Recombinant avian influenza virus inactivated vaccine and application thereof

Abstract

The invention discloses a recombinant avian influenza virus inactivated vaccine, a recombinant avian influenza virus in the vaccine comprises an HA gene of an H9 subtype avian influenza virus, and two repeated P29 genes are inserted behind a signal peptide sequence of the HA gene. The invention also discloses an application of the recombinant avian influenza virus inactivated vaccine. The recombinant avian influenza virus inactivated vaccine can rapidly induce good humoral immunity and cellular immunity response in immune animal bodies, can overcome interference of maternal antibodies, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the field of bioengineering technology, and in particular to a recombinant avian influenza virus inactivated vaccine stably expressing H9 subtype avian influenza virus HA and two repeated complement protein C3d minimum binding region P29 fusion proteins and its application. Background Art

[0002] H9N2 avian influenza virus (AIV) is the most widespread and harmful low pathogenic avian influenza virus in the world. It can infect poultry including chickens, ducks, quails, etc. It can also provide internal genes for other subtypes such as H5 and H7 highly pathogenic avian influenza. It can also directly infect humans across hosts, causing huge economic losses to my country's poultry farming industry every year, and also causing huge damage to public health. For safety reasons, inactivated vaccines are mainly used to prevent and control avian influenza viruses worldwide. In my country, although the use of H9N2 AIV inactivated vaccines has been more than 25 years, due to problems such as interference of maternal antibodies with the immune effect of vaccines, the immune effect of these vaccines in local poultry farms is poor, which in turn leads to the continuous spread of H9N2 AIV in local poultry farms in my country.

[0003] Maternal antibodies (MDAs) are maternal antibodies derived from the placenta (humans and mammals) or yolk sac (poultry). On the one hand, MDAs can protect young animals from infection with many pathogenic microorganisms during the vulnerable period at the beginning of life. On the other hand, MDAs can interfere with the immune response of young animals to vaccines. MDAs decrease with age, which can lead to the formation of an immune window period because MDAs interfere with the immune effect of vaccines. Considering the convenience and cost-effectiveness of vaccinating young animals, it is crucial to develop a new inactivated avian influenza vaccine to overcome the interference of MDAs in young animals.

[0004] P29 is the minimal binding domain of C3d, the final degradation product of avian complement protein C3, and has been shown to enhance antibody responses and Th2 immune responses when bound to antigens, but it is unclear whether P29 or multiple repeats of P29 can help vaccines overcome the interference of MDAs. Summary of the invention

[0005] The present invention aims to solve the technical problem of the current lack of safe and effective inactivated avian influenza vaccines that can overcome the interference of MDAs, and provides a recombinant inactivated avian influenza virus vaccine that can stably express a fusion protein of the major antigen protein HA of the H9 subtype avian influenza virus and two repeated minimum binding regions P29 of the complement protein C3d. In the body of an immunized animal, even in the presence of high-titer MDAs, the vaccine can still induce good humoral and cellular immune responses, and is suitable for use as an avian influenza vaccine that overcomes the interference of MDAs.

[0006] In order to solve the above technical problems, the present invention is achieved through the following technical solutions:

[0007] In one aspect of the present invention, a recombinant avian influenza virus is provided, wherein the recombinant avian influenza virus comprises an HA gene of an H9 subtype avian influenza virus, wherein two repeated P29 genes are inserted after the signal peptide sequence of the HA gene, and the amino acid sequence of the protein encoded by the P29 gene is shown in SEQ ID NO.1.

[0008] Preferably, the nucleotide sequence of the P29 gene is shown as SEQ ID NO.2.

[0009] Preferably, a linker sequence is provided between the two P29 genes, and at the front end of the first P29 gene and the end of the second P29 gene, respectively.

[0010] In a specific embodiment of the present invention, in the recombinant avian influenza virus, two repeated P29 genes are inserted after the signal peptide sequence of the HA gene of the H9 subtype avian influenza virus, and a linker sequence is provided between the two P29 genes, and at the front end of the first P29 gene and the end of the second P29 gene, respectively. The amino acid sequence of the linker sequence is shown in SEQ ID NO.3.

[0011] Preferably, the amino acid sequence of the fusion protein expressed by the H9 subtype avian influenza virus HA gene and the inserted two repeated P29 genes is shown in SEQ ID NO. 4. The nucleotide sequence of the H9 subtype avian influenza virus HA gene and the inserted two repeated P29 genes is shown in SEQ ID NO. 5.

[0012] In another aspect of the present invention, a recombinant avian influenza virus inactivated vaccine is provided, comprising the inactivated recombinant avian influenza virus and an adjuvant.

