CN112500458A

CN112500458A - Novel variant subunit vaccine of chicken infectious bursal disease virus, preparation method and application thereof

Info

Publication number: CN112500458A
Application number: CN202011473614.3A
Authority: CN
Inventors: 祁小乐; 王笑梅; 高玉龙; 高立; 李凯; 崔红玉; 刘长军; 潘青; 张艳萍; 刘爱晶
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2021-03-16
Anticipated expiration: 2040-12-15
Also published as: CN112500458B

Abstract

The invention discloses a novel variant subunit vaccine of chicken infectious bursal disease virus, a preparation method and application thereof. Aiming at the prevalence of the novel IBDV variant, the invention designs a gene expression box of main protective protein VP2 of the novel IBDV variant representative strain SHG19, introduces an element beneficial to the formation of the correct conformation of the IBDV variant, constructs a recombinant prokaryotic expression plasmid, prepares recombinant Escherichia coli genetic engineering bacteria, efficiently prepares novel IBDV variant virus-like particles through the exploration of expression conditions and purification conditions, and emulsifies the virus-like particles and adjuvants to prepare a vaccine, and test results show that the vaccine not only generates 100 percent immune protection on homologous novel variant strains, but also generates good immune protection on lethal attack of heterologous vvIBDV. The invention provides an effective technical means for preventing and treating novel IBDV variant strains and ultra-virulent strains.

Description

Novel variant subunit vaccine of chicken infectious bursal disease virus, preparation method and application thereof

Technical Field

The invention relates to a novel variant subunit vaccine of chicken infectious bursal disease virus, a preparation method and application thereof, in particular to a subunit vaccine consisting of novel variant VP2 protein virus-like particles of chicken infectious bursal disease virus, a preparation method and application thereof. The invention belongs to the technical field of veterinary medicines.

Background

Infectious Bursal Disease (IBD) is an important immunosuppressive disease of chickens, the etiology of which is Infectious Bursal Disease Virus (IBDV). The damage of IBDV to the poultry industry is divided into direct and indirect damage. Direct hazard: destroy the bursa of Fabricius of central immune organs and can kill directly. Indirect hazard: the low immunity of chicken infected and endured by IBDV causes vaccine immunity failure of other important diseases such as avian influenza, Newcastle disease and the like, and is easy to cause secondary infection, influence the production performance of chicken groups and cause serious economic loss. In conclusion, IBD seriously affects the health development of the poultry industry, and its preventive situation is very severe.

There are two serotypes of IBDV: serogroup I and serogroup II. The type I serum causes diseases to the chicken, and the type II serum does not cause diseases to the chicken. Over the past 60 years, serous type I IBDV has undergone two major mutations, one after the other of classical, variant and virulent strains. Since the last 90 s, the world is covered by Very virulent IBDV (vvIBDV) which is mainly characterized by acute and high lethality, and huge losses are caused to the poultry industry. Over the last 30 years of effort, vvIBDV infections are gradually being controlled based on factors such as increased levels of feed management and widespread use of vaccines. However, in recent years, new changes of IBD appear, atypical IBD is discovered in parts of chicken farms in China successively, has no obvious appearance symptoms, but the bursa of fabricius of the central immune organ is seriously shrunk, so that the severe immunosuppression and the reduction of production performance are caused, and the healthy development of the poultry industry is seriously threatened. Since 2017, the laboratory has first identified that the pathogen of the epidemic is a novel IBDV variant strain, the prevalence of the novel IBDV variant strain is detected in main poultry farming areas in China, and the epidemic is spreading and expanding continuously (Fan et al,2019,2020; Xu et al, 2019). Recently, novel IBDV variants have also become prevalent in Japan (Myint et al, 2020).

We found in previous researches that the novel IBDV variant not only causes damage to central immune organs and immunosuppression, thereby significantly affecting productivity, but also causes some vaccines against vvIBDV to have unsatisfactory protective effect due to the significant difference between antigenicity and vvIBDV. Therefore, it is necessary and urgent to develop vaccines against novel IBDV variants.

The subunit vaccine which does not contain complete virus particles and virus nucleic acid components has no risk of virulence reversion and gene recombination and high safety, and becomes a new direction for developing novel vaccines. Therefore, aiming at the prevalence of the novel IBDV variant strain, the invention designs the gene expression cassette of the main protective protein VP2 of the novel IBDV variant strain representative strain SHG19, introduces an original piece beneficial to the formation of the correct conformation of the IBDV variant strain, constructs a recombinant prokaryotic expression plasmid, prepares recombinant Escherichia coli genetic engineering bacteria, and efficiently prepares the novel IBDV variant strain virus-like particles through exploration of expression conditions and purification conditions, wherein the novel IBDV variant strain virus-like particles have good immunoreaction activity. Therefore, the proposal of the invention provides an effective technical means for preventing and treating novel IBDV variant strains and super virulent strains.

Disclosure of Invention

Aiming at the urgent problem that no vaccine is available for the prevention and control of the novel IBDV variant, the invention aims to provide a novel variant subunit vaccine of chicken infectious bursal disease virus, a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical means:

the invention designs a gene expression box of main protective protein VP2 of IBDV novel variant strain representative strain SHG19, introduces elements beneficial to the formation of correct conformation, constructs recombinant prokaryotic expression plasmids, prepares recombinant Escherichia coli genetic engineering bacteria, efficiently prepares IBDV novel variant strain virus-like particles (SHG19-VLP) through the exploration of expression conditions and purification conditions, emulsifies the SHG19-VLP with adjuvant to prepare vaccine, and evaluates the effectiveness of the vaccine on SPF chickens through immune response detection and challenge protection tests. The results of the experiments show that the subunit vaccine of the novel IBDV variant (SHG19-VLP) not only gave 100% immune protection against the homologous novel variant, but also gave good immune protection against lethal challenge with heterologous vvIBDV.

Specifically, the novel variant subunit vaccine of Infectious Bursal Disease Virus (IBDV) of the invention contains virus-like particles composed of optimized novel variant VP2 protein of infectious bursal disease virus, and the amino acid sequence of the optimized novel variant VP2 protein of infectious bursal disease virus is shown as SEQ ID NO. 2.

Preferably, the virus-like particle composed of the protein of the novel chicken infectious bursal disease virus variant VP2 is obtained by inserting a nucleotide sequence coding the optimized protein of the novel chicken infectious bursal disease virus variant VP2 into an expression vector, then transforming escherichia coli, and performing induced expression and purification.

Wherein, preferably, the nucleotide sequence for coding the optimized chicken infectious bursal disease virus novel variant VP2 protein is shown as SEQ ID NO. 1.

Preferably, the expression vector is a pCold I expression vector.

Among them, preferred Escherichia coli is E.coli Transetta (DE3) expression engineering bacteria.

Further, the invention also provides a method for preparing the subunit vaccine, which comprises the following steps:

(1) synthesizing to obtain a nucleotide sequence for coding the optimized novel variant strain VP2 protein of the chicken infectious bursal disease virus, and adding restriction enzyme cutting sites at two ends of the nucleotide sequence to obtain a target fragment;

(2) carrying out homologous recombination reaction on the target fragment obtained in the step (1) and a pCold I expression vector linearized by the same enzyme cutting site by using a homologous recombination kit, taking a homologous recombination reaction product to convert DH5 alpha escherichia coli after the reaction is finished, picking a monoclonal after 12 hours of conversion, screening a positive clone by using bacterial liquid PCR, carrying out sequencing identification on the positive clone, and naming the correctly identified recombinant expression plasmid as pCo-HHT28-SHG19VP 2-466;

(3) pCo-HHT28-SHG19VP2-466 is transformed into E.coli Transetta (DE3) expression engineering bacteria, a monoclonal colony is selected 12 hours after transformation and inoculated into an ampicillin resistance LB liquid culture medium for culture, a bacterial liquid is harvested, RT-PCR identification is carried out, the correctly identified positive bacteria are Escherichia coli genetically engineered bacteria Transetta (DE3) -SHG19VP2 strain strains, and the strains are stored at the temperature of minus 80 ℃;

