control group administered with PBS, 2 chickens died on the third day after infection, while the other one suffered from depression, poor appetite, tarnished feathers, erected, yellowish white and green squats, and unable to stand. Significant symptoms such as constant sorrow, sleepiness, and detection of viral antigens in the Fahrenheit sac, indicating that the PBS that is administered does not induce effective antibodies, and the effect of protecting the chickens is achieved.

From the above results, it is inferred that the point mutation of His249 and His253 does not affect the formation of subviral particles, and in terms of immunogenicity, the point mutation of His249 and His253 causes the adsorption capacity of monoclonal antibodies No.1 and No.4 to be changed. High; and H253A can induce chickens to produce higher neutralizing antibody titers than other point mutation proteins or the original VP2-441 protein; and whether the VP2-441 protein of the mutant His249 or His253 is immunized, there are 100 for the chicken. The protection effect of % can be effectively applied to the diagnosis and vaccine prevention of infectious bursal disease virus.

Figure 1 shows the performance of recombinant VP2 protein variants in insect cells. After the recombinant virus infects the insect cell Hi-5, the cell pellet is collected, and the cells are broken by an ultrasonic oscillator. After centrifugation, the supernatant (lysate) is diluted 100 times, and 1 and 2 are taken separately for 20 μl and 30 μl. 12.5% SDS-polyacrylamide gel electrophoresis was then stained with Coomassie blue.

Figure 2 shows the results of observing the secondary virus particles by a transmission electron microscope. The protein purified by Sucrose gradient centrifugation was concentrated in a 100 kDa concentrated membrane, and the sample was dropped on a copper mesh and negatively stained with 2% uranyl acetate using a transmission electron microscope (JEOLJEM 1200 EX-2). For observation, Figure A is VP2-441; Figure B is H249A; Figure C is H253A; and Figure D is H249.253A. From the figure, it can be observed that the appearance of the icosahedral subviral particle structure is about 23~25 nm and the scale is 50 nm.

Figure 3 shows the adsorption analysis of recombinant proteins and monoclonal antibodies. The non-neutralized monoclonal antibody No.1 and the neutralizing monoclonal antibody No.4 and the VP2-441 protein without point mutation and the recombinant protein after point mutation were subjected to adsorption experiments, and BSA was a negative control group. Figure A shows the adsorption of monoclonal antibody No. 1 with different dilution factors; Figure B shows the adsorption of monoclonal antibody No. 4 with different dilution factors; Figure C shows the experimental group diluted 16000 times with No. 1 antibody and No. 4 antibody.

Figure 4 shows the antibodies produced by chickens immunized with various VP2-441 proteins. should. Each group was immunized with VP2-441 virus particles or subviral particles after VP2-441 point mutation, and the negative control group was immunized PBS. Serum samples were collected weekly before immunization and after immunization for a total of four weeks. The VP2-specific antibody titer was determined by adsorbing the subviral particle (VP2-441) antigen on a 96-well plate and measuring by ELISA.

Figure 5 shows that the chickens were immunized with various VP2-441 proteins to produce a neutralizing antibody response. Each group was immunized with VP2-441 virus particles or subviral particles after VP2-441 point mutation, and the negative control group was immunized PBS. Serum samples were collected weekly before immunization and after immunization for a total of four weeks. The titer of the neutralizing antibody was determined by the dilution of the serum sequence and the addition of IBDV P3009 for 1 hour and then the addition of DF-1 cells for 69 hours. The highest dilution factor of the cytopathic effect (CPE) was not observed in the DF-1 cells.

Figure 6 shows the presence of IBDV virus in Fahrenheit fluid after challenge using Western blotting. From left to right are marker and Lane 1 is VP2-441 internal marker; Lane 2 is vvIBDV virus; Lane 3 is only for injection of PBS without antigen; Lane 4 is for VP2-441 as antigen; Lane 5 is for application H249A is an antigen; Lane 6 is an application of H253A as an antigen; and Lane 7 is an application of H249.253A as an antigen. The primary antibody used was a multi-drug antibody α-VP2 CNC, and the secondary antibody was an AP-conjugate goat anti-rabbit antibody.

<110> National Chung Hsing University

<120> ELISA diagnostic kit for chicken infectious bursal disease (IBD) and method of use thereof

<160> 1

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 441

<212> PRT

<213> Infectious Bursal Disease Virus

<400> 1

Claims (8)

A chicken infectious bursal disease virus (IBDV) VP2 protein variant in which the histidine at position 249 and/or 253 of the mature VP2 (VP2-441) protein amino acid is replaced with another amino acid, wherein The VP2 protein variant still retains the ability to assemble into a secondary virus particle (SVP), wherein the mature amino acid sequence of the mature VP2 (VP2-441) protein is set forth in SEQ NO. The VP2 protein variant according to claim 1, wherein the histidine at position 249 and/or 253 of the amino acid is substituted with alanine. The VP2 protein variant according to item 2 of the patent application is selected from the group consisting of the mutant proteins VP2-H253A, VP2-H249A and VP2-H249.253A. A diagnostic reagent for detecting the presence of an infectious bursal disease virus (IBDV) antibody comprising chicken infectious bursal disease virus (IBDV) VP2 protein variant H249A, H253A or H249.253A, and a buffer for detection And a secondary antibody (anti-chicken serum antibody), wherein the amino acid sequence of the VP2 (VP2-441) protein is as shown in SEQ NO. The diagnostic reagent according to claim 4, wherein the VP2 protein variant system is selected from the group consisting of the mutant proteins VP2-H253A, VP2-H249A and/or VP2-H249.253A. A vaccine for protecting chicken against infectious bursal disease virus (IBDV) comprising histidine at position 249 and/or 253 of mature chicken infectious bursal disease virus VP2 protein amino acid a VP2 protein variant of an amino acid, and a veterinary acceptable adjuvant or excipient, wherein the amino acid sequence of the mature chicken infectious bursal disease virus VP2 (VP2-441) protein is SEQ NO. 1 is shown. A vaccine according to claim 6 wherein the histidine of the amino acid position 249 and/or 253 located in the VP2 protein variant is alanine. A vaccine according to claim 6 wherein the VP2 protein is a system of VP2-H249A, VP2-H253A or VP2-H249.253A.