A heat-resistant infectious bursal disease virus vaccine and its preparation method and application

Abstract

The present invention discloses a heat-resistant infectious bursal disease virus vaccine and a preparation method and application thereof, and belongs to the technical field of vaccine preparation. The technical problem to be solved by the present application is how to improve the thermal stability of infectious bursal disease virus VP2 virus antigen and improve the immune effect. In order to solve the above technical problems, the present application provides a protein whose amino acid sequence is the 1-507th position of sequence 2 or a fusion protein whose amino acid sequence is sequence 2, a chicken bursal disease virus vaccine based on the fusion protein, and its application in preventing chicken bursal disease virus. The present application fuses the chicken bursal disease virus VP-2 protein with the rubredoxin of Thermotoga bacteria to obtain a fusion protein rb2-vp2 with high thermal stability. The fusion protein rb2-vp2 can withstand high temperature treatment, and the prepared vaccine has a higher immune titer after immunizing chicken seedlings, and the adverse reactions are significantly reduced, which can be used for the prevention of chicken bursal disease virus.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine production technology in biotechnology, and in particular to a heat-resistant infectious bursal disease virus vaccine and a preparation method and application thereof. Background Art

[0002] Infectious bursal disease (IBD) is an acute, highly contagious and lympholytic infectious disease of chickens caused by infectious bursal disease virus (IBDV) of the genus BisRNA virus of the family BisRNA virus. Its genome consists of two segments, A and B. Segment A encodes four proteins, VP5, VP2, VP4 and VP3, and segment B encodes VP1 protein. Among them, VP2 protein is the main structural protein and host protective antigen of IBDV. Studies have shown that the mutation of amino acids in the hypervariable region of VP2 is one of the important factors in the change of viral antigen and virulence. IBDV mainly attacks the bursa of Fabricius, causing degeneration and necrosis of pre-B lymphocytes in the bursa of Fabricius in chicks, resulting in varying degrees of immune suppression and inability to produce a normal immune response to vaccines, causing immune failure of multiple vaccines (such as Newcastle disease and avian influenza), and causing secondary infections with other pathogens (coccidia, Escherichia coli, Salmonella, adenovirus). Vaccination is one of the key links in controlling chicken bursal disease, however, there are many types of bursal virus vaccines, with different strains of different strengths, as well as live vaccines, whole virus inactivated vaccines and genetically engineered vaccines.

[0003] There are many reasons for the failure of IBD vaccination, including unreasonable selection of vaccines (strains), incorrect vaccination methods, uncertain vaccination effects, and factors of the chicken itself (such as the presence of immunosuppressive diseases and uneven levels of maternal antibodies in chickens). These are all factors that lead to vaccination failure, but the root causes of IBD vaccination failure are mainly the following two aspects: First, the whole virus inactivated vaccine has the disadvantages of slow antibody production and long immune blank period; second, the antigen of the traditional VP2 protein subunit vaccine is the recombinant VP2 protein, which has a single structure and its antigenic determinant is conformation-dependent. Detergents and heating can easily destroy its conformation and lose the ability to bind to neutralizing antibodies. In addition, E. coli miscellaneous proteins can easily cause immune saturation, thus failing to provide good protection for the chickens.

[0004] Hyperthermophiles are organisms that can live in extremely hot environments (above 60°C). Their optimum growth temperature is usually between 80 and 110°C, and an archaeon strain "strain 121" discovered in 2003 can even double its cell number within 24 hours at 121°C, the same temperature as a sterilizing pot. Most hyperthermophiles belong to archaea, but some bacteria (including some cyanobacteria) can tolerate temperatures of 70 to 80°C. Many hyperthermophiles can also withstand other extreme environments, such as high acidity or radiation intensity.

[0005] The proteins of hyperthermophiles need to have strong thermal stability, which depends on the stability of their structure at high temperatures so that their functions remain stable. These proteins are homologous to the corresponding proteins of organisms living at lower temperatures, but their strongest functions are to be exerted at much higher temperatures. The proteins of hyperthermophiles often have the following characteristics: more salt bonds are formed by internal amino acid residues, and there is a tightly folded hydrophobic core. In addition, some hyperthermophiles produce a lot of intracellular solutes, such as di-inositol phosphate, diglycerol phosphate, mannosyl glycerate, etc., to help proteins resist thermal degradation. Most homologous proteins at low temperatures will denature at 60°C, so these heat-stable proteins have high potential commercial value. For example, the ring structure in Thermotoga-stabilized erythrodoxin and ferredoxin is very small, the a-helix is ​​longer, and both the amino and carboxyl ends of the amino acids promote solubility and thermal stability.

