INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The Sequence listing, named “SEQUENCE LISTING”, created by Nov. 2, 2021 with 14,855 size in bytes is incorporated by reference in this application.

TECHNICAL FIELD

The disclosure relates to the technical field of veterinary biological products, particularly to an avian influenza and fowl adenovirus type 4 bi-combined genetic engineering subunit vaccine and a method for preparing the same.

BACKGROUND OF THE DISCLOSURE

Avian influenza virus (AIV) is an important pathogen of a respiratory system for birds and some mammals, and belongs to orthomyxoviridae influenza virus in taxonomy. According to different protein antigenicities on the surface, the avian influenza virus is divided into 17 subtype hemagglutinins (HA) and 10 subtype neuraminidases (NA) in which H9N2 subtype avian influenza virus is widely epidemic all over the world, which can cause significant reduction in the egg yield of hens and decrease in feed reward, thereby leading to huge economic loss of poultry industry and bring about danger to human health. The pathogenesis of influenza virus on a host depends on multiple genes, wherein HA genes and proteins encoded by HA genes are extremely important, which play a crucial role in virus adsorption and transmembrane processes, are important surface antigens, stimulate an organism to generate a neutralizing antibody neutralizing the infection of viruses.

Fowl adenovirus (FAV) is distributed all over the world and has many serum types. The hydrocardia syndrome of broiler chickens caused by this virus is caused by fowl adenovirus-C′ serum type 4 adenovirus infection. There are reports about this disease across the country. The morbidity of 3˜5-week-old broiler chickens is particularly harmful, the mortality suddenly rises to over 80% when being serious, thereby seriously harming the healthy development of aviculture industry in China.

At present, prevention and control of vaccines is the most effective way for the above two diseases that are extremely harmful to poultry farming aviculture industry. However, in the prior art, a production process of preparing avian influenza vaccines using chick embryo breeding viruses relates to live virus operation which threatens the biological safety of the environment. In the meantime, when the avian influenza is epidemic, production of vaccines has the defects of long production period, low yield and the like due to limitation of chick embryo supply. The fowl adenovirus is not easy to culture, the virus having high titer is difficultly obtained, especially, different isolated strains have great difference, which results in a fact that the prepared vaccine often difficultly provides an ideal immune effect.

Based on the above problems, a selective means to solve the above problem is genetic engineering fermentation. The applicant, Qingdao Yibang Bioengineering Co., Ltd, applied patent application CN201710674039.5 in 2017, which discloses a newcastle disease, avian influenza, bursa fabricius and fowl adenovirus quadruple vaccine containing antigens and a vaccine adjuvant, wherein the antigens are a newcastle disease Lasota virus strain, H9 subtype avian influenza strain with conservation number CCTCCNO:V201517, a bursa fabricius VP2 Protein and an fowl adenovirus Hexon protein having an amino acid sequence of SEQ ID NO: 2 and a chicken α-interferon protein.

Therefore, production of antigens based on genetic engineering fermentation is a biological technology which is widely being researched by those skilled in the art at present.

In the avian influenza and adenovirus bi-combined vaccine, the commonly used methods are to use avian influenza fluid and adenovirus fluid as a mixed antigen to produce the bi-combined vaccine, for example the applicant, Qingdao Yibang Bioengineering Co., Ltd, applied patent application CN201610099573.3 in 2016, which discloses a avian influenza virus and fowl adenovirus bi-combined inactivated vaccine. The H9 subtype avian influenza virus QDY strain used in the vaccine of the disclosure and TCID50/EID50 of I group 4 type fowl adenovirus YBAV-4 new strain are high in valence and good in immunogenicity, and can withstand attacks from isolated viruses of H9 subtype and fowl adenovirus diseases in different places. The vaccines prepared from the above viruses have good safety, and there are no any local and systemic adverse reactions caused by the vaccines. In storage life tests, through character and safety tests and analysis on potency test data, the results are compared with those of the single vaccine of the similar product, the bi-combined vaccine has no significant difference, they are both stable and effective; the results of the potency test prove that the bi-combined vaccine and two single vaccine antibodies are all maintained in high level, can quickly generate antibodies compared with the similar products, and the antibody in control group is negative.

