CN113264989A

CN113264989A - Preparation method and application of H9N2 subtype avian influenza chimeric virus-like particle

Info

Publication number: CN113264989A
Application number: CN202110534239.7A
Authority: CN
Inventors: 丛彦龙; 孙艺学; 宿甲子; 邓效禹; 李佳新; 凌蒙蒙
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-05-17
Filing date: 2021-05-17
Publication date: 2021-08-17

Abstract

The invention relates to a preparation method and application of H9N2 subtype avian influenza chimeric virus-like particles (VLPs), which is characterized by comprising the following steps: VLPs are protein particles of a hollowed structure formed by self-assembly of HA protein of H9N2 subtype avian influenza virus and gag protein of Mouse Leukemia Virus (MLV). Because the chimeric VLPs can only stimulate an organism to generate HA antibodies of specific H9N2 subtype avian influenza virus and gag antibodies of MLV after immunization, but cannot generate antibodies against PB2, PB1, PA, NP, NA, M and NS of the H9N2 subtype avian influenza virus, the chimeric VLPs can be used as a marker vaccine for distinguishing wild virus infection and inactivated vaccine immunization of the H9N2 subtype avian influenza virus clinically. Therefore, the chimeric VLPs are used for immunization, and technical support can be provided for purification of H9N2 subtype avian influenza.

Description

Preparation method and application of H9N2 subtype avian influenza chimeric virus-like particle

Technical Field

The invention relates to a preparation method and application of H9N2 subtype avian influenza chimeric virus-like particles, and belongs to the field of vaccine development, wherein the preparation method is used for preparing H9N2 subtype avian influenza VLPs.

Background

Avian influenza is a highly contagious disease of poultry and wild birds caused by infection with influenza a virus. According to the pathogenicity of the avian influenza virus, the avian influenza can be classified into high pathogenicity, low pathogenicity and non-pathogenicity. The pathogeny causing H9N2 subtype avian influenza belongs to low pathogenic influenza virus, although the lethality rate is not high, respiratory tract symptoms and laying rate of poultry are often caused to be reduced, and the poultry is easy to cause concurrent infection, thereby causing serious economic loss to poultry industry. As a local epidemic infectious disease in China, H9N2 subtype avian influenza has already become popular in poultry flocks in China since the last 90 th century, and vaccine immunization is the most effective and economic means for preventing avian influenza and has positive significance for improving the prevention and control level of avian diseases in China and ensuring the healthy development of poultry industry in China. At present, the domestic vaccine for preventing H9N2 subtype avian influenza is mainly inactivated vaccine. The preparation technology of inactivated vaccine has advantages, but has great defects. Because the avian influenza virus is easy to mutate and genetic material recombination and antigen drift are easy to occur between double and multiple subtype viruses, the long-term safety and effectiveness of the inactivated vaccine are greatly challenged, and the infection of new mutant avian influenza viruses is difficult to deal with, so that the development of a more effective vaccine for preventing and controlling H9N2 subtype avian influenza is very important.

Virus-like particle (VLPs) vaccines are considered to be a focus of research in recent years because they are superior to inactivated vaccines in terms of their immune efficacy and safety. VLPs are highly structured hollow protein particles that are self-assembled from one or more structural proteins of a certain virus, do not contain viral nucleic acids, are incapable of autonomous replication, do not have the potential for genetic recombination or reassortment and virulence reversion, are highly safe, can mimic the natural structure of virions, can activate cellular and humoral immunity, and induce more comprehensive immune protection. The use of a baculovirus expression system enables large scale production of VLP vaccines in insect suspension cells. Because the baculovirus expression system does not depend on chick embryo production, the adequate supply of vaccines during avian influenza pandemics can be ensured, and the method has the advantages of low cost, environmental friendliness, short production period, high safety and the like, the VLP vaccine produced by using the baculovirus expression system has great application potential.

The size of the virus particle mainly depends on the diameter of the matrix protein, and since the average diameter of gag protein of Mouse Leukemia Virus (MLV) is 150-180 nm, which is significantly larger than the diameter of M1 of influenza virus (about 80-120 nm), it is predicted that gag will have a larger surface area as the framework protein of influenza virus VLPs, thereby accommodating more surface protein of influenza virus and producing better immune effect.

Disclosure of Invention

The invention aims to provide a preparation method of H9N2 subtype avian influenza chimeric virus-like particles, which utilizes an insect-baculovirus expression system to enable HA protein of H9N2 subtype avian influenza virus and gag protein of mouse leukemia virus MLV to be automatically assembled in the system to form VLPs with hollow structures; because the chimeric VLPs can only stimulate an organism to generate HA antibodies of specific H9N2 subtype avian influenza virus and gag antibodies of MLV after immunization, and cannot generate PB2, PB1, PA, NP, NA, M and NS antibodies aiming at the H9N2 subtype avian influenza virus, the chimeric VLPs can be used as a marker vaccine for distinguishing wild virus infection and inactivated vaccine immunization of the H9N2 subtype avian influenza virus clinically; therefore, the chimeric VLPs are used for immunization, and technical support can be provided for purification of H9N2 subtype avian influenza.

