Composite multi-epitope expression cassette, a recombinant virus composed thereof and application thereof

Abstract

The present application relates a composite multi-epitope expression cassette, a recombinant virus composed thereof and application thereof, and in particular to a chimeric recombinant Newcastle disease virus inserted with an IBV epitope cassette and a vaccine prepared by using the virus. The expression cassette comprises: (a) T cell epitopes derived from S1 proteins of avian infectious bronchitis virus Holte strain and avian infectious bronchitis virus QX-like strain; and (b) B cell epitopes derived from S1 protein of avian infectious bronchitis virus Australian T strain. In the present application, the multi-epitope chimeric ST/B gene of avian infectious bronchitis virus is inserted into the backbone of LaSota strain, so that the LaSota strain can express S1-T/B protein. Thus, the purpose of preventing both ND and IB diseases is achieved. In addition, the T cell epitopes and B cell epitopes act synergistically to produce an earlier and more comprehensive immune response against virus.

Claims

1. A composite multi-epitope expression cassette comprising: (a) T cell epitopes derived from S1 proteins of avian infectious bronchitis virus Holte strain and avian infectious bronchitis virus QX-like strain; and (b) B cell epitopes derived from S1 protein of avian infectious bronchitis virus Australian T strain.

2. The composite multi-epitope expression cassette according to claim 1, wherein the T cell epitopes have amino acid sequences as shown in SEQ ID NOs. 1-4.

3. The composite multi-epitope expression cassette according to claim 2, wherein the B cell epitopes have amino acid sequences as shown in SEQ ID NOs. 5-7.

4. The composite multi-epitope expression cassette according to claim 2, wherein different epitopes among the T cell epitopes and the B cell epitopes are linked by a flexible small molecule linker.

5. The composite multi-epitope expression cassette according to claim 1, wherein an enzyme cleavage site is further included in front of and behind the expression cassette.

6. A recombinant virus comprising a gene encoding the composite multi-epitope expression cassette according to claim 1.

7. A composite multi-epitope vaccine comprising the recombinant virus according to claim 6.

8. A method for treating Newcastle disease and/or avian infectious bronchitis in a chicken, comprising administrating an effective amount of the composite multi-epitope vaccine according to claim 7 to the chicken.

9. The composite multi-epitope expression cassette according to claim 4, wherein the flexible small molecule linker is KAA, AAY, AAA, GAAA, KAAA, and has the nucleotide sequence as shown in SEQ ID NOs. 8-12.

10. The composite multi-epitope expression cassette according to claim 5, wherein the cleavage site is any one of Spe I, Xho I, BamH I, EcoR I, Nde I, Pst I or Xho I.

11. The composite multi-epitope expression cassette according to claim 1, wherein a KOZAK sequence which has the nucleotide sequence as shown in SEQ ID NO. 13 is further included behind the cleavage site that is located in front of the expression cassette.

12. The composite multi-epitope expression cassette according to claim 1, wherein the expression cassette has the amino acid sequence as shown in SEQ ID NO. 14 and the nucleotide sequence as shown in SEQ ID NO. 15.

Description

FIGURE LEGEND

(1) FIG. 1 is a schematic diagram of construction of a viral infectious clone of the present application, wherein the target gene 1 is HN gene of TS09-C strain; the target gene 2 is IBV S1 gene T/B cell multi-epitope gene cassette; T7 RNA polymerase promoter sequence; IRL: internal long repeat; TRL: terminal long repeat; TRS: terminal short repeat; IRS: internal short repeat;

(2) FIG. 2 (A) shows the restriction enzyme digestion of a plasmid expressing NP (nuclear protein); FIG. 2 (B) shows the restriction enzyme digestion of a plasmid expressing P (phosphoprotein); FIG. 2 (C) shows the restriction enzyme digestion of a plasmid expressing L (large polymerase protein);