However, the problems existing in the above solution are that there are many process steps of producing avian influenza viruses and adenoviruses alone; if the subunit vaccine is prepared based on the genetic engineering principle, how to screen effective antigenic determinants, especially screen two effective antigenic determinants at the same time, is still a problem, although the process steps can be simplified.

Further problem lies in that even though two or more effective antigenic determinants are screened, how to improve their exhibition effect in the subunit vaccine is a very difficult realistic problem.

SUMMARY OF DISCLOSURE

The objective of the disclosure is to provide an avian influenza and fowl adenovirus type 4 bi-combined genetic engineering subunit vaccine and a method for preparing the same. This vaccine contains a fusion protein which can induce poultries to generate high-level specific antibodies to protect the poultries from avian influenza and fowl adenovirus infection.

In order to achieve the above objective, the technical solution provided by the disclosure is to provide an avian influenza and fowl adenovirus type 4 bi-combined genetic engineering subunit vaccine, wherein an antigen in the vaccine is a fusion antigen; the fusion antigen has:

a) an avian influenza virus HA protein;

b) a fowl adenovirus fiber2 protein;

c) a specific linker peptide located between the avian influenza virus HA protein and the fowl adenovirus fiber2 protein; the amino acid sequence of the specific linker peptide is as shown in SEQ ID NO: 2.

The amino acid sequence of the specific linker peptide is as follows:

PEVLPPLPKESRISEGEAVVVG

In the above subunit vaccine, the amino acid sequence of the fusion antigen is as shown in SEQ ID NO: 3.

The amino acid sequence of the fusion antigen is as follows:

MEVVSLITILLVATVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDLSLEGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIWNVSYDGTSTACSGSFYRSMRWLTRKNGDYPVQDAQYTNNQGKNILFMWGINHPPTDTTQRNLYTRNDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLRPSQTLRIKSDGNLIAPWYGHILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEVIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICIPEVLPPLPKESRISEGEAVVVGMLRAPKRRHSENGKPETEAGPSPAPIKRAKRMVRASQLDLVYPFDYVADPVGGLNPPFLGGSGPLVDQGGQLTLNVTDPIIIKNRSVDLAHDPSLDVNAQGQLAVAVDPEGALDITPDGLDVKVDGVTVMVNDDWELAVKVDPSGGLDSTAGGLGVSVDDTLLVDQGELGVHLNQQGPITADSSGIDLEINPNMFTVNTSTGSGVLELNLKAQGGIQAASSGVGVSVDESLQIVNNTLEVKPDPSGPLTVSANGLGLKYDTNTLAVTAGALTVVGGGSVSTPIATFVSGSPSLNTYNATTVNSSANAFSCAYYLQQWNIQGLLVTSLYLKLDSATMGNRPGDLNSANAKWFTFWVSAYLQQCNPSGIQAGTVSPSTATLTDFEPMANRSVTSPWTYSANGYYEPSIGEFQVFSPVVTGAWNPGNIGIRVLPVPVSASGERYTLLCYSLQCTNASIFNPNNSGTMIVGPVLYSCPAASLP

In the above subunit vaccine, the content of the fusion antigen in the vaccine is not less than 75 μg/ml.

In the above subunit vaccine, the vaccine is formed by mixing and emulsifying an oil phase and a water phase; the water phase contains the fusion antigen.

Meanwhile, the discloses also discloses a method for preparing the avian influenza and fowl adenovirus type 4 bi-combined genetic engineering subunit vaccine as described above, wherein the vaccine is obtained by mixing and emulsifying the water phase containing the fusion antigen and the oil phase.