The technical scheme of the invention is realized as follows: an H9N2 subtype avian influenza VLPs is a protein particle with a hollow structure formed by self-assembly of HA protein of H9N2 subtype avian influenza virus and gag protein of mouse leukemia virus MLV; the method is characterized in that: the nucleotide sequence of the HA gene corresponding to the HA protein is SEQ ID NO.1, and the nucleotide sequence of the gag gene corresponding to the gag protein is SEQ ID NO. 2.

A preparation method of H9N2 subtype avian influenza chimeric virus-like particles is characterized by comprising the following steps:

(1) designing primers according to the HA gene of A/chicken/Jilin/DH109/2012(H9N2), namely SEQ ID NO.1, and amplifying the HA gene;

(2) constructing a recombinant baculovirus expressing structural protein of H9N2 subtype avian influenza virus, which specifically comprises the following steps:

cloning the HA gene and the gag gene in the step (1) into a vector pFastBac1 respectively to obtain recombinant plasmids pFastBac1-HA and pFastBac 1-gag;

recombinant plasmids pFastBac1-HA and pFastBac1-gag were transformed respectivelyEscherichia coliDH10Bac competent cell, through screening of blue-white spot 3 times, get recombinant bacmid-HA and rBacmid-gag;

respectively transfecting a host Sf9 insect cell with recombinant bacmid-HA and rBacmid-gag by using a liposome mediated transfection method to obtain recombinant baculovirus rBV-HA and rBV-gag;

(3) the preparation and purification of H9N2 subtype avian influenza VLPs specifically comprise the following steps:

the recombinant baculovirus rBV-HA and rBV-gag are inoculated with host Sf9 insect cells together by MOI =5, after the two structural proteins are expressed and self-assembled, the formed VLPs are secreted into cell culture supernatant, after cell debris is removed by primary centrifugation, purified VLPs are obtained by ultracentrifugation and sucrose density gradient centrifugation.

(4) The H9N2 subtype avian influenza VLPs pass through hemagglutination test and Western bThe lot and the transmission electron microscope are used for detection, and the result shows that the blood coagulation titer reaches 8 logs₂The HA protein and the gag protein can be simultaneously expressed, and the particle morphology structure of the virus-like particles is observed under a transmission electron microscope.

The application of the avian influenza VLPs in preventing H9N2 subtype avian influenza.

The prepared VLPs are used for immunizing SPF chickens once at the age of 10 days, and blood is respectively collected every 7 days after immunization to monitor the antibody titer. Three weeks after immunization at 10⁶EID₅₀The virus counteracting dosage is used for counteracting the virus, and immune protection effect evaluation is carried out by detecting in-vitro toxin expelling and in-vivo virus carrying of the virus and weight change of the chickens.

The method HAs the advantages that based on a Bac-to-Bac insect-baculovirus expression system, HA genes encoding H9N2 subtype avian influenza virus A/chicken/Jilin/DH109/2012 strains and gag genes of MLV are cloned to a baculovirus expression vector to form recombinant baculovirus, expanding culture is carried out after Sf9 cells are transfected, and VLPs of H9N2 subtype avian influenza are obtained through sucrose gradient centrifugation. The advantages are embodied in the following aspects:

1. VLPs can be assembled by themselves, the shape and size of the VLPs are closer to those of natural virus particles, the VLPs can stimulate organisms to generate better immune response, and the immune effect is better.

2. The diameters of VLPs containing gag protein are larger than the average diameter of influenza virus particles, so that more surface proteins of avian influenza virus can be accommodated, and a better immune effect is generated.

3. The H9N2 subtype avian influenza chimeric VLPs do not contain proteins of PB2, PB1, PA, NP, NA, M, NS and the like of avian influenza viruses, so that when serum is detected, animals immunized by the VLPs do not produce antibodies specific to PB2, PB1, PA, NP, NA, M and NS, and therefore the VLPs can be used as a novel marker vaccine for distinguishing wild virus infection and inactivated vaccine immunization of the H9N2 subtype avian influenza viruses.

4. Since VLPs do not contain viral genomes, such VLPs having an empty shell structure are safe in immunizing animals without causing integration of viral genes with host chromosomal genes, and are not exposed to live infectious viruses in the whole production process.

Drawings

FIG. 1 shows the result of HA gene PCR identification of H9N2 subtype avian influenza virus A/chicken/Jilin/DH 109/2012. Wherein lane M is Trans2K DNA Marker; lane 1 shows the result of PCR amplification of the target gene HA using HA forward primer/HA reverse primer, and the fragment size is about 1700 bp.

FIG. 2 shows the restriction identification of the shuttle plasmid. Wherein Lane M is DL5000 DNA Marker; lane 1 shows the HindIII and BamH I digestion of the shuttle plasmid pEASY T1-Simple-HA, resulting in a fragment size of about 1700 bp.

FIG. 3 shows the PCR identification results of two recombinant bacmid specific primers. Wherein lane M is Trans2K DNA Marker; lane 1 is the result of PCR amplification of recombinant bacmid-HA using HA upstream/HA downstream primers with a fragment size of about 1700 bp; lane 2 shows the result of PCR amplification of recombinant bacmid-gag with gag forward primer/gag reverse primer, and the fragment size was approximately 1560 bp.

FIG. 4 shows the PCR identification results of the universal primer M13 upstream primer/M13 downstream primer of two recombinant bacons. Wherein lane M is Trans2K plus DNA Marker; lane 1 shows the result of PCR amplification of recombinant bacmid-HA using M13 upstream primer/M13 downstream primer, with a fragment size of about 4050 bp; lane 2 shows the result of PCR amplification of recombinant bacmid-gag using M13 upstream primer/M13 downstream primer, and the fragment size was about 3780 bp.