[0006] Prokaryotic expression systems are often used to express exogenous proteins because of their high yield, low cost, and convenient production. However, the IBDV VP2 gene has many rare codons, and the VP2 protein is toxic to Escherichia coli. Full-length expression has always been difficult. Even if it can be expressed, endotoxins and impurities are high, and post-processing is troublesome. If they are not removed thoroughly, it will lead to adverse reactions at the immune site, so improvements are needed. Summary of the invention

[0007] The technical problem to be solved by the present application is how to improve the thermal stability of infectious bursal disease virus VP2 virus antigen and improve the immune effect. Specifically, the existing infectious bursal disease virus VP2 virus antigen has poor immune effect, and the effective removal of endotoxins and foreign proteins during production leads to the defect of toxic and side effects immunity.

[0008] In order to solve the above technical problems, the present application provides a fusion protein rb2-vp2, wherein the fusion protein rb2-vp2 can be any one of the following A1)-A3):

[0009] A1), a protein whose amino acid sequence is the 1st to 507th position of SEQ ID No. 2 and / or a protein whose amino acid sequence is the SEQ ID No. 2;

[0010] A2) A protein related to infectious bursal disease virus VP2 obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in A1) having more than 80% identity with the protein shown in A1);

[0011] A3), a recombinant protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0012] In the present application, the connection in A3) may be a peptide bond connection.

[0013] In the present application, the protein can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0014] In the above-mentioned protein, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues; or a substitution and / or deletion and / or addition of no more than 9 amino acid residues; or a substitution and / or deletion and / or addition of no more than 8 amino acid residues; or a substitution and / or deletion and / or addition of no more than 7 amino acid residues; or a substitution and / or deletion and / or addition of no more than 6 amino acid residues; or a substitution and / or deletion and / or addition of no more than 5 amino acid residues; or a substitution and / or deletion and / or addition of no more than 4 amino acid residues; or a substitution and / or deletion and / or addition of no more than 3 amino acid residues; or a substitution and / or deletion and / or addition of no more than 2 amino acid residues; or a substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0015] The protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag protein tag, a His protein tag, an MBP protein tag, an HA protein tag, a myc protein tag, a GST protein tag and / or a SUMO protein tag, etc.

[0016] In some embodiments of the present application, SEQ ID No. 2 consists of 515 amino acid residues.

[0017] The present application also provides a biological material, which may be any one of the following B1)-B4):

[0018] B1), a nucleic acid molecule, the nucleic acid molecule is a nucleic acid molecule encoding the fusion protein rb2-vp2, the nucleic acid molecule is any one of the following g1)-g3),

[0019] g1), the nucleotide sequence of the coding strand is the DNA molecule shown in positions 5071-6618 of SEQ ID No.1;

[0020] g2), the coding sequence of the coding strand is the DNA molecule shown in positions 5071-6618 of SEQ ID No.1;

[0021] g3), a DNA molecule having 80% or more identity with the nucleotide sequence defined in g1) or g2), and encoding the fusion protein rb2-vp2 of claim 1;

[0022] B2), an expression cassette, wherein the expression cassette contains the nucleic acid molecule described in B1);

[0023] B3), a recombinant vector, the recombinant vector contains the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0024] B4), a recombinant microorganism, wherein the recombinant microorganism contains the nucleic acid molecule of B1), or contains the expression cassette described in B2), or contains the recombinant vector described in B3).

[0025] In some embodiments of the present application, the coding sequence of the fusion protein rb2-vp2 encoding gene is shown in SEQ ID No. 1, 5071-6618, wherein SEQ ID No. 1, 5071-5219 is the nucleotide sequence of the Thermotoga rubredoxin rb2 encoding gene, 5230-5247 is the nucleotide sequence of the connecting peptide encoding gene, 5448-6591 is the nucleotide sequence of the infectious bursal disease virus VP2 encoding gene, and 6592-6618 is the nucleotide sequence of the xhoI restriction site, the 8His protein tag encoding gene and the termination codon. The Thermotoga rubredoxin rb2 encoding gene and the infectious bursal disease virus VP2 encoding gene must be included.