In the above method for preparing the subunit vaccine, the method for preparing the fusion antigen comprises:

Step 1: synthesizing a fusion expression gene sequence to construct a recombinant expression vector; synthesizing a fusion gene sequence shown in SEQ ID NO:1, and adding restriction enzyme sites ECOR I and NOT I at two ends of the fusion gene sequence, and meanwhile carrying out double digestion onPichia pastorisexpression vector pPic9k using the restriction enzymes ECOR I and NOT I to obtain a fragment with a size of 9263 bp, and linking the synthesized fusion gene sequence with thePichia pastorisexpression vector pPic9k fragment recovered after double digestion using 14 DNA ligase to obtain a ligation product;

the sequence of the fusion gene is as follows:

atggaagtagtatcactaataactatactactagtagcaacagtaagcaatgcagataaaatctgcatcggctatcaatcaacaaactccacagaaactgtggacacactaacagaaaacaatgtccctgtgacacatgccaaagaactgctccacacagagcataatgggatgctgtgtgcaacaagcttgggacaacctcttattttagacacctgcaccattgaagggctaatctatggcaatccttcttgtgatctatcgctggaaggaagagaatggtcctatatcgtcgagagaccatcagctgtcaacggattgtgttaccccgggaatgtagaaaatctagaagagctaaggtcactttttagttctgctaggtcttatcaaagaatccagattttcccagacacaatctggaatgtgtcttacgatgggacaagcacagcatgctcaggttcattctacagaagcatgagatggttgactcgaaagaacggcgattaccctgtccaagacgcccaatacacaaataatcaagggaagaacattcttttcatgtggggcataaatcacccacccaccgatactacgcagagaaatctgtacacgagaaacgacacaacaacgagtgtggcaacagaagaaataaataggatcttcaaaccattgataggaccaaggcctcttgtcaacggtttgatgggaagaattgattattattggtctgtattgagaccgagtcaaacactgcgaataaaatctgatgggaatctaatagctccatggtatggacacattctttcaggagagagccacggaagaattctgaagactgatttaaaaaggggtagctgcacagtgcaatgtcagacagagaaaggtggcttaaacacaacactgccattccaaaatgtaagtaagtatgcatttggaaactgctcaaaatacattggcataaagagtctcaaacttgcagttggtctgaggaatgtgccttctagatctagtagaggactattcggggccatagcagggtttatagagggaggttggtcagggctagttgctggttggtatgggttccagcattcaaatgaccaaggggttggtatggcagcagatagagactcaacccaaaaggcaattgataaaataacatccaaagtgaataatatagtcgacaaaatgaacaagcagtatgaagtcattgatcatgaattcagtgaggtagaaactagacttaacatgatcaataataagattgatgatcaaatccaggatatatgggcatataatgcagaattgctagttctgcttgaaaaccagaaaacactcgatgagcatgacgcaaatgtaaacaatctatataataaagtaaagagggcgttgggttccaacgcggtggaagatgggaaaggatgtttcgagctataccacaaatgtgatgaccaatgcatggagacaattcgaaacgggacctacaacagaaggaagtatcaagaggagtcaaaattagaaagacagaaaatagagggggtcaagctggaatctgaaggaacttacaaaatcctcaccatttattcgactgttgcctcatctcttgtgattgcaatggggtttgctgccttcttgttctgggccatgtccaatgggtcttgcagatgcaacatttgtatataacctgaagtcctgcctcctctgcctaaggaatctagaatttccgaaggtgaagctgtcgtcgtcggtatgctccgggcccctaaaagaagacattccgaaaacgggaagcccgagaccgaagcgggaccttccccggctccaatcaagcgcgccaaacgcatggtgagagcatcccagcttgacctggtttatcctttcgattacgtggccgaccccgtcggagggctcaacccgccttttttgggaggctcaggacccctagtggaccagggcggacagcttacgctcaacgtcaccgatcccatcatcatcaagaacagatcggtggacttggcccacgaccccagtctcgatgtcaacgcccaaggtcaactggcggtggccgttgaccccgaaggggccctggacatcacccccgatggactggacgtcaaggtcgacggagtgaccgtaatggtcaacgatgactgggaactggccgtaaaagtcgacccgtccggcggattggattccaccgcgggtggactgggggtcagcgtggacgacaccttgctcgtggatcagggagaactgggcgtacacctcaaccaacaaggacccatcactgccgatagcagtggtatcgacctcgagatcaatcctaacatgttcacggtcaacacctcgaccggaagcggagtgctggaactcaacctaaaagcgcagggaggcatccaagccgccagttcgggagtgggcgtttccgtggatgaaagcctacagattgtcaacaacactctggaagtgaaaccggatcccagcggaccgcttacggtctccgccaatggcctagggctgaagtacgacactaataccctagcggtgaccgcgggcgctttaaccgtggtcggaggggggagcgtctccacacccatcgctacttttgtctcgggaagtcccagcctcaacacctacaatgccacgaccgtcaattccagcgcgaacgccttctcttgcgcctactaccttcaacagtggaacatacaggggctccttgttacctccctctacttgaaattggacagcgccaccatggggaatcgccctggggacctcaactccgccaatgccaaatggttcaccttttgggtgtccgcctatctccagcaatgcaacccctccgggattcaagcgggaacggtcagcccctccaccgccaccctcacggactttgaacccatggccaataggagcgtgaccagcccatggacgtactcggccaatggatactatgaaccatccatcggggaattccaagtgttcagcccggtggtaacaggtgcctggaacccgggaaacatagggatccgcgtcctccccgtgccggtttcggcctccggagagcgatacacccttctatgctatagtctgcagtgcacgaacgcgagcatttttaatccaaacaacagcggaaccatgatcgtgggacccgtgctctacagctgtccagcggcctccctcccgtaa