FIG. 5 is a schematic representation of normal Sf9 cells transfected with recombinant bacmid to produce diseased Sf9 cells. Wherein (a) is normal Sf9 cell, (b) is Sf9 cell with pathological changes after recombinant bacmid-HA transfection, and (c) is Sf9 cell with pathological changes after recombinant bacmid-gag transfection.

FIG. 6 shows the PCR identification result of the universal primer M13 upstream primer/M13 downstream primer of the recombinant baculovirus genome of generation P1. Wherein lane M is Trans2K plus DNA Marker; lane 1 shows the result of PCR amplification of recombinant bacmid-HA using M13 upstream primer/M13 downstream primer, with a fragment size of about 4050 bp; lane 2 shows the result of PCR amplification of recombinant bacmid-gag using M13 upstream primer/M13 downstream primer, and the fragment size was about 3780 bp.

FIG. 7 shows Western blot identification results of two recombinant baculoviruses. Wherein lane M is the protein standard molecular mass; lane 1 shows the results of rBV-HA assay; lane 2 is the results of rBV-gag identification.

Fig. 8 is a graph of hemagglutination results for H9N2 subtype avian influenza VLPs.

FIG. 9 shows the Western blot identification of H9N2 subtype avian influenza VLPs. Wherein lane M is the protein standard molecular mass; lane 1 shows VLPs identification.

FIG. 10 is a transmission electron micrograph of H9N2 subtype avian influenza VLPs.

FIG. 11 is a graph showing the rule of decrease in titer of hemagglutination-inhibiting antibodies in H9N2 subtype avian influenza VLPs immunized chicken flocks.

FIG. 12 is a graph showing the results of lymphocyte proliferation after immunization of a chicken flock with H9N2 subtype avian influenza VLPs.

FIG. 13 is a graph showing the results of weight gain after challenge in an immunized chicken flock.

Detailed Description

The invention is further described below with reference to the figures and examples. Before describing the specific embodiments, some of the biological materials, reagents, and devices mentioned in the following embodiments will be briefly described as follows.

Biological material:

strains A/chicken/Jilin/DH109/2012, pFastBac1 plasmid and Sf9 insect cells were stored in the laboratory; DH10Bac competent cells were purchased from Invitrogen;

the gag protein gene of mouse leukemia virus is synthesized by Nanjing Kingsrei Biotech, Inc.;

pEASY-T1 Simple vector was purchased from Beijing Quanjin Biotechnology, Inc.

Experimental reagent:

la Taq DNA Polymerase and DL5000 DNA Marker were purchased from Dalibao Bio Inc.;

t4 DNA ligase, Trans2K DNA Marker, Trans2K Plus DNA Marker available from Beijing Quanjin Biotechnology Ltd;

hind III and BamH I were purchased from NEB;

liposome Transfection Reagent Lipo3000 Transfection Reagent for Invitrogen company products;

the plasmid extraction kit and the DNA gel recovery kit are purchased from Corning company;

FastStart Universal SYBR Green Master (ROX) from Roche;

the insect serum-free culture medium SFM-II and the Imject-alum adjuvant are products of Thermo company;

fetal Bone Serum (FBS) was purchased from BI;

commercial inactivated vaccine H9N2 was purchased from Cisco.

Experimental equipment:

ultrapure water used in the experimental process is prepared by an ultrapure water generator (Christ Spetron Line);

low temperature bench top high speed centrifuge, product of ICE corporation, usa;

PCR instrument, product of Thermo Fisher Scientific Co;

UV gel imager, product of Alpha Inotech Corporation.

It should be noted that, during a brief text, operations are not specifically described in the following embodiments, and the operations may be performed with reference to the prior art and the related product specification, which are not described again.

Example 1

The H9N2 subtype avian influenza VLPs provided by the invention are protein particles with hollow structures formed by self-assembly of HA protein of a strain A/chicken/Jilin/DH109/2012 and gag protein of mouse leukemia virus. Wherein the nucleotide sequence of HA gene corresponding to HA protein is shown as SEQ ID NO.1, and the nucleotide sequence of gag gene corresponding to gag protein is shown as SEQ ID NO. 2.

Designing primers according to HA genes of A/chicken/Jilin/DH109/2012(H9N2), wherein the sequences of upstream primers and downstream primers of the HA genes are shown as follows:

H9F：5’-cgggatccatgggagccgtatcatt-3’

H9R：5’-cccaagctttcaaatgcaaatgttgcacc-3’

example 2

The construction process of the recombinant baculovirus expressing structural protein of H9N2 subtype avian influenza virus in the preparation process of H9N2 subtype avian influenza VLPs is briefly described as follows:

(1) amplification of the Gene sequences of the respective structural proteins

According to the target nucleotide sequence shown by SEQ ID NO. 1-2 in the embodiment 1, the HA gene is obtained by Polymerase Chain Reaction (PCR) amplification with reference to the prior art.

When PCR identification is carried out, the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 90 seconds; finally, extension was carried out at 72 ℃ for 10 minutes. The PCR amplification products were analyzed by 1% (m/v) agarose gel electrophoresis, and the results are shown in FIG. 1.