[0013] The method for preparing the recombinant avian influenza virus inactivated vaccine comprises the following steps:

[0014] Insert two repeated P29 genes after the signal peptide sequence of the HA gene of the H9 subtype avian influenza virus to construct a recombinant avian influenza virus, wherein the amino acid sequence of the protein encoded by the P29 gene is shown in SEQ ID NO.1;

[0015] adding beta-propiolactone to the recombinant avian influenza virus to inactivate the recombinant virus;

[0016] The inactivated recombinant avian influenza virus is mixed with an adjuvant to obtain a recombinant avian influenza virus inactivated vaccine.

[0017] In another aspect of the present invention, there is also provided the use of the above-mentioned recombinant avian influenza virus in the preparation of a vaccine for preventing and / or treating avian influenza.

[0018] In another aspect of the present invention, there is also provided the use of the above-mentioned recombinant avian influenza virus inactivated vaccine in the preparation of biological products for preventing and / or treating avian influenza.

[0019] The recombinant avian influenza virus inactivated vaccine of the present invention uses low pathogenicity H9N2 AIV as a carrier, inserts two repeated gene sequences of the minimum binding region P29 of complement protein C3d into the gene sequence of its main antigen HA protein, thereby constructing a recombinant avian influenza virus expressing a fusion protein of H9 subtype avian influenza virus HA fused with two repeated minimum binding region P29 of C3d. The recombinant avian influenza virus (rH9N2-P29.2) inactivated vaccine obtained by the present invention can stimulate animals to produce a high level of immune response in the presence of MDAs, can effectively overcome the interference of maternal antibodies (MDAs), and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0021] Figure 1 This is a schematic diagram of the construction of the fusion gene of HA and two P29s in Example 1 of the present invention;

[0022] Figure 2 This is a diagram of the gene result of PCR detection of HA fused with two repeats of P29 (HA-P29.2) in rH9N2-P29.2 according to Example 2 of the present invention;

[0023] Figure 3 This is a graph showing the results of Western blotting of the recombinant virus rH9N2-P29.2 expressing the HA-P29.2 fusion protein in Example 2 of the present invention;

[0024] Figure 4 This is a graph showing the immunofluorescence results of the recombinant virus rH9N2-P29.2 expressing the HA-P29.2 fusion protein in Example 2 of the present invention;

[0025] Figure 5 It is a graph of antibody changes in animals immunized with MDAs using the recombinant virus rH9N2-P29.2 of Example 3 of the present invention and a graph of virus titer after challenge. DETAILED DESCRIPTION

[0026] In the following examples, the experimental methods without specific conditions are usually carried out according to conventional conditions, such as the methods described in "A Concise Guide to Molecular Biology Experiments" (Edited by FM Osber, RE Kingston, JG Seidman, etc., translated by Ma Xuejun and Shu Yuelong. Beijing: Science Press, 2004).

[0027] Most of the commercial chickens in the current breeding farms contain maternal antibodies, which seriously interfere with the immune effect of the inactivated avian influenza vaccine. In order to overcome the interference of maternal antibodies, the present invention has developed a recombinant avian influenza inactivated vaccine, using low pathogenicity H9N2AIV as a carrier, inserting two repeated gene sequences of the minimum binding region P29 of the complement protein C3d into the gene sequence of its main surface antigen hemagglutinin HA protein, and constructing a recombinant avian influenza virus that expresses a fusion protein of the H9 subtype avian influenza virus HA fused with two repeated minimum binding regions P29 of C3d. The recombinant virus is prepared as an inactivated vaccine, which can induce good humoral immunity and cellular immune response in the target animal, and more importantly, can overcome the interference of MDAs. The recombinant avian influenza virus (rH9N2-P29.2) inactivated vaccine obtained by the present invention can stimulate animals to produce a high level of immune response in the presence of MDAs.

[0028] In a specific embodiment of the present invention, the recombinant avian influenza virus rH9N2-P29.2 can stably express the avian influenza virus HA and two repeated P29 fusion proteins (HA-P29.2) on the cell membrane.

[0029] The HA protein of the present invention also includes its analogs or mutant proteins, or fragments thereof. The terms "analogs" and "mutants" refer to biologically active derivatives of reference molecules, or fragments of these derivatives that retain the desired activity (such as immunoreactivity). Generally, the term "analog" refers to a compound having a natural polypeptide sequence and structure, as well as one or more amino acid additions, substitutions (usually conservative in nature) and / or deletions relative to the natural molecule, as long as the modification does not destroy the immunogenic activity. The term "mutant" refers to a peptide having one or more peptide mimetics ("peptoids"). Preferably, the analog or mutant protein has at least the same activity or function as the natural molecule. Methods for preparing polypeptide analogs and mutant proteins are known in the art.