(4) expression of recombinant proteins

Inoculating Escherichia coli genetically engineered bacterium Transetta (DE3) -SHG19VP2 strain to ampicillin resistant LB culture medium, when OD is reached₆₀₀When the temperature reaches 0.6 ℃, taking out the shake flask from the shaking table, quickly cooling the shake flask on ice, adding IPTG into the shake flask, and continuously culturing on the shaking table;

after induction expression is finished, transferring the bacterial liquid into a centrifuge tube, centrifuging, removing a culture medium, fully suspending the bacterial precipitate by using a PB buffer solution, then performing high-pressure crushing on the bacterial by using a high-pressure cell crusher, centrifuging the crushed bacterial liquid, and removing the precipitate to obtain a solution containing the novel variant strain VP2 protein of the chicken infectious bursal disease virus;

(5) purification of recombinant proteins

The obtained solution containing the novel variant VP2 protein of the chicken infectious bursal disease virus is subjected to ammonium sulfate precipitation to obtain the preliminarily purified novel variant VP2 protein of the chicken infectious bursal disease virus, then the purified novel variant VP2 protein of the chicken infectious bursal disease virus is further obtained by a molecular sieve method, and the negative staining electron microscope result shows that the purified novel variant VP2 protein of the chicken infectious bursal disease virus forms virus-like particles;

(7) preparation of vaccines

And (3) mixing the virus-like particles obtained in the step (5) with a white oil adjuvant according to the volume ratio of 1:2, and then fully emulsifying to obtain the novel variant subunit vaccine of the infectious bursal disease virus.

Wherein, preferably, KpnI restriction enzyme cutting sites and EcoRI restriction enzyme cutting sites are respectively added at two ends of the nucleotide sequence for coding the optimized novel chicken infectious bursal disease virus variant VP2 protein.

Furthermore, the invention also provides application of the subunit vaccine in preparing a medicament for preventing and treating the infectious bursal disease virus.

Preferably, the chicken infectious bursal disease virus comprises a novel IBDV variant strain and an IBDV super virulent strain.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention designs the gene expression box of main protective protein VP2 of IBDV novel variant strain representative strain SHG19, introduces elements beneficial to the formation of correct conformation, constructs recombinant prokaryotic expression plasmid, prepares recombinant Escherichia coli genetic engineering bacteria, and efficiently prepares IBDV novel variant strain virus-like particles (SHG19-VLP) through groping of expression conditions and purification conditions;

(2) the SHG19-VLP is matched with the antigen of the current epidemic IBDV novel variant strain;

(3) the SHG 19-VLP-based subunit vaccine can generate high-efficiency immune protection on novel IBDV variant strains and ultra-virulent strains.

Drawings

FIG. 1a is a representation of a segmented amino acid sequence evolutionary tree of IBDV isolate VP 2;

FIG. 1b shows a tree representation of the amino acid sequence evolution of the IBDV isolate VP 1.

Among them, red, novel variant; purple red, low virulent strain; light purple, classical strain; blue, ultra-strong toxicity; green, early variant; orange, serum type II; black filled circles, isolate strains in this study.

FIG. 2a is a dendron of the amino acid sequence of the polyprotein of SHG 19;

FIG. 2b is a VP1 amino acid sequence evolutionary tree of SHG 19;

FIG. 2c is a characteristic amino acid alignment of VP5, polyprotein, and VP1 of SHG19

Wherein, Var: variant strains; VV: ultra-strong toxicity; AT: a low virulent strain.

FIG. 3 shows the pathogenicity evaluation of SHG19 strain IBDV on SPF chickens;

wherein (a) body weight change; (b) kinetic profile of BBIX; (c) spleen body weight ratio; (d) pathological change of bursal tissue

FIG. 4 is an evaluation of the horizontal transmission ability of SHG19 strain IBDV;

wherein, (a) serum antibody titer; (b) viral RNA copy number in bursa of fabricius; (c) BBIX; (d) spleen body weight ratio;

FIG. 5 shows the immunosuppression of the novel IBDV variant against avian influenza vaccines;

FIG. 6 shows the immunoprotection evaluation of IBDV supervirulent vaccine against novel IBDV variant strains;

wherein, (a) IBDV antibody titres; (b) BBIX; (c) spleen body weight ratio; (d) bursa of fabricius; (e) pathological changes in bursal disease;

FIG. 7 shows the preparation and identification of VLPs of novel IBDV variants;

carrying out Western-blot detection on A.SHG19-VP 2; SHG19-VP2 (M, 1, 2, 3 are Protein marker (stabilized Protein ladder), sample before purification, sample after purification by saturated ammonium sulfate precipitation method, and sample after molecular sieve purification); negative staining electron microscopy of SHG19-VLP; agar diffusion assay results of SHG19-VLP;

FIG. 8 shows the results of challenge immunization test of SHG19-VLP vaccine against novel IBDV variants;

wherein, A, neutralizing antibody to IBDV novel variant antigen (rGtVarVP 2); B. neutralizing antibodies to the IBDV virulent strain antigen (rGtHLJVP 2); C.B/B value; D. bursa of Fabricius and microscopic pathological sections thereof;

FIG. 9 shows the results of the challenge immunization test of SHG19-VLP vaccine against virulent IBDV strain.

Wherein, a neutralizing antibody against IBDV super virulent antigen (rgthhljvp 2); B. survival rate; C. bursa of Fabricius and microscopic pathological sections thereof.

Detailed Description

The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description of the specific embodiments proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

EXAMPLE 1 isolation and characterization of novel variant of infectious bursal disease Virus, SHG19

1. Materials and methods

1.1 clinical sample Collection and handling

In 2017, in 9 months, 36-day-old broilers in a chicken farm in Chuzhou city, Anhui province show suspected clinical symptoms of IBD. The sick chicken shows lassitude, and the bursa of Fabricius is seriously atrophied by the autopsy. Adding a proper amount of PBS into a tested diseased chicken bursa of Fabricius sample, grinding, repeatedly freezing and thawing for three times, centrifuging the prepared suspension for 5min at 4 ℃ at 5000g, and taking the supernatant for subsequent detection.

1.2 SPF chickens, viruses and major agents

Specific-pathogen-free (SPF) chickens and SPF chick embryos are provided by the experimental animal center of harbourne veterinary institute, china agro-scientific institute, and SPF chickens are raised in negative pressure isolators. DT40 cells were maintained by the poultry immunosuppressive disease team (hereinafter referred to as the laboratory) of Harbin veterinary institute of Chinese academy of agricultural sciences.

Representative strains of IBDV (vvIBDV) in China, Gx (Wang et al, 2004) and HLJ0504(Qi et al, 2011), were isolated and identified in the laboratory. Single factor sera of these three viruses, as well as monoclonal antibody to the IBDVVP2 protein (strain 7D 4) were prepared in the laboratory. The avian influenza bivalent inactivated vaccine (H5+ H7) is a product of Harbin Vitaceae biotechnology limited company.

RNAasso Plus is available from TAKARA; the M-MLV reverse transcription kit is purchased from Invitrogen company; prime STARTM HS DNApolymerase, Ex TaqPolymerase, pMD18-T vector, RNAioso Plus, DL2000 DNAmarker are all products of Dalibao bioengineering, Inc. (Takara); the nucleic acid gel recovery kit and the small quality-improving particle kit are AxyPrep products. The fetal bovine serum is an AusBian product; EDTA-pancreatin digestive juice, penicillin two antibiotics for Harbin national biological science and technology products; DMEM, Opti-MEM, 1640 is Thermo Scientific product.

1.3 extraction of RNA and RT-PCR detection

200 μ L of the above-mentioned pathological suspension was taken, total RNA was extracted from the sample according to the instruction of RNAioso Plus, and cDNA was synthesized according to the procedure provided in the M-MLV reverse transcription kit. Detection of the representative segment of IBDVVP2 was performed using the cDNA as a template and the forward primer 2U and the reverse primer 2L (Table 1). The PCR procedure was: at 95 ℃ for 5min, at 95 ℃ for 30s, at 56 ℃ for 30s, at 72 ℃ for 45s, for 35 cycles; 5min at 72 ℃. The expected amplification length is 930bp by detecting the RT-PCR result by using 1% agarose gel electrophoresis.