[0026] In some embodiments of the present application, the coding gene of the fusion protein rb2-vp2 is constructed into the pET28a(+) vector, and the resulting recombinant expression vector is named as the recombinant expression vector PET28a(+)-rb2-vp2. The nucleotide sequence of the recombinant expression vector PET28a(+)-rb2-vp2 is SEQ ID No. 1. The physical map of the recombinant expression vector PET28a(+)-rb2-vp2 is as follows Figure 1 shown.

[0027] Furthermore, the recombinant microorganism may be Escherichia coli.

[0028] The present application also provides virus-like particles containing the above protein (fusion protein rb2-vp2).

[0029] The present application also provides a vaccine for infectious bursal disease virus of chickens, which comprises the above-mentioned protein (fusion protein rb2-vp2) or the above-mentioned virus-like particles.

[0030] The present application also provides a method for preparing the above protein (fusion protein rb2-vp2) and / or the above virus-like particles, comprising: expressing the coding gene of the above fusion protein rb2-vp2 in biological cells to obtain the fusion protein rb2-vp2.

[0031] Furthermore, in the method, the organism is a microorganism, a plant or a non-human animal.

[0032] Furthermore, in the method, the organism may be a microorganism.

[0033] Furthermore, in the method, the microorganism may be Escherichia coli.

[0034] The present application also provides a method for preparing the above-mentioned vaccine, which comprises the step of mixing the above-mentioned protein (fusion protein rb2-vp2) and / or the above-mentioned virus-like particles with an adjuvant.

[0035] Furthermore, in the method, the adjuvant may be an oily adjuvant and / or an aqueous adjuvant.

[0036] Furthermore, in the method described above, the oily adjuvant may be selected from: adjuvant Marcol-52, aluminum stearate and Span. Furthermore, in the method described above, the aqueous adjuvant may be PBS and Tween-80.

[0037] The present application also provides the use of the above protein (fusion protein rb2-vp2) in the preparation of products for preventing infectious bursal virus infection in chickens.

[0038] The present application also provides the use of the above-mentioned biological material in preparing a product for preventing infectious bursal virus infection in chickens.

[0039] The present application also provides the use of the virus-like particles in the preparation of products for preventing infectious bursal virus infection in chickens.

[0040] The present application also provides a method for preventing and / or treating chicken bursal disease, which comprises administering the above-mentioned vaccine, virus-like particle and / or protein (fusion protein rb2-vp2) to chickens.

[0041] Furthermore, in the present invention, the product may be a reagent, a kit or a medicine.

[0042] Furthermore, in the present invention, the medicine may be a vaccine.

[0043] In the present invention, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of identity (%) can be obtained.

[0044] In the present invention, the above 80% identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Those skilled in the art can easily mutate the nucleotide sequence of the nucleic acid molecule of the present invention by known methods, such as directed evolution and point mutation. Those artificially modified nucleotide sequences that have 75% or higher identity with the nucleic acid molecule encoding the protein isolated by the present invention and encode the above protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0045] The term "coding sequence" means a sequence of nucleotides (position including stop codons) corresponding to the amino acid sequence in a protein; the features include a conceptual translation of the amino acids.

[0046] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0047] In the above biological material, the expression cassette refers to a DNA capable of expressing the protein in a recombinant cell, which may include not only a promoter for initiating transcription of the protein encoding gene, but also a terminator for terminating transcription of the protein encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0048] The term "recombinant vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide, and the polynucleotide is operably connected to a regulatory sequence providing for its expression. The recombinant vector comprises a polynucleotide of the present invention, which is connected to one or more regulatory sequences, such as a promoter and transcription and translation termination signals, and the regulatory sequence directs the production of the polypeptide in an expression host. A variety of nucleotides and regulatory sequences can be combined together to produce a recombinant vector, which can include one or more convenient restriction sites to allow the insertion or replacement of the polynucleotide encoding the polypeptide at these sites. Alternatively, the polynucleotide can be expressed by inserting a nucleic acid construct or polynucleotide comprising the polynucleotide in a suitable vector for expression. In the process of preparing the expression vector, the coding sequence is placed in a vector, so that the coding sequence is operably connected to a suitable regulatory sequence for expression. The recombinant vector can be any vector (e.g., a plasmid or a virus) that can conveniently carry out a recombinant DNA step and can produce expression of the polynucleotide. The choice of the vector will generally depend on the compatibility of the vector with the recombinant cell into which the vector will be introduced. The vector can be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication; alternatively, the vector may be a vector that, when introduced into a recombinant cell, is integrated into the genome and replicates together with the chromosome into which the vector is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids may be used that together contain the complete DNA to be introduced into the genome of the recombinant cell, or a transposon may be used.