(2) Construction of shuttle plasmid

According to the prior art, the HA gene obtained by cloning in the step (1) is cloned into pEASY-T1 Simple, and is transformed and sequenced, and is verified by double enzyme digestion of BamH I and Hind III, and the enzyme digestion product is recovered, so that a correctly constructed recombinant clone plasmid pEASY-T1 Simple-HA is obtained, and the result is shown in figure 2.

Connecting the enzyme digestion product with pFastBac1 (double enzyme digestion of BamH I and Hind III) plasmid by using T4 DNA ligase; then the gag gene synthesized by the gene is connected with pFastBac1 (Sal I and Xba I double enzyme cutting) plasmid, and the connecting system is as follows: 4 mu L of recombinant plasmid restriction enzyme product; 1 mu L of pFastBac1 carrier enzyme digestion product; 5 × Buffer 2 μ L; 1 μ L of T4 DNA ligase; ddH₂O2. mu.L. After ligation at 25 ℃ for 30 min, the ligation product was converted toEscherichia coli In DH 5. alpha. competent cells, selection medium containing specific antibiotics (final ampicillin concentration 100. mu.g/mL, final gentamicin concentration 100. mu.g/mL) was used to screen positive colonies.

And amplifying the screened positive colonies, extracting plasmids, adopting enzyme digestion verification and PCR identification to detect whether the HA gene and the gag gene are correctly recombined into a pFastBac1 vector, and identifying the correct plasmids to determine that the shuttle plasmids pFastBac1-HA and pFastBac1-gag are successfully constructed.

(3) Construction of recombinant bacmids

Transforming the recombinant plasmid identified as correct in step (2) intoEscherichia coliIs subjected to homologous recombination in DH10Bac competent cells containingAfter culturing and screening for 48 hours by using an IPTG conditional screening culture medium (the final concentration of IPTG is 24mg/mL, the final concentration of X-gal is 20mg/mL, the final concentration of the screening culture medium is 24mg/mL, the final concentration of the screening culture medium is 20mg/mL, the screening culture medium can judge whether homologous recombination occurs through colony formation color, white colonies represent that the recombination is successful, and blue colonies do not succeed) of a three-antibody (the final concentration of kanamycin is 100 mu g/mL, the final concentration of gentamicin is 50 mu g/mL, and the final concentration of tetracycline is 70 mu g/mL), picking white spots to obtain recombinant bacmid-HA and rBacmid-gag.

The extracted recombinant bacmids were further subjected to PCR identification to confirm that the recombinant bacmids were constructed correctly. For PCR identification, the specific primer and the universal primer M13 (Bac-to-Bac from Invitrogen company) in step (1) were used^®Synthesized in Baculoviral Expression System operating Specification), the sequences of the upstream primer and the downstream primer of M13 are shown as follows:

M13F：5’-gttttcccagtcacgac-3’

M13R：5’-caggaaacagctatgac-3’

when PCR identification is carried out, the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 95 ℃ for 30 seconds, annealing at 60 ℃ for 30 seconds, and extension at 72 ℃ for 90 seconds; finally, extension was carried out at 72 ℃ for 10 minutes.

The PCR amplification products were analyzed by 1% (m/v) agarose gel electrophoresis, and the results of identification using the specific primers in step (1) are shown in FIG. 3. As can be seen from the analysis of FIG. 3, the HA fragment size was about 1700bp and the gag fragment size was about 750bp, which is consistent with the expected results. The results of the identification with the M13 primer are shown in FIG. 4. As can be seen from the analysis of FIG. 4, the HA fragment size was approximately 4050bp, and the gag fragment size was approximately 3788bp, and the results were consistent with the expectation that the HA gene and the gag gene fragment were correctly recombined into bacmid, i.e., the recombinant bacmid-HA and rBacmid-gag were correctly constructed.

(4) Construction of recombinant baculovirus

The correct recombinant bacmid-HA and rBacmid-gag constructed in step (3) were transfected into Sf9 insect cells, respectively, as hosts, using liposome-mediated transfection (transfection according to the Invitrogen corporation Lipo3000 instructions). Culturing at 28 deg.C for 72 hr, and collecting cell supernatant as first generation recombinant baculovirus rBV-HA and rBV-gag after cytopathic effect (as shown in FIG. 5). The first generation of recombinant baculovirus was then inoculated into insect Sf9 cells and the second generation of recombinant baculovirus was collected under the same culture conditions. And by analogy, collecting the fourth generation recombinant baculovirus. For ease of detection and analysis, each generation of recombinant baculovirus may be stored at-80 ℃ for use.

During the culture, the cultures were PCR-characterized with universal primer M13 as appropriate to determine whether the recombinant baculovirus containing the HA gene and gag gene fragment was intact and correct.

When PCR identification is carried out, the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 45 seconds, annealing at 55 ℃ for 40 seconds, and extension at 72 ℃ for 4 minutes for 30 cycles; the extension was continued for 10 minutes at 72 ℃.

The PCR amplification products were analyzed by 1% (m/v) agarose gel electrophoresis, and the results are shown in FIG. 6. As can be seen from the analysis of FIG. 6, the size of the genome containing the HA gene fragment is about 4050 bp; the size of the genome containing the gag gene fragment is about 3000bp, and the result is in accordance with the expectation, namely the recombinant baculovirus has a complete and effective structure and can be further applied.

