TWI233947B - Method for preparing IBDV infectious sub-unit and vaccine and diagnostic reagent made therefrom - Google Patents

Abstract

The present invention relates to a method for preparing a recombinant infectious bursal disease virus (IBDV) structural protein, which comprises using a VP2-gene-carrying recombinant nuclear polyhedrosis virus (NPV) to inject Lepidoptera or Diptera larva for massively expressing the recombinant protein. According to the present invention, the C-terminal of the rVP2H recombinant protein is fused with six histidines and can be purified by an immobilized metal ion affinity analysis method. The vVP2H prepared according to the invention can be assembled into pseudo-virus particles. The present invention also relates to a vaccine composition and a diagnostic reagent containing the rVP2H pseudo-virus particles, as well as the use of the invented recombinant protein in prevention of infection of IBDV and detection of the presence of the virus.

Description

1233947 V. Description of the invention (1) [Background of the invention] Infectious bursal disease virus (IBDV) is a pathogen that causes infectious bursal disease in chickens. The epidemic started in Gambaor County in the United States in 1962. It was found that the disease is also known as Gan Paul's disease, because it is highly pathogenic and lethal to chickens, and has been a major economic problem for agriculture and livestock (Dob os et al., J Virol 32: 593-605 (1 979) and Nick et al., J Virol 1 8: 227-234 (1 976)). Chickens infected with infectious Fahrenheit virus only have symptoms such as diarrhea, lack of energy, loose feathers, poor appetite, and weight loss. The infectious Fahrenheit bursal virus is mainly parasitic in the infected chicken's Fahrenheit sac. The infected Fahrenheit sac may shrink or become swollen or even bleed. Since Fahrenheit sac tissue is a mature organ of chicken B lymphocytes, damage to this organ can result in the chicken's immune system being impaired (Fahey et al., J Gen Virol 70: 1473-1481 (1989)), causing other infectious diseases. Causes such as Newcastle disease and Marek's disease. In 72 years, Newcastle disease caused a pandemic in the province, causing millions of chickens to die. 'Pathological analysis revealed that 80% of chickens infected with Newcastle disease were mixed with infectious Fahrenheit virus. A serum survey of chickens in the province after 1980 found that the infectivity rate of infectious F. bursal virus reached over 90%, indicating that the virus is widespread in all parts of the province. Infectious Fahrenheit virus is highly stable in the natural environment, and is highly infectious. Isolating sick birds or disinfecting the environment to control the disease is not effective. At present, the focus of epidemic prevention of infectious Fahrenheit virus is vaccination of hens. Make it difficult for infants to fight against passive immunization with migrating antibodies within 3 weeks of birth

1233947 V. Description of the invention (2) Infectious Fahrenheit virus infection. However, the effectiveness of passive immunization is difficult to control, and chicks will lose the protection of maternal antibodies at about two weeks of age. The only practical way to prevent epidemics is to immunize chickens with vaccines. Immune virus particle-induced protection of chickens is a common method of immunity. For example, infectious Fahrenheit virus is propagated by chicken embryo cells (CEF), and attenuated virus is obtained after domestication of 10-14 generations, and chickens are immunized with the virus. The report pointed out that it is difficult to take oral or subcutaneous injections of attenuated vaccines and then to infect the strains with virulent strains and the mortality rate is zero. Relative to the immunized group, the mortality rate is 43% (Sheeles # A, AvianDis23: 456— 465 (1979)) 〇3 # 0.2% Formalin does not activate infectious Fahrenheit virus. Chickens were immunized with the inactivated virus orally and detected by milk agglutination-inhibition test (L! T㊀s 丨). 3 I gG type of antiviral antibody, while immunized chickens have good protection and protection (see Ho Shi et al., Vaccine 1 3: 245 —252 (1995)). Sexual Fahrenheit virus vaccines have inactivated vaccines and attenuated vaccines. There are many shortcomings in using virus particles as vaccines: 1. Injecting attenuating force to induce the contraction of chicken Fahrenheit capsules reduces the chicken's immune capacity. : The occurrence of strains such as Newcastle disease and Marek's disease; 2. There will be chickens in vivo sudden love the manufacturing process of the virus danger; 3 dead poison vaccine bad, anti-change =: process could easily lead to the loss of a little protein virus particles outside the virus, = must borrow! The culture of chicken embryos, followed by purification, is easy to infect F. bursal virus: f masters the quality of chicken embryos; and 5. domesticated K sac virus easily loses infection efficacy 1