[0049] The vector preferably contains one or more selective markers, which allow simple selection of cells transformed, transfected, transduced, etc. The vector preferably contains elements that allow the vector to be integrated into the recombinant cell genome or the vector to replicate independently of the genome in the cell. In order to be integrated into the recombinant cell genome, the vector can rely on the sequence of the polynucleotide encoding the polypeptide or any other vector element for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for guiding the precise position of integration into the host cell genome chromosome by homologous recombination. In order to increase the possibility of integration at the precise position, the integration element should contain a sufficient number of nucleic acids, such as 100 to 10000 base pairs, 400 to 10000 base pairs, and 800 to 10000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integration element may be any sequence, which is homologous to the target sequence in the host cell genome. In addition, the integration element may be a non-coding or encoding polynucleotide. On the other hand, the vector may be integrated into the genome of the recombinant cell by non-homologous recombination. For autonomous replication, the vector may further comprise an origin of replication, which enables the vector to replicate autonomously in the recombinant cell. The origin of replication may be any plasmid replicon that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0050] More than one copy of the polynucleotide of the present invention can be inserted into a recombinant cell to increase the production of the polypeptide. The increase in the number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selective marker gene in the polynucleotide, wherein cells containing an amplified copy of the selective marker gene and thus containing additional copies of the polynucleotide can be selected by culturing the cells in the presence of a suitable selection agent. Methods for connecting the above elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art.

[0051] The recombinant microorganisms can be bacteria such as Escherichia coli, mammalian cells, insect cells, or other expression systems such as yeast, algae, and plants. Mammalian cells can be 293, CHO, BHK, MDCK, etc., and insect cells can be SF9 and Hifv5.

[0052] The beneficial technical effects achieved by this application are as follows:

[0053] In the present application, the VP-2 protein of Chicken Bursal Disease Virus is fused with the rubredoxin rb2 of Thermotoga bacteria to obtain a fusion protein rb2-vp2 with higher thermal stability. The fusion protein rb2-vp2 can be treated with high temperature (in some embodiments of the present application, 60°C). The vaccine prepared with the fusion protein after high temperature treatment has a higher immune titer after immunizing chicken seedlings, and the adverse reactions (redness and swelling at the injection site, foreign body sensation) are significantly reduced. The immune challenge test shows that the challenge protection rate of chicken seedlings immunized with the vaccine prepared with the fusion protein after high temperature treatment is 6 / 7, which has a high challenge protection rate and can be used for the prevention of Chicken Bursal Disease Virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The physical map of PET28a(+)-rb2-vp2

[0055] Figure 2 This is the expression and purification result of the target protein of the recombinant strain BL21 / PET28-rb2-vp2 at 55°C.

[0056] Figure 3 The results of SDS-PAGE detection of the filtrate after heat treatment. A shows the effect of high temperature protein removal, lane 1 is the supernatant 35-0 after fermentation, and lane 2 is the supernatant 35-1 after 55 degrees treatment. The left EP tube (35-0) in B shows the clarity of the supernatant stock solution, and the right EP tube (35-1) is the supernatant after 55 degrees treatment.

[0057] Figure 4 These are the experimental results of testing the efficacy of vaccines using the immune challenge method. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0059] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0060] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0061] Example 1. Obtaining the gene encoding the rb2-vp2 protein

[0062] 1.1. Synthesis of target gene

[0063] In order to improve the heat-resistant immunogenicity of wild-type VP2, the rubredoxin rb2 gene of Thermotoga bacteria was fused with the N segment of the infectious bursal disease virus VP2 gene to obtain the coding gene of the fusion protein rb2-vp2.

[0064] The coding gene of the fusion protein rb2-vp2 was commissioned to be synthesized by Qingke Biotechnology, and its nucleotide sequence is shown in SEQ ID No. 1, 5071-6618. Among them, SEQ ID No. 1, 5071-5219 is the nucleotide sequence of the rubredoxin rb2 coding gene of Thermotoga bacteria, 5230-5247 is the nucleotide sequence of the connecting peptide coding gene, 5448-6591 is the nucleotide sequence of the infectious bursal disease virus VP2 coding gene, and 6592-6618 is the nucleotide sequence of the xhoI restriction site, the 8His protein tag coding gene and the termination codon. The coding gene of the fusion protein rb2-vp2 was constructed into the pET28a (+) vector, and the obtained recombinant expression vector was named as the recombinant expression vector PET28a (+) -rb2-vp2. The nucleotide sequence of the recombinant expression vector PET28a (+) -rb2-vp2 is SEQ ID No. 1. The physical map of the recombinant expression vector PET28a(+)-rb2-vp2 is as follows Figure 1 shown.