The VP2 amplified positive samples, further using B464U/B1718L as primers (Table 1), and amplifying VP1 gene representative segment by PCR, the reaction procedure is: 5min at 95 ℃; 35 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 1 min; 10min at 72 ℃. The expected amplification length is 1255 bp.

The positive PCR product is sent to Jilin province Kuumei Biotechnology limited company for sequencing, and the effective sequence of VP2 is determined to be 777bp (547-1323 bp of VP2 gene, corresponding amino acid is 183-441 aa); the effective sequence of VP1 was determined to be 1152bp (511-1662 bp of VP1 gene, corresponding amino acid 134-517 aa). The VP2 representative segment and VP1 representative segment sequences of the determined SHG19 strain are uploaded to GenBank respectively.

1.4 isolation of the Virus

Virus isolation was performed using the SPF chick embryo method. 200 mu L of the disease material suspension is inoculated with 9-day SPF chick embryos through a chorioallantoic membrane path, 5 chick embryos are inoculated in each generation, and the control is performed for 3 days and 5 days. Culturing in 37 deg.C incubator, observing the clinical symptoms and death condition of chick embryo every day, and discarding dead embryo within 24 h. And 5 days after inoculation, performing autopsy on the chick embryos died in the period and the chick embryos which are resistant to the chick embryos, collecting bursa of fabricius, allantoic membranes and allantoic fluid, mixing and grinding the mixture to prepare a suspension, performing RT-PCR detection, and performing passage on new 9-day-old SPF chick embryos for three blind generations. Collecting the bursa of Fabricius, allantoic membrane and allantoic fluid suspension of the third generation chick embryo, and identifying the specificity and purity, namely obtaining the IBDV (chicken embryo virus) obtained by separation, which is named as SHG19 strain.

1.5 amplification and sequencing of the Virus Whole genome

To further characterize the molecular characteristics of SHG19, the present study amplified the viral genome. The IBDV genome A segment was amplified in two sections (two RT-PCR products overlapping each other) using primers AU/A1542L and A1421U2/AL2 (Table 1); the IBDV genome B-segment was amplified in two segments (two RT-PCR products overlapping each other) using primers BU/B1344L and B1344U/BL (Table 1). The RT-PCR products are respectively cloned into pMD18-T vectors after being purified, and the recombinant plasmids are sent to Jilin province Cumei biotechnology limited for sequencing.

TABLE 1 primers

1.6 analysis of the genetic evolution of the Virus

The SHG19 strain virus was subjected to sequence analysis using the software Clustal X program (version 2.0) (Larkin et al, 2007) and MEGA (version 3.1) (Kuma et al, 2004). All reference strains of each type are shown in table 2. The new IBDV variant strains newly isolated in China (all isolated in the laboratory) are shown in Table 3, the isolation regions of the IBDV variant strains cover 13 provinces (cities) such as Heilongjiang, Liaoning, Hebei, Beijing, Shandong, Jiangsu, Anhui, Guangxi, Shanxi, Yunnan, Zhejiang, Fujian, Hubei and the like, and the epidemic of the IBDV variant strains is spreading continuously.

TABLE 2IBDV reference strains

TABLE 3 novel IBDV variant isolated recently in China

1.7 pathogenicity study of novel IBDV variant SHG19

To investigate the pathogenicity of the IBDV novel variant SHG19 on chickens, 50 SPF chickens aged 16 days were randomized into 3 groups, with group 1 (10) and group 2 (15) being infection groups and group 3 (25) being placebo groups. SPF chickens from

groups

1 and 2 infected 8X 10 at 16 days of age by nasal drip⁶Group 3 of viral RNA copies (200. mu.L) of IBDV SHG19 strain were given the same volume (200. mu.L) of sterile PBS by the same route. 1-5 days after infection (day post-infection, dp.i.), 3 chickens in each of the

groups

2 and 3 were dissected randomly each day, the body weights were weighed, bursa and spleen were collected and weighed, pathological changes of the heart, liver, lung, kidney, thymus, leg and chest muscles were observed, and appropriate bursa and spleen samples were taken and soaked in 10% formalin to prepare sections for histological observation. The bursa-weight index (BBIX) of the dissected chickens was calculated from the bursa weight and body weight of Fabricius. The bursa weight ratio (bursal weight/body weight) x 1000; BBIX ═ test group chicken bursa weight ratio/blank control group chicken mean bursa weight ratio; when BBIX<At 0.7, bursal atrophy is judged (Lucio and Hitchner, 1979). The spleen-to-body ratio was calculated from the spleen weight and body weight, which is (spleen weight/body weight) × 1000. Group 2 chickens were used for pathogenicity observation of SHG19 infection, and clinical symptoms (no necropsy) were observed daily after infection, and clinical symptom index, morbidity, and mortality were counted up to 25 days after infection, and weight was weighed for surviving chickens, and changes in weight were observed.

To evaluate the horizontal transmission of the novel IBDV variants, 5 additional uninfected SPF chickens with the same background were co-housed with the existing 10 infected chickens in group 1 isolators and the remaining 10 chickens were dissected out in group 3 and used as controls. The clinical symptoms of the chickens were observed every day, after 25 days of co-residence, 5 chickens were randomly selected per group, the body weights were weighed, the BBIX and spleen body ratios were calculated, the pathological changes of bursa of Fabricius were observed, and the IBDV copy number in bursa of Fabricius was detected using fluorescent quantitative RT-PCR.

1.8 immunosuppressive Studies of the novel IBDV variant SHG19

The immunosuppression of chicken infected by SHG19 IBDV was evaluated by an avian influenza vaccine immunoassay. 30 SPF laying hens aged 16 days were randomly divided into 3 groups of 10 eggs. At 16 days of age, group 1 was infected with SHG19 by nasal drop and eye drop at a dose of 8X 10⁶viral RNAcopies/mouse.

Groups

1 and 2 were immunized by the leg muscle injection route against the avian influenza bivalent inactivated vaccine (H5+ H7) at 4dp.i., 300 μ L per chicken per product instructions. Group 3 is a blank control. At 0, 7 and 14 days after immunization, sera were collected from blood and examined for antibody titers against H5 and H7 by hemagglutination inhibition assay (HI).

1.9 immunoprotection test of IBDV Ultrasvery virulent vaccine against novel IBDV variants

In order to evaluate the immune protection effect of IBDV (very virulent infectious bursal disease Virus) Vaccine on novel IBDV variant strains, three commercial vaccines aiming at the very virulent IBDV (very virulent infectious bursal disease Virus) are selected for carrying out immune protection tests, namely, attenuated Vaccine (Vaccine A), subunit Vaccine (Vaccine B) and multiple Vaccine (Vaccine C). 30 SPF chickens at 1 day of age were randomly divided into 5 groups of 6 chickens. Group 1 immunised VaccineA by nasal drop route at 10 days of age, according to the instructions for use of the vaccine;

groups

2 and 3 were immunized by leg muscle injection at 10 days of age for Vaccine B and Vaccine C, respectively; group 4 was inoculated with PBS at 10 days of age as a non-immune control group; group 5 served as blank control. All experimental chickens were collected offshoot at 17, 24 and 27 days of age, and serum IBDV antibodies were detected using an avian infectious bursal disease Virus antibody detection kit. At 28 days of age, the chickens in groups 1 to 4 were infected with SHG19 by nasal drip and eye drop at a dose of 8X 10⁶viral RNA copies/mouse. 5dp.i. and 10dp.i., 3 chickens per group were randomly necropsied, weighed, the pathological changes of heart, liver, spleen, lung, kidney, thymus and bursa of fabricius were observed, bursa of fabricius and spleen were weighed, and the BBIX and spleen body ratio was calculated. And (3) soaking part of bursa of Fabricius of the cesary-examined chicken in 10% formalin solution, and observing pathological changes after HE staining. Treating bursa of Fabricius sample, extracting RNA, reverse transcribing to synthesize cDNA, and detecting bursa of Fabricius by IBDV fluorescent quantitative PCR methodIBDV in (1).