[0030] Example 1 Establishment of a recombinant avian influenza virus (rH9N2-P29.2) that stably expresses HA and two repeated P29 fusion proteins (HA-P29.2)

[0031] A P29 sequence (P29.2) containing two repeats was synthesized, and the two P29s were connected by a linker (GGGGS), as shown in the sequence SEQ ID NO.4. Primers containing homologous arms and linker (GGGGS) in the 5' and 3' regions of the HA signal peptide gene fragment of H9N2 AIV, as well as primers for the front section of the HA signal peptide and the back section of the HA signal peptide were synthesized, as shown in Table 1, where lowercase letters represent the sequence of the linker. The P29.2 gene sequence containing homologous arms and linker, as well as the gene sequence of the front section of the HA signal peptide and the back section of the HA signal peptide were obtained by PCR amplification. Using homologous recombination operation technology, the P29.2 gene sequence containing the linker was inserted into the signal peptides in the 5' and 3' regions of the HA gene fragment to construct a recombinant HA-P29.2 gene sequence. The schematic diagram of the construction of the fusion gene is shown in the figure. Figure 1 As shown. Using reverse genetics, the HA-P29.2 gene sequence and the other 7 gene fragments of H9N2AIV (PB2, PB1, PA, NP, NA, M, NS) were constructed into the PHW2000 vector, and the constructed 8 plasmids were co-transfected into HEK 293T cells. After 48 hours of transfection, TPCK trypsin was added to the cells, the supernatant and cell precipitate were collected, and inoculated into 9-11 day old SPF chicken embryos to rescue the recombinant avian influenza virus rH9N2-P29.2.

[0032] Table 1 Primer sequences

[0033]

[0034] Example 2 Verification of rH9N2-P29.2 recombinant virus

[0035] 1.PCR identification

[0036] The genomic RNA of the rH9N2-P29.2 recombinant virus was extracted and reverse transcribed into cDNA. The HA-P29.2 gene in the rH9N2-P29.2 recombinant virus was detected by PCR. The results are as follows Figure 2 As shown, the rH9N2-P29.2 recombinant virus can amplify the specific band of HA-P29.2. The rH9N2 AIV expressing HA and the rH9N2-P29.1 recombinant virus expressing HA fused to a single P29 fusion protein (HA-P29.1) were used as controls.

[0037] 2. Western Blot Identification

[0038] LMH cells infected with rH9N2-P29.2 recombinant virus were washed with PBS. Cells were lysed with 1X SDS-PAGE. The cell lysate was boiled at 100°C in a metal bath for 10 min. A 10% separation gel was prepared, and the lysed cells were loaded and run for gel electrophoresis. The proteins in the electrophoresis gel block were transferred to a PVDF membrane, and the PVDF membrane was blocked with PBS-MT (containing 5% skimmed milk powder and 0.05% Tween-20) on a shaker at 4°C overnight. H9 HA polyclonal antibody diluted with PBS-MT was added and incubated at 37°C on a shaker for 1 h. The cells were washed 5 times with PBS-MT, and HRP-sheep anti-mouse IgG (Sigma, 1:2000) diluted with PBS-MT was added and incubated at 37°C on a shaker for 1 h. After washing 5 times with PBS-MT, the developer was added, and the cells were observed and photographed using a gel imaging system (Tanon4200SF). The results are shown in the figure. Figure 3 As shown in the figure, the experimental results show that the recombinant virus rH9N2-P29.2 can stably express the HA-P29.2 recombinant protein, and the expression level of the recombinant protein does not change significantly. Figure 3 In the assay, cells infected with rH9N2 and rH9N2-P29.1 were used as controls.

[0039] 3. Indirect Immunofluorescence Identification

[0040] LMH cells infected with rH9N2-P29.2 recombinant virus were washed with PBS. After the cells were fixed with 4% paraformaldehyde, they were permeabilized with 5% Triton X-100 and then washed with PBS. The cells were incubated with PBS-MT (containing 5% skim milk powder and 0.05% Tween-20) at 37°C for 90 minutes. Washed three times with 200μL PBST containing 0.25% Tween-20, each time for 3 minutes. Add H9 HA polyclonal antibody diluted with PBS-MT, incubate at 37°C for 1h, and wash as above. Add 50μL FITC-goat anti-mouse IgG (KPL, 1:200) diluted with PBS-MT, incubate at 37°C for 1h, and wash as above. Finally, the results were observed with a Zeiss laser confocal microscope (Zeiss LSM880) and photographed and recorded. The results are shown in Figure 4 As shown in the experimental results, it was found that the recombinant virus rH9N2-P29.2 can express HA-P29.2 recombinant protein, and the recombinant protein is abundantly expressed in LMH cells. The amino acid sequence of HA-P29.2 recombinant protein is shown in SEQ ID NO.1. Figure 4 In the assay, uninfected cells (NC), rH9N2-infected cells, and rH9N2-P29.1-infected cells were used as controls.