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This kind of unit vaccine that is both female and effective in preventing and treating infected chickens is considered the only way. Vengeance: The published literature has described the use of different expression systems to produce structural proteins that transmit ΓιιΔcystis virus, and is used in the development of subunit vaccines. E. coli is used to express the infectious bursal virus VP2-VP4-VP3 polyprotein ( Azad et al., Vir iogy 149: 19〇1 98 (1 986)), although E. coli replicates quickly, more protein products can be obtained, but an inclusion body is formed, and its inclusion body Prokaryotes do not have post-transcriptional modification of proteins, which prevents them from effectively triggering immunity in chickens.

The epidemic cannot even interact with the monoclonal antibody against infectious bursal disease virus vp2.

Macreadie has used VP2, a yeast that expresses infectious F. bursal virus (002-73), as a subunit vaccine, and has a protective effect (Macreadie et al., Vaccine 8: 549 ~ 552 (1990)). Bayliss et al. Used Recombinant DNA technology, the development of recombinant vaccines to prevent and control infectious Fahrenheit virus, which puts the infectious Fahrenheit virus VP2 gene into the poxvirus (FV) genome, infecting chickens with rFV to make the virus produce VP2 in the chicken body as Antigens that induce protective antibodies in chickens (BayHss et al.,

J Virol 75: 1 1 974-1 1 982 (1 991)), although the death rate of chickens can be reduced, the Fahrenheit sac still shrinks.

Darteil et al. (Virology 21 1: 48 1 -490 (1 995)) constructed the VP2 gene of the infectious Fahrenheit virus 52/57 strain in the RR2 (ribonuclotide reductase gene) and the "gene, Two recombinant turkey vesicle therapy viruses were obtained, named VJJντο 〇1 and

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VHVT0 0 2. However, chickens immunized with vHVTOOl continued to benefit. J Office again ..., the production of neutralizing antibody production, ίΓί * protection is zero; 8 of 9 chickens immunized with high dose ▽ liver ^ 02 The chickens could successfully induce neutralizing antibodies, and the chickens did not die after challenge. However, in low-dose immunization experiments, 10 chicken pupae had lesions in the Fahrenheit sac after challenge. In 2002, Japanese scholar Tsukamoto constructed two types of recombinant VP2 protein from recombinant turkey herpes virus to produce infectious Fahrenheit virus. Both had different degrees of protection. Chickens inoculated with rHVT-pecVP2 had no lesions. The protective power is 100%, and the protective power of chickens inoculated with 1 to 1 '-(: 1110? 2 is 58%

Ben et al., J Virol 76: 5637-5645 (2002)).

Vakharia et al. (J Gen Virol 74: 1201-1206 (1993)) used insect cell / baculovirus system to produce VP2-VP4-VP3 of infectious Fahrenheit virus (GLS variant), and applied it as a subunit vaccine, its protection It was found that 79% of chickens could produce antibodies to protect chickens.

Pitcovsk et al. (Avian Dis 40: 753-76 1 (1 996)) constructed the infectious Fahrenheit virus VP 2 gene in the insect baculovirus expression vector pAcYMl, transfected it into insect baculovirus, and then infected Sf — 9 Cells used to express recombinant VP2 protein (containing 453 amino acids), the test results showed that the minimum effective injection volume of 100% retention rate was 4.5 micrograms.