1.10 Cross-neutralization assay of novel IBDV variants with ultra-virulent sera

To understand if there is an antigenic difference between the novel IBDV variant and the very virulent strain (vvIBDV), the present study performed in vitro serocross-neutralization assays using representative strains of IBDV, SHG19 and vvIBDV, Gx and HLJ0504, as well as the respective single-factor sera. Serum cross-neutralization assays were performed on DT40 cells.

TCID50 determination of IBDV: TCID of three strains of IBDV (SHG19, Gx, HLJ0504) on DT40 cells₅₀Detection was performed using IBDV monoclonal antibody-mediated indirect immunofluorescence assay (IFA). After inoculating 10-fold dilution of IBDV into DT40 cells and allowing to feel for 24h, the virus titer was determined by the IFA method. The specific operation steps are as follows: discarding the cell culture solution, adding appropriate amount of 4% paraformaldehyde, and fixing at room temperature for 20 min; discarding paraformaldehyde, washing cells with PBS 3 times, adding appropriate amount of IBDVVP2 protein murine monoclonal antibody (7D4, 1:500 dilution), and incubating at 37 deg.C for 1 h; discarding the monoclonal antibody, washing the cells for 3 times by PBS, adding a proper amount of FITC-labeled anti-mouse fluorescent secondary antibody (diluted 1: 200), and incubating for 1h in a wet box at 37 ℃ in the dark; the fluorescent secondary antibody was discarded, and the cells were washed 3 times with PBS, and finally 100. mu.L of PBS was added to each well and observed under an inverted fluorescent microscope. Those with green fluorescence signals are IBDV infection positive wells.

Serum cross-neutralization assay:

the neutralizing titer of SHG19 single factor serum against three strains of IBDV (SHG19, Gx, HLJ0504) was first determined. 2-fold dilution of SHG19 single factor serum; then mixing SHG19, Gx and HLJ0504 of 200TCID50 and diluted SHG19 serum respectively and incubating for 1h in an incubator at 37 ℃; 100 μ L of the virus serum mixture was added to DT40 cells cultured in a 96-well plate, and the cells were further cultured in a cell culture incubator at 37 ℃ for 24 hours, and the neutralization titer of SHG19 single-factor serum against three strains of IBDV was determined by the IFA method, respectively. In addition, the neutralizing titers of the Gx single-factor serum and the HLJ0504 single-factor serum against three strains of IBDV (SHG19, Gx, HLJ0504) were determined in a similar manner.

The cross-antigen correlations (R values) of three single factor sera to three strains of IBDV (SHG19, Gx, HLJ0504) were calculated using the method proposed by Archetti and Horsfall (1950) (Archetti and Horsfall, 1950). R value calculation method between two IBDVs: r2 ═ R1 x R2; heterologous neutralizing titer of virus 2/homologous neutralizing titer of virus 1; r2 ═ heterologous neutralizing titres of virus 1/homologous neutralizing titres of virus 2. A homologous R value is defined as 1, with an R value equal to 1 or close to 1 indicating antigenic similarity between the two test virus strains. The correlation between the R value and the antigenicity of the virus is as follows: 0-0.10, there is serotype difference between the two viruses; 0.11-0.70, the two viruses have subtype difference; above 0.70, there was no or less difference between the antigenicities of the two viruses (Chen et al, 2018; Jackwood and Saif, 1987).

2. Results

Isolation and Gene cloning of SHG19

IBDV strain SHG19 was successfully isolated from the disease material. VP2 of SHG19 represents the segment and VP1 represents the segment GeneBank accession numbers MH879092 and MH879045, respectively; the genome A, B segments GeneBank accession numbers are MN393076 and MN393077, respectively. Sequencing results show that the full length of the A segment of the SHG19 is 3260bp, including 84bp of 5 'non-coding region, 450bp of VP5 protein gene, 3039bp of PP protein gene and 91bp of 3' non-coding region; the full length of the B segment of the SHG19 is 2827bp, including 111bp of 5 'non-coding region, 2640bp of VP1 protein gene and 76bp of 3' non-coding region.

Analysis of genetic evolution of SHG19

The SHG19 strain, different types of reference strains of 27 strains and 81 novel IBDV variant strains isolated and identified in the laboratory are subjected to gene homology and genetic evolution analysis. The amino acid sequence evolutionary tree based on the VP2 gene representative segment shows that the serous type I IBDV is obviously divided into four branches, namely a classical strain, a super-virulent strain, a variant strain and a low virulent strain. Among the variant strains, the 81 IBDV novel variants were in two completely different subgroups from the early US variant; SHG19 was in the same subgroup as the 81 IBDV novel variant (FIG. 1 a). The amino acid sequence comparison of the novel IBDV variants SHG19 and 81 with the American early Variant shows that the homology rates with the early variants such as Variant E, E/Del, Variant A, 9109, GLS are respectively 95.7% -97.3%, 94.1% -95.7%, 95.2% -96.8%, and 92.5% -94.6%, and the novel IBDV variants SHG19 and 81 have 3 unique amino acid sites 217K, 252I and 299S in the high mutation region sequence of VP 2. The evolutionary tree based on the VP1 gene representative segment amino acid sequence shows that IBDV is obviously divided into two branches, namely a super-virulent strain branch and a non-super-virulent strain branch; the non-hyperviscous strains included variants, attenuated strains and classical strains, and the SHG19 and other novel IBDV variants identified in this study also belong to non-hyperviscous strains but form separate subgroups, and 147D and 508K are also different from the early American variants (FIG. 1 b). The above data indicate that SHG19 belongs to the novel IBDV variant and can be used as a representative strain of the novel IBDV variant.

To further explore the molecular characteristics of the novel variants, we cloned the entire genome of the SHG19 strain. Evolutionary trees based on the amino acid sequences of PP (FIG. 2a) and VP1 (FIG. 2b) showed that SHG19 is in the IBDV variant branch. However, SHG19 also exhibits unique molecular characteristics among IBDV variants. SHG19 has 10 different amino acid positions in PP compared to the U.S. reference IBDV variants, including 73I, 77D, 79S, 187V, 221K, 252I, 299S, 451L, 922Q, and 951L. For VP5, SHG19 has only 96.4% to 96.8% homology with us variants with eight different amino acid positions, 7R, 44P, 78L, 92R, 104G, 109R, 116V and 147E, which have a tetrapeptide (MLSL) deletion at the N-terminus of VP5, and no such deletion for SHG 19. In addition, VP1 of SHG19 also presents a unique amino acid position (508K) (fig. 2 c). The biological significance of these characteristic amino acids is to be investigated further.

2.3 pathogenicity of novel variant IBDV strains

Within 25d of SHG19 infection, the group 1 chickens did not die and no obvious clinical symptoms were observed, whereas body weight was significantly reduced at 25dp.i. (P <0.01) (fig. 3 a). Pathological lesions caused by SHG19 were further evaluated by necropsy, with 3 chickens in each of group 2 and group 3 being randomized by necropsy each day. The bursa of Fabricius was observed to have inflammatory exudation, bleeding and yellow staining at 3-5dp.i. in group 2 chickens, with BBIX below 0.7 at 3dp.i. to drop to 0.24 at 5dp.i. (FIG. 3 b). In addition, the splenomegaly ratio of SHG19 infected chickens in group 2 was significantly higher than the control group, especially at 4dp.i. (fig. 3 c). Pathological section observation results show that the bursa of the infected chickens in the group 2 have obvious histopathological injury, starting from 1dp.i., the lymphocytes are reduced, macrophage infiltration is observed in the follicles, proliferation of fibrous tissues is found around the follicles at 3dp.i., and severe atrophy of the follicles is observed at 5 dp.i.; no lesions were found in the control group (fig. 3 d). These data demonstrate that the novel IBDV variant is highly pathogenic in chickens.