(3) FIG. 3 shows PCR identification results of the recombinant Newcastle disease virus inserted with a IBV-T/B epitope cassette gene of the present application;

(4) FIG. 4 is an electron micrograph of the recombinant virus rNDV-IBV-T/B;

(5) FIG. 5 (A) and FIG. 5 (B) show PCR identification results of episode cassette gene of the recombinant virus rNDV-IBV-T/B after 25 passages;

(6) FIG. 6 shows detection of NDV-specific IgG antibodies by ELISA after immunization;

(7) FIG. 7 shows analysis of CD8.sup.+ T cell proliferation;

(8) FIG. 8 shows protective efficacy against avian infectious bronchitis virus challenge.

DETAILED DESCRIPTION

(9) The technical solutions of the present application are further described below by specific embodiments. It should be understood by those skilled in the art that the examples are only to facilitate to understand the present application and should not to be construed as specific limitations to the present application.

(10) The experimental methods used in the following examples are conventional methods unless otherwise specified.

(11) The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Construction of a Recombinant Plasmid Replaced with the HN Gene of TS09-C Strain and Inserted with a IBV S1-T/B Multi-Epitope and Virus Rescue

(12) Three pairs of primers SEQ ID NOs. 18-23 were designed according to the established whole genome sequence of TS09-C strain (GenBank accession number: JX110635.1). During the design of primers, a hepatitis D virus ribozyme sequence and a T7 RNA polymerase terminator sequence were introduced downstream of the 5′ non-coding region, and a T7 RNA polymerase promoter sequence was introduced upstream of the 3′ non-coding region, as shown in Table 1 below:

(13) TABLE-US-00005 TABLE 1 Primer Name Primer Sequence (5′ .fwdarw. 3′) SEQ ID NO. 18 TAATACGACTCACTATAGGGAGAACCAAACAGAGAA TCTGTGAGTTAC SEQ ID NO. 19 AACTCAGTGCCAACATGACTCGGAC SEQ ID NO. 20 TCCCGGTCGGCGCCTTCAAGGTGCA SEQ ID NO. 21 TCTGATGCTCCGCCCTCTCGGGACC SEQ ID NO. 22 AAAAATGTGGGTGGTAGCGGGATAT SEQ ID NO. 23 ACCAAACAAAGATTTGGTGAATGACAATAACTAGCA TAACCCCTTGGGGCCTCTAAACGGGTCTTGATGGCC GGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGG CAACA

(14) Three overlapping whole genome cDNA fragments were amplified from allantoic fluid infected with LaSota strain virus, which were 1-4616 nt, 4517-8417 nt and 8318-15186 nt, respectively. The three cDNA fragments were stepwise connected to a clone vector pBR322 to obtain a full-length cDNA clone pBR-LS.

(15) The HN gene was amplified from allantoic fluid infected with TS09-C strain virus by employing RT-PCR. The HN gene from TS09-C strain was integrated by fusion PCR and was used to replace the HN gene in pLS to obtain a plasmid pBR-LS-THN. In the same method, the multi-epitope chimeric S-T/B gene of avian infectious bronchitis virus was inserted between the M and F genes of LaSota strain to obtain a plasmid pNDV-IBV-T/B which had a plasmid profile as shown in FIG. 1.

(16) The full-length sequences of NP, P and L genes of NDV LaSota strain were amplified separately by RT-PCR by using pLS plasmid as a PCR template and were ligated into a eukaryotic expression plasmid pVAX1 to obtain helper plasmids that could express proteins NDV NP, P and L, respectively, namely pVAX-NP, pVAX-P and pVAX-L.