Dybing et al. Constructed the VP2, VP2 / VP4, and VP2-VP4-VP3 genes of infectious Fahrenheit bursal virus (MD variants) into the insect baculovirus AcNPV (baculovirus), and produced VP2 and VP2-VP4-VP3 from insect cells. Kind of recombinant protein. The chickens injected with these two recombinant proteins all induced high neutralizing antibodies, and they were also resistant to both types of infectious Fahrenheit disease.

Page 7 1233947 V. Explanation of the invention (5) Poison ’However, the chicken scrotum produced yellow swelling, inflammation, and atrophy after infection with the virus. Therefore, the author claims that the vaccine has only partial protection (Avian

Dis 42: 80-91 (1998)). In order to seek a safe, economical and effective vaccine for the prevention and treatment of BDV virus infection, the present invention is devoted to the development of a subunit vaccine of infectious Fahrenheit virus, to construct a vaccine containing infectious Fahrenheit virus (strain p3009). The recombinant insect baculovirus vP3009VP2H of the vp2 gene was orally fed to infected pupae to produce heavy rVP2H to achieve the purpose of preparing rVP2II in large quantities and reduce the cost of subunit vaccine development. [Summary of the Invention] The present invention uses the moth larvae-parasitic larvae as a host to express the infectious Fahrenheit virus VP2 protein. It is confirmed through observation by penetrating electron microscope that the expressed recombinant protein can self-assemble into virus-like particles. In a specific embodiment of the present invention, the protective effect of the chicken against immunization with infectious F. bursal virus infection produced by immunizing chickens with injection and oral methods using the recombinant protein produced by the pseudo-ulcer is also evaluated. Therefore, an object of the present invention is to provide a method for preparing a recombinant infectious Fahrenheit virus VP2 protein, comprising: (1) substituting the infectious Fahrenheit virus P3009) structural protein VP2 gene downstream of a nuclear polyhedron promoter, and Recombinant nuclear polyhedrosis virus is obtained; (") ;: The recombinant nuclear polyhedrosis virus prepared in step 感染 is used to infect the hypothalamus (ϋ collecting the worm body which has expressed the recombinant protein rVP2, separating its body fluid, and purifying it to obtain the Recombinant rVP2 protein V. Description of the invention (6) In a specific embodiment of the present invention, the VP2 gene sequence is from the vp2 starting nucleic acid to the 1 356th nucleic acid, and the c-terminus of the gene sequence is fused with six encoding histamines Acid codon sequence to facilitate the purification of the expressed rVP2H recombinant protein using an immobilized metal ion affinity chromatography resin (IMAC). Another object of the present invention is to provide a recombinant rVP2 protein prepared by the method of the present invention. Recombinant proteins can self-assemble into virus-like particles, and their stable structure is a great advantage for developing subunit vaccines. Another object of the present invention is to provide a method for preventing and treating infectious Fahrenheit. A virus-infected vaccine includes a recombinant rVP2 protein prepared by the method of the present invention. In a specific embodiment of the present invention, the recombinant prepared by the method of the present invention is "?"! Virus-like particles are prepared for injection or oral administration Chickens vaccinated with type V have shown that they can induce a high neutralizing antibody titer. Another object of the present invention is to provide a diagnostic reagent and method for diagnosing the presence of infectious Fahrenheit virus (IBDV). The prepared recombinant protein. In a specific embodiment of the present invention, the antibody prepared from the 4 ^ vp2h protein is used as a diagnostic reagent to determine whether the infectious Fahrenheit virus exists in the chicken Fahrenheit sac, and Use rVP2H protein as the detection reagent to test chicken blood to check whether chickens have been infected with Fahrenheit virus. This diagnostic method can be analyzed by Western blot analysis (W / B), agar gel precipitation method and / or enzyme-linked immunity Analytical method (ELISA) was completed. [Schematic description] 1233947 V. Description of the invention (7) Figure 1 · The western dot analysis results of rVP2H prepared in Example 1 are listed. Recognize rabbit anti-VP2 antibodies. Lane i: quasi-cut humoral body fluid (40-fold dilution) infected with VP3009VP2H; iane 2: precipitation of quasi-cut humoral body fluid infected with 10-50% ammonium sulfate; lane 3: purified by IMAC Figure 2. List the appearance and size of ibdv VP2 virus-like particles produced by the quasi-ruler-based expression system by TEM. Figure 3 List the rvp2H-I gG in the serum of immunized chickens using AC-EL ISA. The number of performances in each subsequent week. Lane a: injection of 20 μg of VLP purified by I MAC produced by Pseudo-ruchi; lane b: injection of 20 μg of VLP purified of IMAC produced by Hi-5 cells; lane c: 500,000 microliters of commercial inactivated infectious Fahrenheit virus were injected; 1 ane d: PBS was injected. Figure 4 • Detection of neutralizing antibody potency induced by the administration of virions. Lane a: injection of 20 micrograms of ILP-purified VLP produced by Pseudosciatus; lane b: injection of 20 micrograms of ILP-purified VLP produced by Hi-5 cells; lane c: injection of 500 microliters of commercial inactivated vaccine; lane d: PBS injection. Figure 5. Listed SPF chickens three days after challenge with an LD50. Western blot analysis (W / B) and agar gel precipitation analysis (AGP) were used to detect infectious Fahrenheit virus in Fahrenheit sac of chickens tested. Residual situation. W / B is rabbit