In chickens co-housed with the SHG 19-infected group, IBDV-specific antibodies were detected by ELISA 25 days after co-housing (FIG. 4a), suggesting that horizontal transmission of novel IBDV variants occurred. The results of fluorescent quantitative RT-PCR at 25 days after the same residence show that the average titer of IBDV in the bursa of Fabricius of the same residence chicken reaches 7.9 multiplied by 10⁷viral RNA copies/gram tissue (FIG. 4b), further confirming the horizontal transmission of the novel IBDV variant. The results of 25 days post-mortem examination of the same chicken showed severe atrophy of the bursa of fabricius with an average BBIX of 0.29 (FIG. 4 c). Furthermore, the splenomes of the co-housed chickens were significantly lower than the control 25 days after co-housing (FIG. 4 d). This suggests that the novel IBDV variant may spread the infection by horizontal transmission.

2.4 immunosuppression of novel IBDV variant on chickens

To evaluate the immunosuppressive effect of IBDV variants on chickens, the effect of SHG19 on the immune efficacy of an avian influenza vaccine was examined after infection of SPF chickens with SHG 19. 16 day old layers were infected with SHG19 and immunized against a bivalent inactivated (H5+ H7) avian influenza vaccine at 4dp.i. At 14d post-immunization, sera were collected for Hemagglutination Inhibition (HI) antibody titers. The HI titers of 14d, H5(P <0.05) and H7(P <0.05) were significantly inhibited in the SHG 19-infected group after vaccination with avian influenza vaccine compared to the uninfected group (fig. 5).

2.5 immune protection evaluation results of IBDV super-virulent vaccine against novel IBDV variant

In order to evaluate the immune protection effect of the existing commercial vaccines on the novel IBDV variant, the present study examined the virus-attacking protection of three vaccines (attenuated Vaccine A, subunit Vaccine B, and concatemeric Vaccine C) against vvIBDV on the novel IBDV variant SHG 19. The results showed that all three vvIBDV vaccine immunization groups were seropositive for IBDV at 27 days of age (FIG. 6 a). At 28 days of age, three vvIBDV vaccine immunised groups were challenged with the novel IBDV variant SHG19, except for group 5, which served as a blank control. Compared with the blank control group, the three immunity counteracting groups show severe bursal damage at 5dp.i. and 10dp.i. and are characterized by atrophy, yellow stain and hard texture; the bursa of Fabricius in group 4 (non-immune challenge control) also exhibited severe atrophy and lesions similar to the immune challenge group (FIG. 6 d). The BBIX assay results also showed that the bursa of Fabricius of the three immuno-challenge groups were atrophic, the BBIX values at 5dp.i for the three Vaccine groups, Vaccine A, Vaccine B, and Vaccine C were 0.453 + -0.188, 0.266 + -0.078, and 0.380 + -0.043, respectively, and the BBIX values at 10dp.i were 0.606 + -0.114, 0.235 + -0.079, and 0.293 + -0.063, respectively (FIG. 6B). The fluorescent quantitative RT-PCR results also showed the presence of SHG19 was detected in diseased bursa of fabricius. In addition, spleen to body ratio results showed spleen swelling in Vaccine C group at 5dp.i. (fig. 6C). Histopathological examination showed a decrease in bursa of fabricius lymphocytes, macrophage infiltration, follicular atrophy, and connective tissue proliferation in the three immunization groups compared to the control group at 5dp.i. (fig. 6 e). This indicates that the three vvIBDV vaccines have unsatisfactory challenge protection effect on IBDV novel variant SHG 19.

2.6 Cross-neutralization test results of novel IBDV variant and ultra-virulent serum

The antigenic association between a novel IBDV variant (SHG19) and a very virulent strain (Gx and HLJ0504) was explored by an in vitro serum cross-neutralization assay. The results of the study showed that the R value of the antigen correlation between the very virulent IBDV strain Gx and HLJ0504 was 1.05 (Table 4), indicating that there was no significant difference in antigenicity between the two. However, the R values of the IBDV novel variant SHG19 and Gx and HLJ0504 were 0.64 and 0.35 (Table 4), respectively, which indicates that SHG19 has a significant difference from the antigenicity of Gx and HLJ 0504.

The challenge protection test and the serum cross-neutralization test of the vaccine show that the antigenicity of the novel IBDV variant is obviously different from that of vvIBDV. The potential molecular basis was revealed by genomic alignment in this study. SHG19 showed a clear difference in both PP and VP1 compared to vvIBDV. In PP, the other 17 characteristic amino acids of SHG19 listed in FIG. 2c, except for amino acid residues 253, 284, 299, 330, 451, 541, 981 and 1005, differ from vvIBDV, and these differences may be associated with antigenic variation. Of the 15 characteristic amino acids listed in FIG. 2c, VP1, 13 of SHG19 differed from vvIBDV, which may be associated with differences in virulence.

TABLE 4 serum cross-neutralization assay of novel IBDV variants with ultra-virulent strains

Serum/strain	SHG19	Gx	HLJ0504
				SHG19	1.00
Gx	0.64	1.00
				HLJ0504	0.35	1.05	1.00

EXAMPLE 2 preparation of novel variant Virus-like particles of IBDV

1. Materials and methods

1.1 Virus, plasmid and Strain

A representative strain of IBDV, SHG19(Fan et al,2019), was isolated and stored as described in example 1. The pCold-I prokaryotic expression plasmid is TaKaRa product. Coli engineering strains DH5 alpha and E.coli Transetta (DE3) are products of Beijing Quanji Biotech, Inc.

1.2 Primary reagents

The M-MLV reverse transcription kit is a product of Invitrogen. RNAioso Plus, Primersar DNA Polymerase is TaKaRa product. Restriction enzymes KpnI and EcoRI, PrestatinedProtein Ladder is TheromoFisher Scientific product. The gel recovery kit is an Axygen product. The homologous group kit is a product of Nanjing NuoZan Biotechnology GmbH. Sepharose 6 Fast Flow column chromatography is GE product. Ammonium sulfate is a product of chemical reagents of national drug group. IBDV VP2 protein monoclonal antibody was prepared and stored in the laboratory. The IBDV standard positive serum and IBDV standard antigen are products of Haerbin national Biotechnology GmbH.

1.3 construction of recombinant expression plasmids

The VP2 gene of a representative strain SHG19 of the IBDV novel variant is selected for prokaryotic expression and VLP preparation.

1.3.1 design of the expression cassette for VP2 of SHG19 Strain

In order to facilitate the soluble expression and the correct assembly of the VP2 gene of the SHG19 strain, a VP2 gene expression cassette is designed. pVP2 encodes 511 amino acids, and during virus maturation assembly, pVP2 becomes mature VP2 (encoding 440 amino acids) by C-terminal self-cleavage. At the N-terminus of pVP2, there are four alpha helices, the first two of which favour the formation of the correct conformation of VP 2. Therefore, SHG19VP2 expressed in this example retained two alpha helices at the C-terminus, and had a total length of 465 amino acids (aa 2-466) (except for the start codon), designated SHG19-VP 2-466. At the N-terminus of SHG19-VP2-466, HT28 was fused. HT28 is a special 6 His-containing tag encoding 27 amino acids (except for the start codon) that mimics the interaction of the C-terminus of IBDV VP3 with VP2 to facilitate the formation of the correct conformation of VP 2. The nucleotide sequence of the VP2 gene expression cassette is shown in SEQ ID NO. 1.

1.3.2 construction of recombinant expression plasmids

1.3.2.1 primer design

Primers (Table 5) were designed based on the gene sequence of SHG19 strain (GenBank accession No.: MN393076) and the HT28 tag sequence, and used for amplification of SHG19-VP2 of SHG19 strain and fusion of HT28 tag. The primers were synthesized by Jilin Kuumei Biotech, Inc.

TABLE 5 primers

Note: the font bold sequence is a homologous arm sequence; HT28 sequence is highlighted underlined; the cleavage sites KpnI (GGTACC) and EcoRI (GAATTC) are indicated in italics.