[0065] The recombinant expression vector PET28a(+)-rb2-vp2 expresses the fusion protein rb2-vp2, and the amino acid sequence of the fusion protein rb2-vp2 is SEQ ID No. 2. Among them, SEQ ID No. 2 Nos. 1 to 53 are rubredoxin rb2 of Thermotoga bacteria, wherein Nos. 54 to 59 of SEQ ID No. 2 are the connecting peptide sequence, Nos. 60 to 507 are the infectious bursal disease virus VP2 protein, and Nos. 508 to 515 of SEQ ID No. 2 are the xhoI restriction site encoding protein and the His protein tag.

[0066] 1.2. Expression of fusion protein

[0067] The recombinant expression vector PET28a(+)-rb2-vp2 was transformed into the host bacteria E. coli BL21 (DE3). The specific steps include: adding 0.5 μL of the recombinant expression vector PET28-rb2-vp2 to the competent (full gold) EP tube of E. coli BL21 (DE3), ice bathing for 30 minutes, heat shocking at 42°C for 90S and ice bathing for 2 minutes, adding 500 μL of SOC medium and culturing at 37°C for 1 hour, then coating on a Kana (kanamycin) resistant LB plate, 37°C overnight, picking a single bacterium and inoculating it in the LB medium, shaking and culturing at 37°C, 200 rpm for 6 hours to obtain a bacterial solution containing the recombinant strain BL21 / PET28a(+)-rb2-vp2, mixing the bacterial solution and 50% glycerol aqueous solution at a ratio of 7:3 and storing at -80°C.

[0068] The composition of SOC culture medium is: 20g / L Tryptone, 5g / L Yeast Extract, 0.5g / L NaCl, 2.5mM KCl, 10mM MgCl2, 20mM glucose, and the rest is water.

[0069] 1.3. Product expression and determination of expression form

[0070] The recombinant strain BL21 / PET28a(+)-rb2-vp2 preserved in glycerol in 1.2 was inoculated at 1% into 100mL LB medium with 25μg / mL kana resistance for 6 hours, and 1mM TPTG was added to induce in a 250mL Erlenmeyer flask for 12 hours to obtain a culture named fermentation broth. The culture conditions were 16°C and 220rpm. The fermentation broth after induced expression was ultrasonically disrupted to obtain a bacterial liquid named broken bacterial liquid. The broken bacterial liquid was centrifuged at 12000rpm for 10 minutes to obtain a centrifugal supernatant and a centrifugal precipitate. The fermentation broth, broken bacterial liquid, centrifugal supernatant and centrifugal precipitate were taken for SDS-PAGE detection. The results are shown in Figure 2 As shown, MK (LOT#N51130 full-form gold) from bottom to top are 25kDa, 30kDa, 40kDa, 50kDa, 70kDa, 100kDa, and 120kDa, respectively, lanes 1 and 2 are electrophoresis bands of fermentation broth, lane 3 is an empty vector control (PET28a(+)), lane 4 is an electrophoresis band of centrifugal precipitate, and lane 5 is an electrophoresis band of centrifugal supernatant. Figure 2 The results showed that the fusion protein was expressed in the supernatant.

[0071] Example 2. Fermentation and purification of fusion protein rb2-vp2

[0072] 2.1. High-density fermentation of E. coli in fermenters

[0073] 2.1.1. Seed solution preparation or plate culture

[0074] The recombinant strain BL21 / PET28a(+)-rb2-vp2 was cultured in LB medium containing kanamycin at 30-37° C. and 250-280 rpm for 10-12 hours.

[0075] The composition of LB medium containing kanamycin is: 10 g / L peptone + 50 g / L NaCl + 5 g / L yeast powder, kanamycin 25 μg / mL, and the rest is water.

[0076] 2.1.2. Secondary seed preparation

[0077] The strain was cultured in modified LB medium containing kanamycin at 30-37°C and 220 rpm for 12 hours.