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1233947 V. Description of the invention (8) Anti-rVP2H antibody recognition, and AGP was detected by using anti-yeqing from chickens in the experimental group as the antibody source. [Detailed description of the invention] The infectious F. bursal virus belongs to Brinaviridae, its virus particle size is about 65 nm, and it contains two double-stranded RNA genomes, of which the small-strand rnA fragment is about 2.9 kb, which translates a 90 kDa protein product νρι, It is an RNA-dependent polymerized large RNA fragment of approximately 3.2 kb with two open reading regions, of which ORF 2 translates a protein product VP5 with a molecular weight of approximately 17 kDa. At present, this protein is considered to be infectious Fahrenheit disease virus (Brandt and Vakharia, J Virol 75: 11974-11982 (2001); and Mundt et al., J Virol 71: 5647-56 5 1 (1 997)) ° 0RF 1 110 kDa protein, which is a VP2 (~ 40 kDa), VP3 (~ 32 kDa), and VP4 (~ 28 kDa) precursor protein. VP2 and VP3 are the main viral structural proteins, and VP4 is the protein? Although 1 (Azad et al., 1987; and Fahey et al., 1989).

Fahey et al. Used affinity resins to isolate the structural proteins VP3 and VP-2a / 2b of infectious F. bursal virus. After immunoassay and analysis of neutralizing antibodies, they confirmed that VP2 protein of F. bursal virus induced the host to produce neutralizing antibodies. Major antigens have opened up the idea of using recombinant vp2 protein in subunit vaccine development. The expression of exogenous proteins can be divided into eukaryotic and prokaryotic systems. The prokaryotic expression system used at this stage is mainly E. coli. However, many foreign genes are derived from eukaryotic organisms.