(17) Plasmid co-transfection was carried out by employing a calcium phosphate method. According to the instructions, four plasmids pNDV-IBV-T/B, pVAX-NP, pVAX-P and pVAX-L (mass ratio of 4:2:1:1) were co-transformed into BHK-21 cells that had been pre-infected with vTF7-3. After 6 h of transfection, cells were washed twice, and DMEM nutrient solution containing 1% of double antibiotics and 0.2 μg/mL TPCK-trypsin was added, followed by incubation for another 3 days. Repeated freeze-thaw was performed for 3 times and the cell culture was harvested. After removing the poxvirus vTF7-3 by filtration through a filter with a pore size of 0.22 μm, serial passage was performed in 9-11 day old SPF chicken embryos for 3 times, and then allantoic fluid was harvested for virus detection.

(18) The infectious bronchitis virus S1 protein T and B cell epitope cassette IBV-T/B was inserted between P and M genes in the full-length cDNA backbone of LaSota strain containing the thermostable HN gene from TS09-C strain to obtain a recombinant plasmid pNDV-IBV-T/B. Electrophoresis after enzyme digestion of helper plasmid pVAX-P showed target fragments of 5.5 kb and 1.2 kb, electrophoresis after enzyme digestion of pVAX-NP with Xho I and Xba I showed two target fragments of 5.5 kb and 1.5 kb and digestion of pVAX-L with EcoR I showed three target fragments of 8.2 kb, 2.9 kb and 1.0 kb. Graphs of the restriction enzyme digestions were shown in FIG. 2(A)-FIG. 2(C).

(19) The pNDV-IBV-T/B was co-transfected with three helper plasmids pVAX-NP, pVAX-P and pVAX-L (mass ratio of 4:2:1:1) into BHK-21 cells that had been pre-infected with vTF7-3, and then incubated for 72 hours. The supernatant was harvested, filtered, and was used to inoculate SPF chicken embryos. The allantoic fluid was collected, and RNA was extracted therefrom to reverse transcribe into cDNA. A RT-PCR amplification was performed with a upstream primer located at the P gene and a downstream primer located at the M gene to obtain PCR product of S-T/B with a size of 1365 bp. The PCR identification result was shown in FIG. 3. The sequencing result showed 100% matching with the expected sequence.

(20) It was indicated that the T/B epitope sequence of infectious bronchitis virus had been inserted at the correct position of NDV, and an expected recombinant virus was obtained.

Example 2

Identification and Purification of Recombinant rNDV-IBV-T/B

(21) (I) Virus Titer and Growth Kinetics

(22) The virus titer of recombinant Newcastle disease vector live vaccine was determined by a HA test, a 50% tissue culture infective dose (TCID.sub.50) assay on BHK-21 cells in the presence of 0.2 μg/ml TPCK-trypsin and a 50% egg infection dose (EID.sub.50) assay in 10 day old SPF chicken embryos.

(23) To determine the growth kinetics of recombinant virus, BHK-21 monolayer cells were infected with 0.1 MOI of recombinant Newcastle disease vector live vaccine for 1.5 hours, then washed with phosphate buffered saline (PBS) three times and covered with medium containing 2% fetal bovine serum (FBS). Supernatant was collected from the medium with infected cells at indicated time points and virus titration from the medium was performed by using a TCID.sub.50 assay.

(24) The following primers were designed based on the position of the replaced HN gene and the inserted S1-T/B epitope cassette: SEQ ID NOs. 24-25 were designed for TS09-C HN gene and SEQ ID NOs. 26-27 were designed for S1-T/B epitope cassette. The specific sequences are shown in Table 2:

(25) TABLE-US-00006 TABLE 2 Primer Name Primer Sequence (5′ .fwdarw. 3′) SEQ ID NO. 24 ATGGACCGCGCAGTTAGCCAAGTTG SEQ ID NO. 25 TTATGGCCAACTGGCAGCGTAGGAC SEQ ID NO. 26 CACTCGGCATCACACGGAATC SEQ ID NO. 27 GTCCACAAGTCAAGGCGCTG

(26) The specific PCR system is shown in Table 3 below, as follows:

(27) TABLE-US-00007 TABLE 3 Reaction System Reagent Components mx [μl] PCR Reaction PCR mix 25 Upstream primer 1 Downstream primer 1 DNA template 5 ddH.sub.2O 18 Total 50

(28) The specific PCR conditions are shown in Table 4 below, as follows:

(29) TABLE-US-00008 TABLE 4 Reaction Procedure Number of Cycles Amplification  .sup. 94° C. 2 min 1 Procedure 94° C. 30 s 30 55° C. 30 s 68° C. 40 s  .sup. 72° C. 3 min 1 4° C.  .sup.  1

(30) HN fragment and S1-T/B gene fragment were amplified.