1.3.2.2IBDV genome extraction and cDNA Synthesis

200. mu.L of SHG19 strain virus liquid was taken, total RNA was extracted with RNAioso Plus (Takara) according to the product specification, and the extracted RNA was reverse transcribed into cDNA using M-MLV reverse transcription kit (Invitrogen) according to the product specification.

1.3.2.3 amplification of fragments of interest

The desired fragment was obtained by two-step PCR. First, a first PCR was performed using the cDNA of 1.3.2.2 as a template and primers HT28-F2 and SHG19-VP 2-R. The PCR reaction system is shown in Table 6. The reaction procedure is as follows: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃, 1min at 72 ℃ and 30s for 35 cycles; 10min at 72 ℃. After the reaction product was identified by 1% agarose gel electrophoresis, the target fragment was recovered using a gel recovery kit (AxyPrep) according to the instructions. The second PCR reaction takes the first PCR product as a template and utilizes primers HT28-F1 and SHG19-VP2-R for amplification. The reaction system is as shown in Table 6; the reaction procedure was the same as the first step PCR reaction procedure. After the reaction product was identified by 1% agarose gel electrophoresis, the target fragment was recovered using a gel recovery kit (AxyPrep) according to the instructions.

TABLE 6 PCR reaction System

1.3.2.4 construction of expression plasmids and identification of Positive clones

And (3) carrying out homologous recombination reaction on the target fragment recovered from the gel in 1.3.2.3 and the pCold I expression vector linearized by KpnI and EcoRI by using a homologous recombination kit. The reaction system is shown in Table 7, and the reaction condition is 37 ℃ for 30 min. And after the reaction is finished, 10 mu L of homologous recombination reaction product is taken to transform DH5 alpha escherichia coli, monoclonal antibody is picked up 12 hours after transformation, positive clone is screened by using bacterial liquid PCR, and sequencing identification is carried out on the positive clone. The correctly identified recombinant expression plasmid was designated pCo-HHT28-SHG19VP 2-466.

TABLE 7 homologous recombination reaction System

1.4 preparation of recombinant engineering bacteria

pCo-HHT28-SHG19VP2-466 is transformed into E.coli Transetta (DE3) expression engineering bacteria, a monoclonal colony is selected 12 hours after transformation and inoculated into an ampicillin resistant LB liquid culture medium, the culture is carried out for 12 hours at 37 ℃ and 220rpm, a bacterial liquid is obtained, and the bacteria are subjected to RT-PCR identification by using primers HT28-F1 and SHG19-VP2-R according to a method of 1.3.1. The positive bacteria are Escherichia coli genetically engineered bacteria Transetta (DE3) -SHG19VP2 strain, mixed with 50% glycerol freeze-drying protective agent in equal amount, subpackaged, stored at minus 80 ℃ in 1 mL/tube.

1.5 expression of recombinant proteins

Inoculating the recombinant strain with LB culture medium with ampicillin resistance at a ratio of 1%, culturing at 37 deg.C and 220rpm for 2 hr, and measuring OD with spectrophotometer₆₀₀. When OD is reached₆₀₀When the temperature reached 0.6, the flask was taken out of the shaker and placed on iceAnd rapidly cooling for 10 min. IPTG was added to the flask to a final concentration of 0.2mM and the cultivation was continued on a shaker at 22 ℃ and 180rpm for 20 h.

After the induction expression is finished, transferring the bacterial liquid into a centrifuge tube, centrifuging for 10min at 8000g and 4 ℃, removing the culture medium, and fully suspending the bacterial sediment by using a PB buffer solution (pH6.5) with the bacterial weight of 10 times. The cells were then disrupted by high pressure using a high pressure cell disruption apparatus (homogenic Systems Ltd.) at a disruption pressure of 120 Pa. And centrifuging the crushed bacterial liquid at 8000g and 20 ℃ for 10min, discarding the precipitate, and carrying out Western-blot detection on a supernatant sample, wherein the antibody is an anti-IBDVVP 2 monoclonal antibody.

1.6 purification of recombinant proteins

1.6.1 ammonium sulfate precipitation method for crude and pure target protein

The protein solution was placed on a magnetic stirrer and stirred slowly while an equal volume of saturated ammonium sulfate solution was added slowly to the protein solution. After stirring for 5min, the protein solution was transferred to a centrifuge tube and centrifuged at 1000g at 25 ℃ for 10 min. After centrifugation, the supernatant was discarded (the supernatant was drained to remove ammonium sulfate as much as possible), and the pellet was thoroughly resuspended in PB buffer (pH 6.5). The insoluble protein was removed by centrifugation at 1000g for 10 minutes at 25 ℃. After completion of the centrifugation, the supernatant was filtered through a 0.22 μm filter, and the filtrate was used for the next purification.

1.6.2 molecular Sieve method for further purification of proteins of interest

Balancing: a PB solution (pH6.5) was used to equilibrate the Sepharose 6 Fast Flow column at a Flow rate of 2.5mL/min for 2 column volumes. Loading: the protein sample prepared in 1.6.1 was injected into a 10mL sample cup using a syringe, the system Buffer flow path was switched, and 10mL of the protein sample in the sample cup was injected into a well-balanced Sepharose 6 Fastflow chromatography column at a constant flow rate (2.5 mL/min). Collecting: frac950 automatically started collection when UV280 signal of the UV detector was above 20mAU, every 14mL, and then SDS-PAGE detection was performed on the collected samples.

1.7 identification of Virus-like particles

1.7.1 Electron microscopy identification

The samples were negatively stained with uranyl acetate and observed with a transmission electron microscope (HITACHI H-7650) to determine whether the expressed VP2 samples formed IBDV virus-like particles (VLPs), designated SHG 19-VLP.

1.7.2 agar diffusion test identification

The specificity and the agar diffusion potency of the SHG19-VLP are detected by using agar diffusion test (agar diffusion for short). The specific method comprises the following steps: firstly, agar diffusion test plates are prepared by a conventional method. And secondly, punching by using a hexagonal puncher, wherein the aperture is 4mm, and the hole interval is 3 mm. After picking up the agar in the well, the bottom of the dish was slightly heated to slightly melt the agar at the bottom of the well and the bottom was sealed. ③ adding 20 mu L of IBDV positive serum into the middle hole; 2-fold dilution is carried out on the SHG19-VLP sample, and 22, 23, 24 and 25-fold diluted samples are sequentially and respectively added into peripheral wells; and simultaneously setting a negative control (PBS) and an antigen standard positive control. And fourthly, inverting the plate, putting the plate in a wet box, incubating the plate for 36 hours at 37 ℃, and observing the result.

2. Results

2.1 construction of recombinant expression plasmids and preparation of recombinant engineering strains

Sequencing results show that the recombinant expression plasmid pCo-HHT28-SHG19VP2-466 is successfully constructed, two alpha helical sequences are reserved at the C end of the VP2-466 gene of the IBDV novel variant strain SHG19, a label HT28 is fused in the N section, the total length of the inserted exogenous gene is 1482bp, the nucleotide sequence is shown as SEQ ID No.1, 493 amino acids are coded, and the amino acid sequence is shown as SEQ ID No. 2. The recombinant Escherichia coli genetically engineered bacterium Transetta (DE3) -SHG19VP2 strain RT-PCR is positive in detection, 100 tubes (1 ml/tube) are prepared in total, and the strain is stored at minus 80 ℃.

2.2 expression and purification of the recombinant protein SHG19-VP2

The recombinant Escherichia coli genetically engineered bacterium Transetta (DE3) -SHG19VP2 strain is induced and expressed, and Western-blot detection results show that the recombinant SHG19-VP2 protein is successfully expressed and has the size of about 55kDa (figure 7A). SDS-PAGE detection results show that the recombinant SHG19-VP2 protein is purified in series by a saturated ammonium sulfate method-molecular sieve method, and a good purification effect is obtained (figure 7B).

2.3 identification of Virus-like particle SHG19-VLP

The negative electron microscope results showed that the recombinant protein efficiently formed virus-like particles (SHG19-VLP) around 25nm (FIG. 7C). The results of the agar diffusion test showed that SHG19-VLP reacted specifically with IBDV-positive sera with an agar diffusion titer of 4log2 and a good immunoreactivity (FIG. 7D).