The system does not produce the same biologically active protein. Prokaryotic performance system is missing

Page 11 1233947 V. Description of the invention (9) In the case of lack of post-translational modification, the protein of inclusion body type is often produced, resulting in loss of protein activity and inconvenience of purification. Since Smith et al. Successfully established an insect baculovirus expression system to produce human / 3 interferon (Smith et al., MoI CeU B, 3.2. 56-21 65 (1 983)), the use of rod-shaped The virus has increased the second benefit of foreign proteins. The method is to directly infect the second insect spores with the recombinant insect baculovirus, and express the recombinant protein through the growth and proliferation of the virus in the cells. Nuclear polyhedrosis virus (NPV) belongs to a group of baculoviruses. After Npv is in the host, the polyhedrin gene in the virus produces a polypeptide protein, which is activated in the late stage of viral infection and produces Protein (PIB) embeds baculovirus, and each ρΐβ can embed a virus particle with f. The function of PIB is mainly to encapsulate virus particles, to prevent the diseased body 2 from reducing its biological activity due to the environment, because the protein will be activated in a large amount in the late stage of infection. Therefore, the heavy dinucleotide gene promoter can be used to Heavy performance: f = baculovirus because the polykeratin gene has been replaced by a foreign gene; if method is correct! The production process is carried out for inclusion, and the long-term infection effect between the host and the virus caused by the infection of infected insects is caused to produce a larger amount of recombinant protein. With double fruit, although the use of insect cells to express exogenous I, but the production is convenient, low cost and use for many years, the use of individual insects to produce foreign proteins has gradually been adopted: the quality of the insects through the recombinant protein And Diptera larvae are more appropriate to use =

1233947 V. Description of the invention (10) :: Larvae Trichoplusia η. And Bombyx mori (B⑽ ... m0r.) Spot ^ There are many kinds of proteins that can be expressed by insect expression system. The types of proteins include extraviral proteins, hormones , Growth factors, cytokines, etc. In this month, the recombinant baculovirus was used to infect the pseudolarvae to produce the infectious Fahrenheit virus 2 protein from intact individuals. In addition to the present invention, the recombinant VP2 will be produced by the pseudolarvae. In addition to the pioneering invention applied to subunit vaccines, the purification of recombinant vp2 protein has also been improved. One of them is the fusion of six histidine genes at the C-terminus of the VP2 gene to facilitate the use of immobilized metal ion affinity chromatography. Resin (IMAC) purification showed rVp2H weight = protein; and another feature is the use of vitamin c as an antioxidant combined with sulfur ^ application 'to solve the problem of easy blackening of insect body fluids, extend the life of affinity resin, reduce Cost of Purifying rVP2H Recombinant Protein. [Examples] ^ The present invention will further illustrate the technical content of the present invention by the following examples. However, the listed examples are for illustration only. It is not intended to limit the scope of the present invention. Any person skilled in the art can make any modifications & modifications to the present invention and specific embodiments without departing from the spirit and scope of the present invention. However, it should still be included in the scope of the present invention. ~ / Example 1 · Preparation of recombinant VP2 protein (rVP2H) A · Preparation of recombinant insect baculovirus Recombinant insect baculovirus VP3009VP2H is based on the original baculovirus Of virus

1233947 V. Description of the invention (11) The VP2 gene of the structural protein of infectious Fahrenheit virus (P30 09) is substituted into the downstream of the nuclear polyhedron promoter. The gene sequence is from the VP2 starting nucleic acid to the 1356th nucleic acid, and the gene The C-terminus of the sequence was constructed by fusing the six histamine codons. B. Infection with a recombinant virus (1 ^ (: 11〇01113 丨 3 111) was provided by Director Gao Shengsheng, Director of the Biological Pharmacy Department of the Agricultural Drugs Toxicology Laboratory of the Agricultural Committee of the Executive Yuan, and was bred at 25 ° c. Boxes, artificial food for artificial feeding. Recombinant virus infections are divided into three methods based on the artificial rule. 1. Hole-fed breeding and infection: 30 microliters of vP3009VP2H virus solution is dripped on an average of 0.25 cubic centimeters of artificial feed. Ruler. 2. Large-scale infection: Drip 3 ml vP3009VP2H virus droplets onto 25 g (approximately 81 cubic centimeters, 1 cm in height) of artificial feed on 300 artificial rulers. 3. Mixed virus feed infection: artificial feed production During the process, when the feed was cooled to 40 ° C, 3 ml of VP3 009VP2H virus solution was mixed in the feed, and 300 pupae were fed. After being infected, the pupae were all starved for 6 hours and then fed with feed containing recombinant virus VP3 00 9VP2H Infected worm age is 4 years. C. Purification of recombinant rVP2H protein. After infecting Pseudotussus larvae with VP3009VP2H for 120 hours, individuals infected with Pseudotussus larvae were collected, and the body fluids were crushed directly by gravity compression at 10,000 r pm ( (KUBOTA RA-200-J) Centrifuge for 30 minutes twice to isolate the worm body. Take the worm body fluid and add 1% (w / v) vitamin C to the worm body supernatant. This vitamin c