(31) II) Purification of Recombinant Virus

(32) The virus strain which had been identified as positive and was a single plaque was used for purification. After digesting cells within the well containing this virus, half thereof was absorbed to dilute 100 times, and finally diluted into 10 ml with 2% FBS DMEM medium. The 10 ml of virus mixture was dispensed into a 96-well plate with 100 μl per well and incubated at 37° C., 5% CO.sub.2 for 5 days. Then the 96-well plate was observed to select individual plaques for identification and storage.

(33) III) Thermal Stability Test

(34) 1.0 ml of undiluted allantoic fluid containing recombinant Newcastle disease virus was sealed in a sterile vial. The sterile vial was immersed in a water bath at 56° C. and transferred to ice water at designated time points to stop the heat inactivation. The infectivity and HA activity of heat-inactivated recombinant Newcastle disease virus were titrated by performing a TCID50 assay and a standard HA assay in BHK-21 cells, respectively. Regression lines were plotted based on the infectivity and HA activity of virus over time and by monitoring recombinant Newcastle disease virus and parent strain LaSota at different time points. The time points of the heat-resistant virus for HA activity were 30, 60, 90 and 120 minutes, respectively.

(35) In order to verify whether the rescued recombinant virus rNDV-IBV-T/B had similar heat-resistance characteristics to the parent TS09-C strain, a heat-resistance test was carried out for rTS09-C: the allantoic fluid from infected chicken embryo was placed in a water bath at 56° C. for heat treatment for 30, 60, 90 and 120 minutes, respectively, and then it was taken out and immediately placed in an ice bath. In order to determine whether the heat treated virus was still infectious, the heat-treated allantoic fluid was inoculated into SPF chicken embryos (5 embryos/sample). After 5 days of infection, the allantoic fluid was harvested for detection of HA activity. The results are shown in Table 5 below:

(36) TABLE-US-00009 TABLE 5 Heat treatment time/min Strain Name 30 60 90 120 TS09-C + + − − LaSota − − − − rNDV-IBV-T/B + + + − Note: + indicates infectious to chicken embryos; − indicates non-infectious to chicken embryos.

(37) It can be seen from Table 5 that the TS09-C strain was still infectious after heat treatment at 56° C. for 60 min, and the period for rNDV-IBV-T/B strain was slightly longer, which was up to 90 min, while the LaSota strain was non-infectious after heat treatment for 30 min, indicating that the recombinant rNDV-IBV-T/B strain had heat-resistant properties.

(38) IV) Observation by Transmission Electron Microscope (TEM)

(39) The recombinant virus was largely propagated in 9-11 day old SPF chicken embryos. The harvested allantoic fluid was centrifuged at 2000 r/min for 20 min. Supernatant was collected and centrifuged at 28 000 r/min for 2 h. The supernatant was discarded and the precipitate was resuspended in PBS. The resuspended sample was added to the upper layer of 20%, 40%, 60% discontinuous density gradient of sucrose, centrifuged at 28000 r/min for 2.5 h, and 40-60% of intermediate protein layer was collected. The collected protein layer was diluted and mixed with PBS, and centrifuged at 28000 r/min for 2 h. The precipitate was resuspended in PBS, and 1 to 2 drops of resuspension were added to a copper mesh. After negative staining with tungsten phosphate, observed under TEM at 10,000 times magnification. The results are shown in FIG. 4.