EXAMPLE 3 preliminary evaluation of the immune Effect of the novel variant subunit IBDV vaccine (SHG19-VP2)

1 materials and methods

1.1 Primary reagents, consumables and instruments

The fetal bovine serum is an Ausbian product; EDTA-pancreatin digestive juice, penicillin two antibiotics for Harbin national biological science and technology products; DMEM and 1640 culture medium is Sigma product; the cell culture plate is a NEST product; the multifunctional microplate reader is a PE product.

1.2 cells and viruses

DF1 cells were maintained by the poultry immunosuppressive disease Innovation team (hereinafter referred to as the laboratory) of Harbin veterinary institute, Chinese academy of agricultural sciences. Representative strain SHG19 of IBDV novel variant is isolated and identified in example 1, and representative strain HLJ0504 of IBDV super-virulent strain is isolated and identified in the laboratory. IBDV recombinant virus rGtVarVP2 (with attenuated strain Gt as parent skeleton, expressing main protective antigen protein VP2 of novel variant SHG19) for detecting neutralizing antibody of IBDV novel variant; IBDV recombinant virus rGtHLJVP2 (with attenuated strain Gt as parent skeleton and expressing main protective antigen protein VP2 of hyper-virulent strain HLJ0504) is used for detecting IBDV hyper-virulent neutralizing antibody; both strains were rescued and prepared in the laboratory.

1.3 test animals

Specific-pathogen-free (SPF) chickens were purchased from the laboratory animal center of Harbin veterinary institute, Chinese academy of agricultural sciences, and housed in negative pressure isolators at that center.

1.4 preparation of subunit vaccines

SHG19-VLP stock was diluted with PBS to agar titers of 3log2 and 1log2, respectively. And mixing the SHG19-VLP with two antigen contents with a white oil adjuvant according to the volume ratio of 1:2, and fully emulsifying.

1.5 evaluation of immunoprotective Effect against novel IBDV variants

1.5.1 immunization

20 SPF chickens at the age of 14 days were randomly divided into four groups of 5 chickens each. Group 1 is blank control group, group 2 is non-immune and toxic counteracting control group, and

groups

3 and 4 are immune group.

Groups

1 and 2 were intramuscularly injected with 200 μ L PBS;

groups

3 and 4 were each intramuscularly injected with 200 μ L of each of the two SHG19-VLP vaccines (antigen content 3log2, 1log2 agar titers, respectively).

1.5.2 neutralization Titers assay

And on the 13 th day after immunization, collecting all chicken sera for determination of neutralizing titer, and respectively detecting neutralizing antibodies aiming at the novel IBDV variant strain and the IBDV super-virulent antigen.

1.5.2.1 detection of neutralizing antibodies against IBDV novel variant antigens

Neutralizing antibody titers against the antigens of the novel IBDV variant were detected in the sera with the rGtVarVP2 strain on DF1 cells. The serum samples were inactivated in a 56 ℃ water bath for 30min and then filtered through a 0.22 μm filter. Serum samples were then diluted 2-fold with DMEM containing 2% fetal bovine serum (serum samples tested starting at 24 dilutions). Mixing 100 μ L diluted serum with 100 μ L diluted serum containing 200TCID₅₀The rGtVarVP2 was mixed well and incubated in an incubator at 37 ℃ for 1 h. The culture medium of DF1 cells cultured in 96-well plate was discarded, 100. mu.L of the above virus-serum mixture was added to DF1 cell culture wells, and the mixture was cultured in a 37 ℃ incubator for 72 hours. A negative control and a positive control were set simultaneously. Serum sample neutralization titers were determined by observing cytopathic effects.

1.5.2.2 detection of neutralizing antibodies against very virulent strains of IBDV

Neutralizing antibody titers against the antigen of the virulent IBDV strain were detected in the serum on DF1 cells using the rGtHLJVP2 strain.

1.5.3 challenge protection test

On day 14 after immunization, challenge protection tests were performed. Using SHG19 strain to attack the chickens of

groups

2, 3 and 4 by nose-drop eye-drop method, wherein the dose of the attack is 10CID₅₀(50% chicken infection dose); group 1 SPF chickens were inoculated with 200. mu.L PBS in the same manner. The medicine is applied day by day after toxin attackAnd (5) observing the bed, and counting the disease occurrence condition. All SPF chickens were euthanized and subjected to a necropsy on day 7 after challenge. The body weight and the weight of the bursa were weighed, and the bursa/body weight ratio (B/B) was calculated as (bursa/body weight) × 1000. In each group, 3 bursa of Fabricius were randomly selected and immersed in 10% formalin solution, and subjected to HE staining for pathological observation.

1.6 evaluation of the immunoprotective Effect against IBDV Supervirulent strains

15 SPF chickens at 14 days of age were randomly divided into 3 groups of 5 chickens each. Group 1 is blank control group, group 2 is non-immune and toxic counteracting control group, and group 3 is immune group.

Groups

1 and 2 were intramuscularly injected with 200. mu. LPBS, and group 3 was intramuscularly injected with SHG19-VLP vaccine (semi-finished antigen content of 1log2 agar titer). On day 13 after immunization, all chicken sera were collected and subjected to neutralization titer determination by 1.5.2 method. On day 14 after immunization, challenge protection tests were performed. The virus for attacking is IBDV super virulent strain HLJ0504, the approach for attacking is eye-drop and nose-drop, and the dose for attacking is 10CLD₅₀(50% chicken lethality). Group 1 SPF chickens were inoculated with 200. mu.LPBS in the same manner. After the toxic materials are attacked, clinical observation is carried out day by day, and death and morbidity conditions are counted. On day 7 after challenge, all SPF chickens were euthanized and subjected to a caesarean section according to 1.5.3.

2. Results

2.1 immunoprotective Effect against novel IBDV variants

After the SHG19-VLP vaccine is used for immunizing chickens, the growth state of the chickens is normal. Two different doses (3log2 and 1log2 agar titer) of the SHG19-VLP vaccine stimulated the production of neutralizing antibodies in chickens. On day 13 post-immunization, the neutralizing antibody titers against the IBDV novel variant antigen were 10.80. + -. 1.79log2 and 10.60. + -. 0.89log2 for both immunization dose groups, respectively (FIG. 8A); the neutralizing antibody titers against IBDV very virulent antigen were relatively low, 7.40. + -. 1.82log2 and 7.40. + -. 1.67log2, respectively (FIG. 8B); the neutralizing antibody potency value of the non-immune group to both IBDVs was below 4log 2. Subsequent challenge protection experiments showed that at day 7 after challenge, the bursa of fabricius of the challenge control group yellowed, shriveled, and the B/B value was significantly lower than that of the blank control group (fig. 8C); pathological sections also showed atrophy of bursal follicles, interstitial hyperplasia, and a significant decrease in lymphocytes (FIG. 8D). No clinical symptoms were seen after challenge in both immunization groups, bursa of Fabricius was normal without any lesions as seen by autopsy (FIG. 8D), and the B/B values were not significantly different from those of the blank control group (FIG. 8C). This indicates that challenge with the novel IBDV variant with the SHG19-VLP vaccine resulted in 100% (5/5) immune protection.

2.2 immunoprotective Effect against very virulent IBDV strains

The experimental data of 2.1 also show that after the SHG19-VLP vaccine is used for immunizing chicken, serum antibodies also have certain neutralizing activity on the IBDV super-virulent strain HLJ0504, and the research further tests the immunoprotection effect of the SHG19-VLP vaccine (1log2 agar titer) on the IBDV super-virulent strain HLJ 0504. In this experiment, at day 13 after immunization, the immunized group produced both neutralizing antibodies against the antigen of the novel variant IBDV strain (11.75. + -. 0.50log2) and neutralizing antibodies against the antigen of HLJ0504 (8.75. + -. 0.96log2) (FIG. 9A); the neutralizing antibody potency value of the non-immune group to both IBDVs was below 4log 2. The result of the challenge protection test based on the IBDV super virulent strain shows that 100% (5/5) of the challenge control group chickens have the disease; depression and loss of appetite started from day 3 after challenge to day 7 after challenge, resulting in 60% (3/5) deaths (fig. 9B); the 2 chickens that survived the day 7 after challenge were examined by dissection and were seen to have atrophy, yellowing of bursa of fabricius, atrophy of follicular fluid, interstitial hyperplasia, and significant decrease in lymphocytes (fig. 9C). After challenge, the immunized group of chickens had no clinical symptoms as the blank control group of chickens, and the bursal disease examination was normal without any lesions (fig. 9C). This indicates that lethal challenge with virulent IBDV after immunization with the SHG19-VLP vaccine also resulted in 100% (5/5) immune protection.