Page 14 1233947 V. Description of the invention (12) The addition can effectively prevent the blackening of the insect body fluid.

After determining the content of rVP2H protein in rabbit anti-rVP2H antibody by Western blotting method, the pseudo-ulcer body fluid was diluted 10-fold with deionized water, and the protein was dehydrated with ammonium sulfate at 10% saturation, and the supernatant was taken. RVP2H was salted out with 1 Q ~ 50% sulfuric acid, and the precipitated protein was dissolved in deionized water to the original volume, adjusted to pH 7.8 and reacted with Ni-NTA agarose (QIAGEN Inc ·) at 4 ° C for 4 hours, The ratio of Ni-NTA agarose volume to the volume of the original worm fluid was 丨: 3. After the reaction was completed, the protein not bound to Ni-NTA was released at room temperature, and then buffered with 30 times the volume of Ni-NTA at pH 7 · 8. Solution, PBS 6. 3 buffer solution to extract non-specific binding protein, and finally 10 times Ni-NTA volume of pH 4.0 buffer solution to extract the rVP2H protein. Example 2: Characteristic analysis of the recombinant VP2 protein (rVP2H) A. Quantitative analysis of the expression protein rVP2H. 108 pfu of VP3 00 9VP2H was fed to the infected scaleworm, and the insects were collected after 120 hours. Linked immunoassay (ELISA) was quantified by individual antibodies, and the average body fluid contained approximately 75 mg of rVP2H protein per ml of body fluid. It was estimated that approximately 5 mg of rVP2H protein could be produced per pseudo-ulcer. The VP2H solution treated with ammonium sulfate precipitation was purified with immobilized metal ion affinity resin (IMAC). The purity of the obtained VP2H was above 90% (Figure 1). Quantitative analysis of individual antibodies by ELISA, an average of 1 ml of worm body expression solution, after purification by IMAC, 12 mg of rVP2H protein can be obtained, purified and recovered