(40) It can be seen from FIG. 4 that the virion was in an irregular shape with non-fully identical size, about 200-300 nm in diameter, and a capsule located on the surface layer of virus can be clearly observed, which is consistent with the morphology of NDV. The above results fully demonstrated that the recombinant virus rNDV-IBV-T/B had been successfully rescued.

Example 3

Determination of Biological Characteristics of Recombinant rNDV-IBV-T/B

(41) The above allantoic fluid that had been collected at each generation was subjected to determination of biological characteristics of the recombinant virus including mean death time of minimum lethal dose (MDT/MLD), intracerebral pathogenicity index (ICPI) virulence index and hemagglutination test (HA test) according to the OIE standard.

(42) (I) MDT Assay

(43) 10 day old chicken embryos were taken. The vaccine was 10-fold serially diluted to five dilutions 10-7, 10-8, 10-9, 10-10, 10-11. 0.1 ml of each dilution was inoculated into the allantoic cavity of each of 5 SPF chicken embryos, and incubated in an incubator at 37.5° C. The eggs were irradiated twice a day for 7 consecutive days. Death time of each chicken embryo, minimum lethal dose which referred to the maximum dilution at which all inoculated chicken embryos were dead and MDT which was the mean time (h) required for the minimum lethal dose to kill all chicken embryos were recorded.

(44) Time judgment criteria: immediate (acute/velogenic)<60 h, middle (subacute, mesogenic) 61-90 h, slow (low-virulence)>90 h. The results are shown in Tables 6-7:

(45) TABLE-US-00010 TABLE 6 Determination of minimum lethal dose 10.sup.−7 10.sup.−8 10.sup.−9 10.sup.−10 10.sup.−11 LaSota 5/5 5/5 3/5 1/5 0/5 rNDV-IBV-T/B 5/5 5/5 8/5 2/5 0/5

(46) TABLE-US-00011 TABLE 7 Determination of mean death time 1 2 3 4 5 6 7 8 MDT LaSota 165 170 155 162 172 152 173 165 164.25 rNDV-IBV-T/B 166 153 169 155 152 172 168 159 161.75

(47) It can be seen from Table 6 that the minimum lethal dose of recombinant Newcastle disease rNDV-IBV-T/B and its parent strain LaSota was 10.sup.−8 and it can be seen from Table 7 that the mean death time of recombinant Newcastle disease virus to chicken embryos at the minimum lethal dose was 161 hours, which was not significantly different from the parent strain.

(48) (II) ICPI Assay

(49) The recombinant NDV propagated from chicken embryo allantoic fluid was diluted 1:10 with sterile saline. 0.05 ml of each strain was inoculated intracerebrally into each of 8 susceptible chicks hatched 24-36 hours ago via a micro-syringe (0.25 ml). A control group of 4 chicks was set up at the same time, and each chick was inoculated with 0.05 ml of sterile saline. Chickens were fed separately. The health status of chickens was observed every day at time points corresponding to the inoculation time, and the test chickens were evaluated: normal=0, sick=1, dead=2. Observed for 8 consecutive days, and the ICPI was finally obtained according to the equation. The specific results are shown in Table 8:

(50) TABLE-US-00012 TABLE 8 Determination of intracerebral pathogenicity index in 1 day old chicks Symptoms after Number of Observation Days Strain Inoculation 1 2 3 4 5 6 7 8 Total Integration ICPI LaSota normal 0 0 0 0 0 0 0 0 0 0 0 onset 0 0 0 0 0 0 0 0 0 0 death 0 0 0 0 0 0 0 0 0 0 rNDV-IBV-T/B normal 0 0 0 0 0 0 0 0 0 0 0 onset 0 0 0 0 0 0 0 0 0 0 death 0 0 0 0 0 0 0 0 0 0

(51) It can be seen from Table 8 that the recombinant virus had an intracerebral pathogenicity index of 0.00 in 1 day old SPF chickens.