Sequence listing

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Thr Thr Gly Pro Ala Ser Ile Pro Asp Asp Thr Leu Glu Lys His Thr

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Ser Gly Leu Ile Val Phe Phe Pro Gly Phe Pro Gly Ser Ile Val Gly

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Ala His Tyr Ile Leu Gln Ser Asp Gly Ser Tyr Lys Phe Asp Gln Met

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Leu Leu Thr Ala Gln Asn Leu Pro Ala Ser Tyr Asn Tyr Cys Arg Leu

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Val Ser Arg Ser Leu Thr Val Arg Ser Ser Thr Leu Pro Gly Gly Val

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Tyr Ala Leu Asn Gly Thr Ile Asn Ala Val Thr Phe Gln Gly Ser Leu

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Ser Glu Leu Thr Asp Val Ser Tyr Asn Gly Leu Met Ser Ala Thr Ala

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Asn Ile Asn Asp Lys Ile Gly Asn Val Leu Val Gly Glu Gly Val Thr

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Val Leu Ser Leu Pro Thr Ser Tyr Asp Leu Gly Tyr Val Arg Leu Gly

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Asp Pro Ile Pro Ala Val Gly Leu Asp Pro Lys Met Val Ala Thr Cys

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Leu Val Ile Pro Thr Ser Glu Ile Thr Gln Pro Ile Thr Ser Ile Lys

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Leu Glu Ile Val Thr Ser Lys Ser Asp Gly Gln Ala Gly Glu Gln Met

340 345 350

Ser Trp Ser Ala Ser Gly Ser Leu Ala Val Thr Ile His Gly Gly Asn

355 360 365

Tyr Pro Gly Ala Leu Arg Pro Val Thr Leu Val Ala Tyr Glu Arg Val

370 375 380

Ala Thr Gly Ser Val Val Thr Val Ala Gly Val Ser Asn Phe Glu Leu

385 390 395 400

Ile Pro Asn Pro Glu Leu Ala Lys Asn Leu Val Thr Glu Tyr Gly Arg

405 410 415

Phe Asp Pro Gly Ala Met Asn Tyr Thr Lys Leu Ile Leu Ser Glu Arg

420 425 430

Asp Arg Leu Gly Ile Lys Thr Val Trp Pro Thr Arg Glu Tyr Thr Asp

435 440 445

Phe Arg Glu Tyr Phe Met Glu Val Ala Asp Leu Asn Ser Pro Leu Lys

450 455 460

Ile Ala Gly Ala Phe Gly Phe Lys Asp Ile Ile Arg Ala Leu Arg Arg

465 470 475 480

Ile Ala Val Pro Val Val Ser Thr Leu Phe Pro Pro Ala

485 490

Claims

1. a chicken infectious bursal disease virus (infectious bursal disease virus, IBDV) novel variant strain subunit vaccine, it is characterized in that, in the described vaccine, contain the new type of chicken infectious bursal disease virus after optimization The virus-like particle composed of the VP2 protein of the variant strain, the amino acid sequence of the VP2 protein of the new variant strain of the chicken infectious bursal disease virus after optimization is shown in SEQ ID NO.2.

2. subunit vaccine as claimed in claim 1 is characterized in that, the virus-like particle that described chicken infectious bursal disease virus novel variant VP2 protein is formed is to encode the described optimized chicken infection The nucleotide sequence of the VP2 protein of the new variant strain of bursal disease virus is inserted into the expression vector, and then transformed into E. coli, and obtained after inducing expression and purification.

3. subunit vaccine as claimed in claim 1 is characterized in that, the nucleotide sequence of coding described the VP2 protein of novel variant strain of chicken infectious bursal disease virus after optimization is as shown in SEQ ID NO.1 Show.

4. The subunit vaccine of claim 1, wherein the expression vector is a pCold I expression vector.

5. The subunit vaccine of claim 1, wherein the Escherichia coli is E.coli Transetta (DE3) expressing engineered bacteria.

6. a method for preparing the subunit vaccine described in any one of claim 1-5, is characterized in that, comprises the following steps:

(1) synthesizing the nucleotide sequence encoding the VP2 protein of the novel variant strain of IBDV described in claim 1, and adding the two ends of the nucleotide sequence Restriction endonuclease cut site to obtain the target fragment;

(2) use the homologous recombination kit to carry out the homologous recombination reaction between the target fragment obtained in step (1) and the pCold I expression vector linearized by the same restriction site, and after the reaction, take the homologous recombination reaction product and transform it into DH5α Escherichia coli, single clones were picked 12 hours after transformation, positive clones were screened by bacterial liquid PCR, and the positive clones were sequenced and identified, and the correctly identified recombinant expression plasmid was named pCo-HHT28-SHG19VP2-466;

(3) transforming pCo-HHT28-SHG19VP2-466 into E.coli Transetta (DE3) expression engineering bacteria, picking monoclonal colonies 12 hours after the transformation and inoculating them into ampicillin-resistant LB liquid medium for culture, and harvesting the bacterial liquid, RT-PCR identification was carried out, and the correct positive bacteria were identified as Escherichia coli genetically engineered bacteria Transetta (DE3)-SHG19VP2 strains, which were stored at minus 80°C;

(4) Expression of recombinant protein

The transetta (DE3)-SHG19VP2 strain of Escherichia coli genetically engineered bacteria was inoculated into ampicillin-resistant LB medium. When the OD ₆₀₀ reached 0.6, the shake flask was taken out of the shaker, placed on ice for rapid cooling, and placed in the shaker flask. Add IPTG and continue to cultivate on the shaker;

After the induction and expression, the bacterial liquid was transferred to a centrifuge tube, centrifuged, the medium was discarded, and the bacterial cell pellet was fully resuspended with PB buffer, and then the bacterial cell was subjected to high-pressure fragmentation using a high-pressure cell crusher, and the fragmented bacterial liquid was crushed. Centrifuge, discard the precipitation to obtain a solution containing the VP2 protein of the new variant of chicken infectious bursal disease virus;

(5) Purification of recombinant protein

The obtained solution containing the VP2 protein of the new variant strain of IBDV is subjected to ammonium sulfate precipitation to obtain the VP2 protein of the new variant strain of IBDV after preliminary purification, and then further obtained by the molecular sieve method. The VP2 protein of the new variant of IBDV after purification, the negative staining electron microscope results showed that the purified VP2 protein of the new variant of IBDV formed virus-like particles;

(6) Preparation of vaccines

The virus-like particles obtained in step (5) are mixed with white oil adjuvant in a volume ratio of 1:2 and then fully emulsified to obtain a novel variant subunit vaccine of infectious bursal disease virus.

7. The method according to claim 6, wherein the two ends of the nucleotide sequence of the VP2 protein of the novel variant strain of chicken infectious bursal disease virus described in claim 1 are respectively Add KpnI and EcoRI restriction sites.

8. method as claimed in claim 6 is characterized in that, the nucleotide sequence of the VP2 protein of the novel variant strain of chicken infectious bursal disease virus after encoding described in claim 1 is such as SEQ ID NO. 1 shown.

9. Use of the subunit vaccine of any one of claims 1-5 in the preparation of a medicament for preventing and treating IBDV.

10. The use according to claim 9, wherein the infectious bursal disease virus of chickens comprises IBDV novel variant strains and IBDV super-virulent strains.