Page 12 1233947

The rate is about 16%. B. Observation of Virus Hke particles (VLP) with a transmission electron microscope (TEM) Further observation was made with a transmission electron microscope to see if rVP2H was able to self-contain and become virus-like particles. Take 20 microliters of I MAC-purified sample. Long 'has been treated with carbon for 3 to 5 minutes on a copper wire, remove excess sample with filter paper, and negatively stain with 5% uranium acetate solution for 2) seconds. Then, observation was performed with a transmission electron microscope (EOL JEM 1 200 EX-2) at a magnification of 50,000 to 10,000. The results show that the rVP2H protein prepared in Example 1 can be self-assembled into virus-like particles with a particle size of about 20-30 nm (Figure 2). '' Example 3 Discussion on the immune efficiency of recombinant rVP2H virion A. rVP2H-IgG The potency is to confirm that the rVP2H administered in the test can induce antibodies in chicken blood to be specialized in the chickens. ”This example uses AC-ELISA to measure immune chickens. "IgG in blood only?" IgG. Intramuscular injection of virus-like chickens produced by Hi-5 cells, only 20% (1/5) of chickens induced rVP2H-IgG in the first week after immunization, the average of the experimental group as a whole The force value is 0, the average force value 2 weeks after the injection reached 272,000, the week 3 reached 7 6800, and the week 4 reached 73600 (Figure 3, lane b). The intramuscular injection was produced by the basal body Similar to virus particle chickens, chickens did not induce rVP2H-IgG in the first week after immunization, and the average valence of the antibody was 9600 in the second week after immunization. The antibody valence measured in the third week after immunization was 392. (), And reached 75200 by the fourth week (Figure 3, lane a). Inject chickens with commercial inactivated infectious F. bursal virus by intramuscular injection at 1233947 V. Description of the invention (14) Serum was detected at 2 weeks after immunization There was rVP2H-IgG, the average force value was 1 666.7, the value detected in the third week was 1 28 0, and the fourth week was 440 (Figure 3, lane c). Table The current valence showed a downward trend over time. B. Neutralizing antibody test The chickens immunized with H i -5 cells producing virus-like particles produced the neutralizing antibody valence induced by the i-th week after immunization was 212.2. The neutralizing antibody potency reached 396 · 2 in 2 weeks, 1 280 in week 3, and the neutralizing antibody increased to 2764.8 in week 4 (Figure 4, 1 ane b). SPF was produced on a pseudo-scale basis and SPF was applied. For chickens, the neutralizing antibody power detected in the blood of chickens at the first week after immunization was 467.2. As the immunization time increased, the neutralizing antibody power was measured in the blood of self-immunized chickens at the second week. The price increased to 665.6, and the valence of the 3rd week after immunization reached 2124.8, and the valence of the 4th week was 1945.6 (Figure 4, lane a). When SPF chickens were inoculated with the inactivated virus, they were induced in the gold fluid in the 1st week. The neutralization antibody power value was 0, and it was increased to 31.2 in the second week, until the neutralization antibody power value reached 64 in the third week after immunization, and in the fourth week was 483.5. Vaccine chickens were low (Figure 4, lane c).

C. Determination of protection The protection of SPF chickens was tested 4 weeks after immunization with a lethal dose of LD50 of a virulent infectious Fahrenheit virus. The chickens in the challenge control group died at the third day of the challenge, and the chickens in the various test vaccine immunization groups did not die. The weight of the difficult group in the immunization group was 497 ± 22.3 grams (commercial vaccine group), 1233947 V. Description of the invention (15) 475 ± 49.2 grams (iarvae VLP) and 50 4 ± 15 were higher than the challenge The average body weight of the chickens in the control group (385 + 501) After the protection of the chickens in each group, the color of the Fahrenheit sac was good and the weight was heavy ^, and the Fahrenheit sac in the challenge control group showed inflammation and atrophy ...., Human's positive-analyzed Hua: Liquid: Western blot analysis and soil lipid gel Shen Dianzhi's Fahrenheit's sac were all negative, while the Fahrenheit's sac of the challenge control virus were all positive (Figure 5). Therefore from the above data It can be confirmed that inoculation of 20 μg of rVP2H virion-like particles purified by IMAC by intramuscular injection can indeed induce 100% protection of chickens. Example 4 · Immune efficacy of oral rVP2H virion-like vaccines prepared according to the present invention The obtained rVP2Η can self-assemble into virus-like particles. After physical property analysis, the protein particles can stably exist in the solution of ρΗ ^^ for more than 8 hours, so it has the potential to develop oral vaccines. All oral vaccines are diluted to 500 microliters. Mix 3% sodium bicarbonate solution It neutralizes gastric acid and reduces protein cleavage. Chickens were immunized orally at 10, 15, 20, and 25 days of age in SPF chickens. The number of experimental chickens in each group was 4 and the immune antigen dose was sequentially from 25 micrograms. Increase by a factor of two until the immunization dose of 200 micrograms is given to the chickens at the age of 25. The control group of chickens were orally administered with the wild bodyworm and the PBS solution. At first, 5 microliters of worm body fluid was given, and then the oral dose was doubled every 5 days, until the fourth page 1233947. V. Description of the invention (16) 40 microliters of worm fluid was immunized; PBS group was given 500 micrograms orally to a single chicken. Litres of PBS solution, the time of oral administration is the same as that of immunized chickens. The results are shown in the table below. Table · Oral subunit vaccine test chicken protective mortality mortality weight (g) B / B ratio (*) Western ink agar Gel rVP2H- Insect body fluid ammonium sulfate precipitation rVP2H Purified rVP2H wild type body fluid PBS PBS (not challenged) Spot method 4.92 ± 0.821 3.9010.386 3.42 ± 0.545 4.07 ± 1.043 5.41 4.25 ± 0.762 475.6 ± 48.75 448.8 + 33.13 519.1 ± 93.01 446.0 ± 63.85 438 514.9 ± 38.94 (*) B / B ratio refers to: (Fahrenheit sac net weight / chicken net weight) χ 100, each group of chickens separately estimates the average and standard deviation of the B / B ratio, between samples The β / β ratio was not different by AN0VA analysis (ρ > 0 1). According to the test results in the table above, it was found that chickens administered orally to rVp2jj (group C) were in a LD50 infectious Fahrenheit In the case of bursal virus infection, the mortality rate on the 3rd day was 25%, while the mortality rate of unimmunized chickens was 100% (group E). As a result of the detection of the virus in the Fahrenheit sac, 25% of the positive responses were obtained by oral administration of purified rVP2H chickens with the best protection (group C). Oral protection of rVP2II chickens without IMAC purification, 3 days after measurement Page 19 1233947