[0078] Improved LB medium containing kanamycin: 5g / L glycerol + 10g / L peptone + 5g / L NaCl + 10g / L yeast powder, kanamycin 25μg / mL, and the rest is water.

[0079] 2.1.3. Fermentation production

[0080] Fermentation culture is divided into three stages:

[0081] (1) Bacteria culture stage

[0082] After sterilization, 3 L of initial fermentation medium was placed in a 5 L fermenter, and kanamycin was added at a rate of 25 μg / mL. The seed solution prepared in 2.1.2 was inoculated at a rate of 10% (volume ratio), and cultured at 30°C with aeration and stirring for 7 hours. During the culture process, as the strain grew, the sugar in the culture medium was gradually consumed. When the carbon source was consumed, the bacteria stopped growing and the dissolved oxygen (DO) returned to 10%. Ammonia water was used to maintain the pH value at 7.0 during the culture process.

[0083] Among them, the formulas of the initial fermentation medium and the feed medium are as follows:

[0084] The initial culture medium composition is: glucose 5g / L, peptone 5g / L, yeast powder 5g / L, KH2PO4 2g / L, K2HPO44g / L, Na2HPO4 . 12H2O 3g / L, (NH4)2SO4 3g / L, NH4Cl 1.2g / L, MgSO4 . 7H2O 3g / L, defoamer 0.2g / L, and the rest is water.

[0085] (2) Source feeding stage

[0086] After confirming that the sugar in the previous step is consumed (with DO rising by 20% and pH rising without adding acid as the standard), start this step. Use feed medium to control DO between 10-20%. Use ammonia water to maintain the pH at 7.0. After this step is maintained for 3 hours, start inducing expression.

[0087] The composition of the feed medium is (g / L): glycerol 80g / L, glucose 80g / L, peptone 36g / L, yeast powder 36g / L, MgSO4 . 7H2O 2.5g / L, add distilled water to 1L, sterilize at 105℃ for 10min before use.

[0088] (3) Induction expression stage

[0089] Start induction after 3 hours of feeding, lower the temperature to 16°C, add IPTG with a final concentration of 0.1mM for 12 hours of induction, control DO between 10-30% by feeding, maintain pH at 7.0 with ammonia water during the whole induction process, collect the fermentation broth after 12 hours of induction, let it stand for 3-5 hours to let the bacteria settle, discard the supernatant and centrifuge the bacteria.

[0090] The composition of the feed medium is (g / L): glycerol 80g / L, glucose 80g / L, peptone 36g / L, yeast powder 36g / L, MgSO4 . 7H2O 2.5g / L, add distilled water to 1L, sterilize at 105℃ for 10min before use.

[0091] 2.2. Purification process of rb2-vp2 protein

[0092] 2.2.1. Centrifuge the fermentation broth obtained in 2.1.3 at 5000 rpm for 10 min, collect the cells and store at -20°C;

[0093] 2.2.2. Suspend in a 10% mass ratio in a buffered lysis solution, and lyse the cells by high pressure homogenization in an ice bath for 2-3 times. Divide the lysate into two parts, heat one part to 60°C, maintain for 10 minutes, and quickly cool to 37°C. Centrifuge the unheated 35-0 and heated 35-1 fermentation broths at 10,000 rpm for 20 minutes to separate the cell debris. Pass the supernatant through a 0.45 μm filter membrane, and collect the filtrate as the antigen to be used.

[0094] The composition of the buffered lysis solution is: 10 mM Tris-HCl, 200 mM NaCl, 10 mM glycine, 5 g / L glycerol, and the rest is water, adjusted to pH 8.0;

[0095] 2.2.3. The filtrate prepared in step 2.2.2 was subjected to SDS-PAGE detection. The results were as follows: Figure 3As shown in A, protein gel No. 1 (EP tube on the left side of B in the figure, marked 35-0) is not heated, and protein gel No. 2 (EP tube on the right side of B in the figure, marked 35-1) is 35-1 after heat treatment at 55°C. The protein gel image shows that the bacterial impurity protein bands after heat treatment are significantly reduced, but the target protein (indicated by the arrow) has no obvious change. In the EP tube, the fermentation broth after treatment changes from light yellow 35-0 to transparent solution 35-1.

[0096] Example 3 Preparation of vaccine

[0097] 3.1. Potency test using 35-0 and 35-1 as antigens

[0098] 3.1.1 The avian bursal IBV fusion protein rb2-vp2 protein is provided by our company. For specific information, see Table 1.