(5) Western blot identification

Taking a proper amount of cell supernatant, uniformly mixing the cell supernatant with a protein loading buffer solution according to the proportion of 5:1, boiling for 10 minutes, centrifuging for 5 minutes at 12000g, adding a sample into a loading hole, transferring in a semi-dry manner, and operating at a constant pressure of 80V for 2 hours. Cutting the adhesive tape to a proper size, cutting the PVDF membrane and the filter paper to be as large as the adhesive tape, sensitizing the PVDF membrane in methanol for 15 seconds, and then transferring the adhesive tape, the PVDF membrane and the filter paper to a membrane transferring buffer solution to be soaked for 15 minutes. When the membrane is transferred, the anode carbon plate, the filter paper, the PVDF membrane, the gel, the filter paper and the cathode carbon plate are sequentially placed and aligned from bottom to top, bubbles are removed, a power supply is turned on, the pressure is kept constant for 20V, and the membrane is transferred for 20 minutes. After the transfer was completed, the membrane was taken out, PBS containing 5% skim milk powder was added, and shaking-table incubation was performed at 37 ℃ for 2 hours. Discard blocking solution, wash membrane 3 times with PBST, 15 minutes each time. Because of the different antibodies used, the membranes were cut open and primary antibody was added at 1:1000, 4 ℃ overnight. Discard primary antibody, wash membrane 3 times with PBST, 15 minutes each time. Secondary antibodies were added at a concentration of 1:5000, respectively, and incubated at 37 ℃ for 1 hour. Discard the secondary antibody, wash the membrane 3 times with PBST for 15 minutes each time, develop with ECL luminescence solution, and check the results. As a result, as shown in FIG. 7, Western blot was detected with bands of approximately 65kDa and 55kDa, which was in agreement with the expectation.

Example 3

The present example briefly describes the expression, assembly and purification process of structural proteins in H9N2 subtype avian influenza VLPs as follows:

(1) expression of structural proteins in VLPs

The supernatant containing the fourth generation of recombinant baculoviruses rBV-HA and rBV-gag collected in example 2 was co-infected with Sf9 suspension cells at MOI =5 for 96 hours. After infection, the infected cells were subjected to shaking culture at 28 ℃ at 120 rpm. In this culture process, the structural proteins assemble themselves after being expressed separately in the host cells, and the assembled VLPs are secreted into the cell culture supernatant.

(2) Purification and identification of H9N2 subtype avian influenza VLPs

Collecting the cell culture supernatant obtained in the step (1), and centrifuging for 30 minutes at 3000 r/m to primarily remove large cell debris; and ultracentrifugation and sucrose density gradient centrifugation are carried out, a white band is formed in the middle of a sucrose layer, baculovirus is precipitated at the bottom, other small foreign proteins stay at the top layer, and the middle white band layer is the purified VLPs sample.

The hemagglutination titer of the prepared H9N2 subtype avian influenza VLPs is detected, the result is shown in figure 8, and the hemagglutination titer of the purified H9N2 subtype avian influenza VLPs is 8log₂(ii) a Then, Western blot identification is carried out on the particles, the process is the same as that (5) in the example 2, the result is shown in FIG. 9, bands detected by the Western blot are about 65kDa and 55kDa, and the result is consistent with the expectation; and finally, observing the purified VLPs by a transmission electron microscope, wherein the forms of the VLPs are shown in FIG. 10. The results all show that H9N2 subtype avian influenza VLPs are correctly constructed.

Example 4

In this example, the immunization effect of VLPs prepared in example 3 when used as a vaccine was briefly evaluated.

(1) Immunization program and challenge protocol

Referring to '2020 national animal epidemic disease mandatory immunization program' of the Ministry of agricultural rural China, VLPs vaccine immunization components are divided into 3 immunization doses of high (40 mug/300 muL), medium (30 mug/300 muL) and low (15 mug/300 muL), and the doses are 0.3 mL/feather; the commercial vaccine group for H9N2 subtype avian influenza was also injected with 0.3 mL/plume according to the instructions. The immunization route is intramuscular injection in the legs. Performing nasal cavity challenge on day 21 after immunization, and performing challenge by using A/chicken/Jilin/DH109/2012 strain at a dose of 10 mL/nostril⁶EID₅₀The specific immunization schedule and challenge protocol are shown in table 1.

TABLE 1 Experimental animal grouping and immunization challenge protocol

(2) HI antibody titer assay

Blood is collected from the infrawing vein every other week from the first week after immunization, and serum is separated and stored at-20 ℃ for later use.

The positive antigen of H9N2 subtype avian influenza virus hemagglutination inhibition test is used as detection antigen, after serum samples are diluted in multiple proportion, 25 mu L of four-unit antigen is added and mixed in equal volume, the mixture acts for 30 minutes at 37 ℃, 25 mu L of 1% chicken erythrocyte is added and acts for 30 minutes on ice, and HI titer is the highest serum dilution multiple of complete erythrocyte agglutination inhibition.