V. Explanation of the invention (17) The mortality rate is 25%, and the western blot analysis is used to detect the residual Fahrenheit virus in the range of 50 ~ 75%. The results show that protein purity is extremely important for oral immunization. Based on the survival rate of chickens and the status of infectious Fahrenheit virus in the Fahrenheit sac, the protective power was defined. The chickens with orally purified rVP2H achieved 75% complete protection, and the orally unpurified rVP2H and chickens had protection efficacy of 25-50%. It has been shown that administration of the recombinant rVP2H protein of the present invention in the form of an oral vaccine is also effective in preventing and treating infectious Fahrenheit virus infection. Therefore, the present invention is based on the intended purpose of the present invention, and the present invention has practical effects. Therefore, the above-mentioned creations, according to the law, filed an application for the invention. The end of the chapter β The high creation of the rule of nature, which can be achieved is a design that has not been seen before. It is in line with the requirements of a high degree of invention patents, and please grant a patent as soon as possible.

Claims (1)

1233947_ VI. Scope of patent application 1. A method for preparing a recombinant infectious Hua I bursal virus VP2 protein, comprising:: ιί; (1) Substitution of an infectious Hua bursal virus (P 3 0) downstream of a nuclear polyhedron promoter 0 9) a structural protein VP2 gene to prepare a recombinant nuclear polyhedrosis virus; wherein the C-terminus of the gene sequence is fused with six codon sequences encoding histidine; (2) the recombinant prepared in step (1) Nucleopolyhedrosis virus infection of Pseudomonas rufasciatus; and (3) Collecting Pseudomonas rufasalis bodies that have expressed recombinant VP 2 protein, isolate their body fluids, and purify the expressed rVP2H recombinant protein by immobilized metal ion affinity chromatography resin (I MAC) The recombinant protein was virus-like in shape. 2. The method according to item 1 of the scope of patent application, wherein the VP2 gene sequence is from the VP2 starting nucleic acid to the 136th nucleic acid. 3. The method according to item 1 of the scope of patent application, wherein vitamin C is added to the separated body fluid to prevent blackening of the body fluid. Page 24 1233947 3 figure 1 1235947 PBS Inactive IBDV Larvae VLP PBS Hi-5 VLP AGP MN1212345123 45PMN1212345