[0099] Table 1. Protein information

[0100]

[0101] 3.1.2 Challenge virus strain: Avian bursal disease virus strain BC6 / 85 (purchased from AVI, China Veterinary Drug Administration), chicken embryo virus F4 generation virus, virus content 10 8.1 EID 50 / 0.1mL.

[0102] 3.2. Vaccine preparation

[0103] The total amount of vaccine is 36mL, and when the water-oil ratio is 1:2, the oil phase is 24mL (white oil 23.04mL, aluminum stearate 0.36g, and 1.44mL of Span); the water phase is 12mL. To ensure that the pure protein agar expansion index in the vaccine reaches 1:8, 1.125mL of 35-solution needs to be added to 36mL of vaccine, and 10.395mL of PBS solution is added at the same time. The specific addition ratio is shown in Table 2.

[0104] Table 2. Vaccine ratio and dosage

[0105] Target protein Dosage PBS Twain-80 Adjuvant Marcol-52 Aluminum Stearate Siban Total vaccine volume 35-0 1.125mL 10.395mL 0.48mL 23.04mL 0.36gg 1.44mL 36mL 35-1 1.125mL 10.395mL 0.48mL 23.04mL 0.36gg 1.44mL 36mL Comparison -- 12mL 0.48mL 23.04mL 0.36gg 1.44mL 36mL

[0106] 3.2.2 Emulsification process

[0107] 1) Place a 100 mL beaker in an ice box (low temperature ice bath).

[0108] 2) First, add 24 mL of oil phase adjuvant (adjuvant Marcol-52, aluminum stearate and Span) to a 100 mL beaker, place a 6G shear head on the liquid surface, stir the oil phase at a low speed with an emulsifier, add the aqueous phase protein with a pipette, and pre-emulsify for 1 minute. The aqueous phase (protein solution, PBS and Tween-80) and the oil phase are mixed and emulsified in a ratio of 1:2 (aqueous phase: oil phase = 12 mL: 24 mL).

[0109] 3) Run at high speed 2 (10000rpm) for 1 minute, pause for 1 minute; high speed 2 for 1 minute, pause for 1 minute; high speed 2 for 1 minute, pause for 1 minute; high speed 2 for 1 minute, emulsify for a total of 5 minutes. (Emulsification standard: take a small amount of drops on the surface of clean cold water, except for the first drop, it should not spread.)

[0110] 4) Packaging: 8 mL / bottle, pack into 4-5 bottles, label them, and store at 4℃.

[0111] 5.2.3 Sterility test

[0112] Take the vaccine to be tested and inoculate 1 tube of thioglycollate medium TG (purchased from Beijing Zhonghai Biotechnology Co., Ltd.) with a volume of 0.6 mL / tube, and incubate at 37°C for 3 days. Pipette the culture and transfer it to a new tube of TG and 1 tube of trypticase soy bean liquid TSB (purchased from Beijing Zhonghai Biotechnology Co., Ltd.) (specification 4 mL / tube), with a volume of 0.2 mL / tube. The culture method is the same as above, and the results are observed after 7 days of culture.

[0113] 5.3 Immune efficacy test

[0114] 5.3.1 Immunization Grouping

[0115] A total of 30 4-5 week old SPF chickens (purchased from Beijing Boehringer Ingelheim Weitong Biotechnology Co., Ltd.) were randomly divided into 3 groups, with 10 chickens in each group. One group was immunized with the non-heat-treated 35-0 vaccine, one group was immunized with the heat-treated 35-1 vaccine, and the other group was the control group. The vaccine was injected into the neck muscle at 0.3 mL / head, and the control group was injected with normal saline at 0.3 mL / head through the neck muscle. After the injection, the injection site was observed for adverse reactions, and blood was collected and serum was separated on the 14th and 21st days after immunization to detect serum antibody values. The method for detecting the antiserum titer by agar diffusion experiment is as follows:

[0116] 1. Weigh 1.0 g agarose and dissolve it in 100 mL PBS (pH 7.4) or 0.85% saline, and heat to melt.

[0117] 2. Pour the hot agarose onto a clean glass slide or dish. The thickness of the agar should be about 3 mm.

[0118] 3. After it cools down, make small holes with a diameter of 3 to 5 mm in a plum blossom shape and pick out the agar in the holes.

[0119] 4. Heat slowly over flame to melt the bottom edge of the well slightly to seal the bottom and place it in a place to prevent leakage after adding sample.