The results are shown in fig. 11, and the antibody levels of each dose group and commercial vaccine group continuously increased after immunization, reached a peak 21 days after immunization, and slightly decreased after challenge, but the antibody levels were always above the protective line (2 log)₂). Wherein the 30 μ g VLPs group was not significantly different from the commercial vaccine group (p> 0.05), the 40 μ g VLPs group was significantly higher overall than the commercial vaccine group (p<0.001) and HI antibody titers appeared dose-dependent, i.e., 40 μ g VLPs panel > 30 μ g VLPs panel > 15 μ g VLPs panel. Therefore, the experiment shows that each dose group and the commercial vaccine group can induce organisms to generate good HI antibody titer and provide good immune protection effect, and the 40 mu g VLPs group inducesThe highest level of humoral immunity was produced, with the 30 μ g VLPs group being able to induce comparable levels of humoral immunity to the commercial vaccine group.

(3) Lymphocyte proliferation assay

Spleens of chickens were harvested by aseptic manipulation, ground thoroughly on a 200 mesh screen, and resuspended in an appropriate amount of lymphocyte isolate. The slurry was transferred to a 15mL centrifuge tube and centrifuged at 2000 rpm for 10 minutes to obtain the middle white lymphocyte layer. The red blood cell lysate was added to the centrifuge tube containing the white lymphocytes and resuspended, after 5 minutes at room temperature, centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded, an appropriate amount of PBS solution was added and gently mixed, centrifuged at 2000 rpm for 10 minutes, and repeated 2 times. The supernatant was discarded, and the lymphocyte concentration was adjusted to 1X 10 by using 1640 medium⁶one/mL. Add 100. mu.L of lymphocyte suspension to each well of 96-well cell culture plate, put in 5% CO at 37 ℃₂After 2 hours of culture in a cell incubator, adding ConA, inactivated H9N2 subtype avian influenza virus and PBS as stimulators, and arranging two groups of controls, wherein one group is added with 1640 culture medium, the other group is added with lymphocyte suspension, and each group is provided with three multiple wells. Put in 5% CO at 37 DEG C₂The cell culture box was cultured for 24 hours. Adding 10 μ L of CCK-8 solution into each well, and adding 5% CO at 37 deg.C₂The cell incubator was incubated for 2 hours. OD was measured at 490nm using a microplate reader.

And (4) judging a result: SI = (sample OD value-blank OD value)/(negative OD value-blank OD value)

The results are shown in FIG. 12, with increasing SI values in each immunization group, peaking at day 21 post-immunization and showing a dose-dependent relationship, i.e., 40 μ g VLPs > 30 μ g VLPs > 15 μ g VLPs. Furthermore, the lymphocyte activation capacity of the 30 μ g VLP-gag group has not significantly differed from that of the commercial vaccine group (p> 0.05); at 7, 14 days after immunization, the 40 μ g VLP-gag group did not significantly differ from the commercial vaccine group: (p> 0.05), but the lymphocyte activation capacity of the 40 mug VLP-gag group is higher than that of the commercial vaccine group at 21 and 28 days after immunization, and the difference is very significant (p< 0.0001); all groups were significantly higher than the PBS group. This indicates that the 40. mu.g group of VLPs induced the highest level of cellular immunity, 30. mu.gThe VLPs group induced a comparable level of cellular immunity to the commercial vaccine group.

(4) Weight monitoring

Animals were monitored for weight daily in the morning after challenge and prior to feeding. The results are shown in FIG. 13, and there is no significant difference in the rate of weight gain between the 30 μ g VLPs and the commercial vaccine group: (p> 0.05), while the group with 40 μ g VLPs had a higher rate of weight gain than the group with the commercial vaccine, the difference was very significant (p< 0.001), and this difference persists until day 14 after challenge. The 15 μ g VLPs group and the commercial vaccine group differed very significantly on day 5 after challenge (p< 0.001), but the body weight growth rates of the two were not significantly different from each other overall (p> 0.05). Thus, all dose groups and commercial vaccine groups provided effective protection for experimental animals.

(5) In vitro detoxification time detection

Collecting oropharynx and cloaca cotton swabs every other one day from the 3 rd day after the challenge, putting the swabs into 500 mu L PBS solution containing 1% double antibody (penicillin and streptomycin), centrifuging for 10 minutes at 2000g, taking the supernatant, sucking 100 mu L of sample liquid from each sample, inoculating the sample liquid into the allantoic cavity of SPF chick embryos aged 9-11 days, collecting the allantoic liquid after 48 hours, carrying out hemagglutination test to detect the toxin expelling condition, and excluding the chick embryos dead within 24 hours.

TABLE 2 detection of in vitro toxicity discharge time after H9N2 subtype avian influenza VLPs immunization challenge

The results are shown in Table 2, and the cloaca detoxification results and the oropharynx detoxification results both show that no detoxification phenomenon is detected in groups of 15 μ g VLPs, 30 μ g VLPs and 40 μ g VLPs on day 3 after challenge; on day 5 after challenge, detoxification was stopped for the commercial vaccine group. The results show that each dose of VLPs group has shorter in vitro detoxification time than the commercial vaccine group, and can better prevent virus replication.

(6) In vivo distribution assay for viruses

From day 1 after challenge, 3 experimental chickens were sacrificed randomly every other day, their brains, tracheas, lungs, spleens, intestines, pancreas, kidneys and muscles were taken, tissues of appropriate size were taken and put into PBS solution containing 1mL for grinding, 2000g was centrifuged for 10 minutes, the supernatant was taken and its RNA was extracted and reverse transcribed to obtain cDNA, qPCR identification was performed using the HA gene specific primers in example 1 to determine whether there was a virus.