(52) Based on the results of Tables 6-8, it is clear that the recombinant Newcastle disease virus was an attenuated strain and was more safe.

(53) (III) HA Assay

(54) The red blood cells used were 1% chicken red blood cells freshly prepared according to a conventional method. Parent LaSota strain was used as a positive control in HA test and subjected to the same test. The HA test-positive allantoic fluid samples were diluted 1:100, 1:500, and 1:1000 times with 0.9% sterile saline, and then 0.2 mL of each dilution was used to inoculate each of 3 SPF chicken embryos. After culturing according to the above method, allantoic fluid was collected and also subjected to a HA test. The HA test-positive allantoic fluid samples were continued to being passaged in chicken embryos after appropriate dilution.

(55) The recombinant Newcastle disease virus was passaged continuously for 25 generations in SPF chicken embryos, and mutations in nucleotide sequence of IBV-T/B epitope cassette was detected by RT-PCR and DNA sequencing. The results are shown in FIG. 5(A)-FIG. 5(B).

(56) It can be seen from FIG. 5(A)-FIG. 5(B) that the IBV-T/B epitope cassette gene was still stably present after multiple passages without mutations in its bases, indicating a good genetic stability.

Example 4

Immunoprotective Efficacy of Recombinant rNDV-IBV-T/B

(57) 60 SPF chickens were randomly divided into 4 groups of 15 in each group. The specific groupings are shown in Table 9. Chickens were subjected to primary immunization at 7 day old and challenged with NDV and IBV at a dose of 5*10.sup.5 ELD.sub.50 at 21 day old, specifically as follows:

(58) TABLE-US-00013 TABLE 9 Experimental groupings and immunization procedures Immunization Immunization Immunization Number of Group Group Dose Route Immunizations 1 rNDV-IBV-T/B 10.sup.6 EID.sub.50 Intranasal 1 2 rNDV-IBV-T/B 10.sup.6 EID.sub.50 Intranasal 1 3 PBS 200 μl Intranasal 1 4 PBS 200 μl Intranasal 1

(59) (I) Detection of IgG Antibodies Specific for Avian Infectious Bronchitis Virus by ELISA

(60) Three chickens were randomly selected from each group to collect blood from the subpteryx vein prior to primary immunization and on 7 d, 14 d and 21 d after immunization, and serum was separated. The antibody titer was detected by an indirect ELISA method which was specifically as follows: The NDV and IBV virus solution were diluted 10-fold with coating solution, and then added to a microplate (100 μl/well) and incubated overnight at 4° C.; the liquid was discarded, the plate was washed 3 times with 300 μl of PBST per well and 3 min for each time, and 200 μl of 10% fetal bovine serum was added into per well to block, left at 37° C. for 2 h, and the plate was washed 3 times with PBST; the serum to be tested that had been diluted 20 times with PBS in advance was added (100 μl/well), placed at 37° C. for 2 h, and then the plate was washed 3 times with PBST; rabbit anti-chicken enzyme-labeled antibody IgG (1:5000 diluted with PBS) was added (100 μl/well), incubated at 37 h ° C. for 1 h, and then the plate was washed 3 times, 100 μl/well of TBM substrate solution was added, incubated at room temperature in dark for 20 min; 100 μl/well of 2N H.sub.2SO.sub.4 was added to terminate the reaction, and the OD values of each well was determined at a wavelength of 450 nm. The results are shown in FIG. 6.

(61) It can be seen from FIG. 6 that the antibody level of vaccine group increased rapidly 7 days after the primary immunization, and reached the peak at 14 d, followed by a decrease and maintained at a high level. The vaccine immunization group was significantly higher than the PBS control group (p<0.05).