[0041] Example 3: Experiment on overcoming maternal antibody interference by recombinant avian influenza inactivated vaccine (rH9N2-P29.2)

[0042] β-Propiolactone was added to the recombinant avian influenza virus rH9N2-P29.2 (1:2000) and incubated in a shaker at 4°C for 16 hours to inactivate the virus. Then, the β-propiolactone was inactivated in a 37°C water bath for 2 hours. The inactivated recombinant virus was inoculated into 9-11 day old SPF chicken embryos to detect whether it was completely inactivated. The inactivated recombinant virus was mixed with 71VG adjuvant (SEPPIC) in a ratio of 3:7 to prepare an inactivated avian influenza vaccine. 0.3 mL of H9N2 AIV-specific antibody IgY was injected intravenously into 1-day old SPF chickens to simulate the presence of maternal antibodies (MDAs), and then 0.1 mL of the above-prepared inactivated vaccine was immediately immunized. Serum was collected every week to observe and record the immune response. After 4 weeks of immunization, 0.1 mL of H9N2 AIV was inoculated by nasal infection. Nose and cloaca cotton wool samples were collected 3 and 5 days after infection to titrate the virus. The results are as follows Figure 5 As shown, the left figure shows that 21 days after immunization with the inactivated rH9N2-P29.2 vaccine, the antibody titer is significantly higher than that of the inactivated recombinant avian influenza virus vaccine without (rH9N2) and with a P29 (rH9N2-P29.1). The right figure shows that immunization with the inactivated rH9N2-P29.2 vaccine can significantly reduce the titer of virus discharged from the nasal cavity 3 days after the challenge. The inactivated rH9N2 and rH9N2-P29.1 vaccines were used as controls.

[0043] The above-mentioned embodiments only express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A recombinant avian influenza virus, characterized in that: The recombinant avian influenza virus comprises the HA gene of the H9 subtype avian influenza virus, and two repeated P29 genes are inserted after the signal peptide sequence of the HA gene. The amino acid sequence of the protein encoded by the P29 gene is shown in SEQ ID NO.

1.

2. The recombinant avian influenza virus according to claim 1, characterized in that The nucleotide sequence of the P29 gene is shown in SEQ ID NO.

2.

3. The recombinant avian influenza virus according to claim 1, characterized in that A linker sequence is arranged between the two P29 genes, at the front end of the first P29 gene and at the end of the second P29 gene.

4. The recombinant avian influenza virus according to claim 3, characterized in that The amino acid sequence of the linker sequence is shown in SEQ ID NO.

3.

5. The recombinant avian influenza virus according to claim 4, characterized in that The amino acid sequence of the fusion protein expressed by the H9 subtype avian influenza virus HA gene and the inserted two repeated P29 genes is shown in SEQ ID NO.

4.

6. The recombinant avian influenza virus according to claim 5, characterized in that The nucleotide sequences of the H9 subtype avian influenza virus HA gene and the inserted two repeated P29 genes are shown in SEQ ID NO.

5.

7. A recombinant avian influenza virus inactivated vaccine, characterized in that: The invention relates to an inactivated virus comprising the recombinant avian influenza virus according to any one of claims 1 to 6 and an adjuvant.

8. The recombinant avian influenza virus inactivated vaccine according to claim 7, characterized in that: The method for preparing the recombinant avian influenza virus inactivated vaccine comprises the following steps: Insert two repeated P29 genes after the signal peptide sequence of the HA gene of the H9 subtype avian influenza virus to construct a recombinant avian influenza virus, wherein the amino acid sequence of the protein encoded by the P29 gene is shown in SEQ ID NO.1; adding beta-propiolactone to the recombinant avian influenza virus to inactivate the recombinant virus; The inactivated recombinant avian influenza virus is mixed with an adjuvant to obtain a recombinant avian influenza virus inactivated vaccine.

9. Use of the recombinant avian influenza virus according to any one of claims 1 to 6 in the preparation of a vaccine for preventing and / or treating avian influenza.

10. Use of the recombinant inactivated avian influenza virus vaccine according to claim 7 or 8 in the preparation of biological products for preventing and / or treating avian influenza.

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