[0120] 5. Add 0.1 mg / mL VP2 antigen to the middle well, filling it up without overflowing into other wells.

[0121] 6. Add antiserum diluted in series with physiological saline to the surrounding wells.

[0122] 7. After adding, place the slide or dish in a humidified box and incubate at 37°C overnight.

[0123] 8. Take out the serum the next day and the highest dilution factor that shows a precipitate band under scattered light is the agar titer.

[0124] The results of antibody determination are shown in Table 3. It can be seen that the fusion protein rb2-vp2 of the present invention can produce protective antibodies greater than 1:8 regardless of whether the impurity protein is removed or not, but the antibody value after removing the impurity protein is higher than that of the group without removing the impurity protein. The presence of impurity protein may cause immune saturation. At the same time, the chickens in the 35-0 group showed redness and swelling at the immunization site after immunization, and felt a foreign body when touched 21 days later. However, the above-mentioned side effects did not occur in the 35-1 and control groups. Therefore, we gave up the 35-0 group and only did the 35-1 and control group.

[0125] Table 3 HI antibody levels in serum of SPF chickens after immunization

[0126]

[0127]

[0128] 5.3.2 Immunotherapy

[0129] On the 28th day after immunization, 7 chickens were selected from each group for the challenge experiment. The virus seed was diluted 1:10 with PBS (0.01M pH7.4) and injected intravenously with 0.2 mL / chicken (about 2.5×10 7 EID 50 ). The animals were dissected 3 days after the challenge and the size of the bursa was observed to see if there were any bleeding spots or atrophy. Figure 4 .

[0130] On the day of virus attack, one chicken in the control group died and was removed from the control group. After autopsy, only one of the 7 chickens in group 35-1 had atrophy and reduction in bursal volume, and the other 6 were normal. However, the bursal volumes of the control group were atrophied and bleeding. The results showed that the efficacy evaluation of the laboratory-produced heat-resistant IBV-vp2 protein subunit vaccine was effective. There was no need to remove endotoxins and purify impurities in the production process. Antigen preparation was completed in one step. The immunized chickens could resist bursal virus attack, and there was no hyperplasia of granulomas at the immunization sites.

[0131] Table 5 Partial sequences in this application

[0132]

[0133]

[0134]

[0135]

[0136] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A protein, characterized in that The protein is a fusion protein rb2-vp2, and the fusion protein rb2-vp2 is the following A1) or A2): A1), a protein whose amino acid sequence is the 1st to 507th position of SEQ ID No.2 or a protein whose amino acid sequence is the SEQ ID No.2; A2), a recombinant protein obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

2. Biomaterial, characterized in that The biological material is any one of the following B1) to B4): B1), a nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid molecule encoding the fusion protein rb2-vp2 of claim 1, and the coding sequence of the coding strand of the nucleic acid molecule is a DNA molecule shown in positions 5071-6618 of SEQ ID No.1; B2), an expression cassette, wherein the expression cassette contains the nucleic acid molecule described in B1); B3), a recombinant vector, wherein the recombinant vector contains the nucleic acid molecule described in B1) or the expression cassette described in B2); B4), a recombinant microorganism, wherein the recombinant microorganism contains the nucleic acid molecule of B1), or contains the expression cassette described in B2), or contains the recombinant vector described in B3).

3. A virus-like particle, characterized in that The virus-like particle contains the protein according to claim 1.

4. A vaccine for infectious bursal disease virus of chickens, comprising the protein of claim 1 or the virus-like particle of claim 3.

5. A method for preparing the protein of claim 1 and / or the virus-like particle of claim 3, comprising: The step of expressing the coding gene of the fusion protein rb2-vp2 according to claim 1 in a biological cell to obtain the fusion protein rb2-vp2.

6. The preparation method according to claim 5, characterized in that: The organism is a microorganism, a plant or a non-human animal.

7. The method for preparing the vaccine according to claim 4, characterized in that: The method comprises the step of mixing the protein of claim 1 and / or the virus-like particle of claim 3 with an adjuvant.

8. Use of the protein according to claim 1 in the preparation of a product for preventing infectious bursal virus infection in chickens.

9. Use of the biological material according to claim 2 in preparing a product for preventing infectious bursal virus infection in chickens.

10. Use of the virus-like particles according to claim 3 in the preparation of a product for preventing infectious bursal virus infection in chickens.

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