(62) (II) Analysis of CD8+ T Lymphocyte Proliferation

(63) Three chickens were randomly selected from each group to be euthanized. The spleens were isolated under sterile conditions and prepared into lymphocyte suspensions. The lymphocytes were stained with 1 ml of pre-warmed PBS containing 2.5 μM CFSE in a water bath at 37° C. for 10 minutes. After treatment, 0.2 mL FBS was used to terminate the reaction. The cell density was diluted to 10.sup.6 cells/ml with RPMI 1640 medium. The stained lymphocytes were divided into two groups and inoculated into 24-well plates at 10.sup.6 cells/well, respectively. The lymphocytes were stimulated with identified functional T cell epitope polypeptides (P8SRIQTATDP, P9SRNATGSQP, P18GAYAVVNV and P19SRIQTATQP) and avian infectious bronchitis virus, respectively, and cultured in an incubator at 37° C., 5% CO.sub.2 for 5 days. The cultured lymphocytes were stained with PE-labeled mouse anti-chicken CD8 T cell monoclonal antibody. The proliferation ratio of CD8.sup.+ T cells was detected by flow cytometry, and the proliferation rate was determined by CXP software. The proliferation rate of CD8.sup.+ T cells of PBS control group after immunization was determined by flow cytometry and was set to 100% as normalization and used for comparison with immunization groups. The results are shown in FIG. 7.

(64) It can be seen from FIG. 7 that the proliferation rate of CD8.sup.+ T cells in rNDV-IBV-T/B group was 133.5±1.8% when stimulated with IBV, which was significantly higher than that in PBS group (100.5±0.6%) (p<0.05). The highest proliferation rate of T cells when stimulated and induced by mixed T cell epitope was also rNDV-IBV-T/B (131.1±0.7%), which was significantly higher than that of PBS control group pV-S1B (100.7±2.1%), exhibiting statistical difference (P<0.05). The results showed that the recombinant multi-epitope live vaccine rNDV-IBV-T/B was able to induce significant T lymphocyte proliferation in chickens.

Example 5

Protection Against Recombinant rNDV-IBV-T/B Challenge

(65) Experimental method: A virus challenge was performed on the 7th day after booster immunization. Each chicken was challenged with 10.sup.6 ELD.sub.50 of IBV Australian T strain via an intranasal route. After the challenge, the incidence of each group of chickens was observed for 10 consecutive days. The results are shown in FIG. 8.

(66) It can be seen from FIG. 8 that the chickens in PBS group began to die on the 4th day after challenge with a mortality rate up to 100%. The lesions were mainly founded in respiratory tracts and kidneys after necropsy, with specific performance of mucus-filled upper respiratory tract, swollen throat, trachea with bleeding points, enlarged kidney, typical tinea kidney. After immunization with vaccine rNDV-IBV-T/B, the provided protective efficacy against NDV and IBV was 100% and 90%, respectively. It is thus obvious that the provided rNDV-IBV-T/B live vaccine can effectively resist the velogenic NDV and velogenic IBV challenges after immunization of SPF chickens with protection rates of 100% and 90%, respectively, proving that the vaccine was safe and effective.

(67) In summary, in the present application, the multi-epitope chimeric ST/B gene of avian infectious bronchitis virus is inserted into the backbone of LaSota strain, so that the LaSota strain can express S1-T/B protein. Thus, the purpose of preventing both ND and IB diseases is achieved. In addition, the T cell epitopes and B cell epitopes can act synergistically to produce an earlier and more comprehensive immune response against virus. Moreover, in the present application, the HN gene of lentogenic TS09-C strain is replaced with that of the LaSota vaccine strain, increasing the thermal stability of LaSota strain without increasing its pathogenicity, and reducing the requirements for vaccine storage conditions and prolonging the shelf life.

(68) The applicant states that the technological methods of the present application are illustrated in the present application through the embodiments described above, however, the present application is not limited to the technological procedures described above, i.e. it does not mean that the application must rely on the technological procedures described above to implement. It should be apparent to those skilled in the art that, for any improvement of the present application, the equivalent replacement of the selected raw materials of the present application, the addition of auxiliary components and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present application.