TABLE 3 in vivo viral load detection after immune challenge of H9N2 subtype avian influenza VLPs

The results are shown in Table 3, and no virus was detected in all tissues to be examined at day 3 after challenge, in the 15. mu.g VLPs group, the 30. mu.g VLPs group, and the 40. mu.g VLPs group; on day 5 post challenge, all tissues tested in the commercial vaccine group were virus-free. The results show that the VLPs groups at each dose had lower viral loads in vivo than the commercial vaccine groups, and were better able to prevent viral replication.

In conclusion, the H9N2 subtype avian influenza VLPs designed and prepared by the invention can stimulate chickens to generate stronger antibody titer, provide effective protection for immunized chickens, and can obviously inhibit toxin expelling and virus replication in vivo. The VLPs can be used as a marker vaccine for distinguishing wild virus infection and inactivated vaccine immunity of H9N2 subtype avian influenza virus clinically. Therefore, the chimeric VLPs are used for immunization, and technical support can be provided for purification of H9N2 subtype avian influenza.

Sequence listing

<110> Jilin university

<120> preparation method and application of H9N2 subtype avian influenza chimeric virus-like particle

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1683

<212> DNA

<213> Influenza (Influenza A virus)

<400> 1

atgggagccg tatcattgat aactatgcta ctagtagcaa cagtaagcaa tgcagacaaa 60

atctgcatcg gataccaatc aacaaactcc acagaaactg tagacacact aacagaaaac 120

aatgtccctg tgacacatgc caaagaattg ctccacacag agcacaatgg gatgctatgt 180

gcaacaaact tgggacatcc tcttattcta gacacctgta ccattgcagg actaatctat 240

ggcaatcctt cttgtgatct attgctggga ggaagagaat ggtcttacat cgtcgagaga 300

ccatcggctg tcaatggatt gtgctacccc gggaatgtag aaaatctaga agaactaagg 360

tcacttttca gttctgctag ttcttatcaa agaatccaga tttttccgga cacaatatgg 420

aatgtgtctt acagtggaac aagcaaagca tgttcagatt cattctacag aagcatgaga 480

tggttgaccc aaaagaacaa cgcttaccct attcaagacg cccaatacac aaataatcga 540

gaaaagaaca ttcttttcat gtggggtata aatcacccac ccaccgagac tacacagaca 600

gatctgtaca caagaaccga cacaacaaca agtgtggcaa cagaagaaat aaataggacc 660

ttcaaaccat tgataggacc aaggcctctt gtcaatggtt tgcagggaag aattgattat 720

tattggtcgg tattgaaacc aggtcaaaca ctgcgagtaa gatccaatgg gaatctaata 780

gctccatggt atggacacat tctttcagga gagagccacg gaagaatcct gaagactgat 840

ttgaaaaggg gtagctgtac agtgcaatgt cagacagaaa aaggtggctt aaacacaaca 900

ttgccattcc aaaatgtaag taagtatgca tttggaaact gctcgaaata tgttggagta 960

aagagtctca aacttgcagt tggtctgagg aatgtgcctt ctaaatctag tagaggacta 1020

tttggggcca tagctggatt catagaggga ggttggtcag gactagttgc tggttggtat 1080

ggattccagc attcaaatga ccaaggggtt ggtatggcag cagatagaga ctcaacccaa 1140

aaggcaattg ataaaataac atccaaagtg aataacatag tcgataaaat gaacaaacag 1200

tatgaaatta ttgatcatga attcagcgag gttgaaaata gacttaacat gatcaataat 1260

aagattgatg atcaaattca agacatatgg gcatataacg cagaactgct agtgctactt 1320

gaaaaccaga aaacactcga tgagcatgat gcaaatgtaa ataatctata taataaagtg 1380

aagagggcat tgggttccaa tgcagtggaa gatgggaaag gatgtttcga gctatatcac 1440

agatgtgatt accagtgcat ggagacaatt cggaacggga cctacaacag gaggaaatat 1500

caagaggaat caaaattaga aaggcagaga atagaggggg tcaagctgga gtctgaagga 1560

acttacaaaa ttctcaccat ttattcgact gtcgcctcat ctcttgtgat tgcaatgggg 1620

tttgctgcct tcttgttctg ggccatgtcc aatgggtctt gcagatgcaa catttgtata 1680

taa 1683

<210> 2

<211> 2577

<212> DNA

<213> Murine leukemia Virus (Murine leukemia virus)

<400> 2

atgggagccg tatcattgat aactatgcta ctagtagcaa cagtaagcaa tgcagacaaa 60

atctgcatcg gataccaatc aacaaactcc acagaaactg tagacacact aacagaaaac 120

aatgtccctg tgacacatgc caaagaattg ctccacacag agcacaatgg gatgctatgt 180

gcaacaaact tgggacatcc tcttattcta gacacctgta ccattgcagg actaatctat 240

ggcaatcctt cttgtgatct attgctggga ggaagagaat ggtcttacat cgtcgagaga 300

ccatcggctg tcaatggatt gtgctacccc gggaatgtag aaaatctaga agaactaagg 360

tcacttttca gttctgctag ttcttatcaa agaatccaga tttttccgga cacaatatgg 420

aatgtgtctt acagtggaac aagcaaagca tgttcagatt cattctacag aagcatgaga 480

tggttgaccc aaaagaacaa cgcttaccct attcaagacg cccaatacac aaataatcga 540

gaaaagaaca ttcttttcat gtggggtata aatcacccac ccaccgagac tacacagaca 600

gatctgtaca caagaaccga cacaacaaca agtgtggcaa cagaagaaat aaataggacc 660

ttcaaaccat tgataggacc aaggcctctt gtcaatggtt tgcagggaag aattgattat 720

tattggtcgg tattgaaacc aggtcaaaca ctgcgagtaa gatccaatgg gaatctaata 780

gctccatggt atggacacat tctttcagga gagagccacg gaagaatcct gaagactgat 840

ttgaaaaggg gtagctgtac agtgcaatgt cagacagaaa aaggtggctt aaacacaaca 900

ttgccattcc aaaatgtaag taagtatgca tttggaaact gctcgaaata tgttggagta 960

atgggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa agatgtcgag 1020

cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac cttctgctct 1080

gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa ccgagacctc 1140

atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc agaccaggtc 1200

ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt caagcccttt 1260

gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc ccttgaacct 1320

cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc tctaggcgcc 1380

aaacctaaac ctcaagttct ttctgacagt ggggggccgc tcatcgacct acttacagaa 1440

gaccccccgc cttataggga cccaagacca cccccttccg acagggacgg aaatggtgga 1500

gaagcgaccc ctgcgggaga ggcaccggac ccctccccaa tggcatctcg cctacgtggg 1560

agacgggagc cccctgtggc cgactccact acctcgcagg cattccccct ccgcgcagga 1620

ggaaacggac agcttcaata ctggccgttc tcctcttctg acctttacaa ctggaaaaat 1680

aataaccctt ctttttctga agatccaggt aaactgacag ctctgatcga gtctgttctc 1740

atcacccatc agcccacctg ggacgactgt cagcagctgt tggggactct gctgaccgga 1800

gaagaaaaac aacgggtgct cttagaggct agaaaggcgg tgcggggcga tgatgggcgc 1860

cccactcaac tgcccaatga agtcgatgcc gcttttcccc tcgagcgccc agactgggat 1920

tacaccaccc aggcaggtag gaaccaccta gtccactatc gccagttgct cctagcgggt 1980

ctccaaaacg cgggcagaag ccccaccaat ttggccaagg taaaaggaat aacacaaggg 2040

cccaatgagt ctccctcggc cttcctagag agacttaagg aagcctatcg caggtacact 2100

ccttatgacc ctgaggaccc agggcaagaa actaatgtgt ctatgtcttt catttggcag 2160

tctgccccag acattgggag aaagttagag aggttagaag atttaaaaaa caagacgctt 2220

ggagatttgg ttagagaggc agaaaagatc tttaataaac gagaaacccc ggaagaaaga 2280

gaggaacgta tcaggagaga aacagaggaa aaagaagaac gccgtaggac agaggatgag 2340

cagaaagaga aagaaagaga tcgtaggaga catagagaga tgagcaagct attggccact 2400

gtcgttagtg gacagaaaca ggatagacag ggaggagaac gaaggaggtc ccaactcgat 2460

cgcgaccagt gtgcctactg caaagaaaag gggcactggg ctaaagattg tcccaagaaa 2520

ccacgaggac ctcggggacc aagaccccag acctccctcc tgaccctaga tgactag 2577

Claims

1. An H9N2 subtype avian influenza chimeric virus-like particle, characterized in that: the HA protein of the H9N2 subtype avian influenza virus and the gag protein of the mouse leukemia virus are contained, the nucleotide sequence of the encoded HA protein is represented by SEQ ID NO.1, and the nucleotide sequence of the encoded gag protein of the mouse leukemia virus is represented by SEQ ID NO. 2.

2. The chimeric virus-like particle of avian influenza subtype H9N2 according to claim 1, characterized by the following preparation method:

(1) the primer design method specifically comprises the following steps:

designing primers according to HA genes of A/chicken/Jilin/DH109/2012(H9N2), wherein the sequences of upstream primers and downstream primers of the HA genes are as follows:

H9F：5’-cgggatccatgggagccgtatcatt-3’

H9R：5’-cccaagctttcaaatgcaaatgttgcacc-3’

(2) constructing a recombinant baculovirus expressing HA protein of the avian influenza virus, which specifically comprises the following steps:

cloning the HA gene and the gag gene in the step (1) into a transfer vector pFastBac1 respectively to obtain recombinant plasmids pFastBac1-HA and pFastBac 1-gag;

recombinant plasmids pFastBac1-HA and pFastBac1-gag were transformed respectivelyEscherichia coliDH10Bac competent cell, screen and obtain recombinant bacmid-HA and rBacmid-gag;

(3) the preparation and purification of avian influenza VLPs specifically comprise the following steps:

co-infecting host Sf9 insect cells with recombinant baculovirus rBV-HA and rBV-gag with MOI =5, expressing and self-assembling two structural proteins, secreting the formed VLPs into cell culture supernatant, centrifuging to remove cell debris, ultracentrifuging and sucrose density gradient centrifuging to obtain purified VLPs.

3. An application of H9N2 subtype avian influenza chimeric virus-like particle as a marker vaccine for distinguishing wild virus infection and inactivated vaccine immunity of clinical H9N2 subtype avian influenza virus.