The above underline part

(SEQ ID NO: 4)cctgaagtcctgcctcctctgcctaaggaatctagaatttccgaaggtgaagctgtcgtcgtcgg
is a gene position of a specific linker peptide fragment.

Step 2: transforming the ligation product in step 1 intoE. coliDH5α, picking colonies, carrying out positive cloning verification using PCR, and carrying out sequencing verification on a target fragment;

The above target fragment refers to a linkage product, sequencing verification of the target fragment is to demonstrate whether the linkage product succeeds in transforming intoE. coliDH5α;

Step 3: after sequencing in step 2 succeeds, extracting plasmids from the colonies to obtain recombinant plasmids, introducing the recombinant plasmids intoPichia pastorisGS11 using an electrotransformation method to obtain thallus;

The above recombinant plasmid refers to the linkage product.

Step 4: collecting the thallus in step 3 and picking transformants capable of normally growing on a resistance plate, extracting genomes from the transformants, and carrying out PCR amplification on the target fragment;

The above transformant is a normally grown bacterium obtained by screening the thallus transformed in Step 3 on the plate of the YPD solid culture medium containing G418 antibiotics.

The target fragment in Step 4 refers to the linkage product, the target fragment contained in a genome undergoes PCR amplification, the existence of the target fragment is verified, if yes, Step 5 is conducted.

Step 5: after the target fragment is determined, activating the transformants again, and carrying out fermentation induction culture to obtain a protein solution containing the fusion antigen; and

Step 6: purifying and precipitating the protein solution to obtain the fusion antigen.

In the above method for preparing the subunit vaccine, the Step 2 is specifically as follows: the ligation product in step 1 is transformed intoE. coliDH5 α, 100 μl ofE. coliDH5α cells molten on ice is put into a 5 ml EP tube with a round bottom, the ligation product in step 1 is added, the resulting mixture is put for 30 s on ice, subjected to heat shock for 45 s at 42° C., and then put on ice for 1-2 min, an soc culture medium is added until the final volume is 1 ml, the resulting mixture is subjected to shaking culture at 37° C. for 1 h, and a proper amount of cultured product is coated on an LB agar plate and cultured overnight at 37° C.; colonies are picked to be subjected to positive cloning verification using PCR, and sequencing verification is performed on the target fragment;

the step 3 is specifically as follows: after sequencing succeeds, plasmids are extracted from the colonies to obtain the recombinant plasmids, the recombinant plasmids are introduced intoPichia pastorisGS11 by using an electrotransformation method, wherein the electrotransformation conditions are as follows: the prepared recombinant plasmids are added into a 1.5 mL centrifuge tube containing 80 μL ofPichia pastorisGS115 competent cells to be subjected to ice bath for 15 min to obtain a plasmid competent mixed solution, the sucked plasmid competent mixed solution is added into a pre-cooled 0.2 cm electroconversion cup and the cup is subjected to electric shock transformation on an electromotor; electric shock is performed for 4.5 ms at 1500 V; after electric shock is completed, 500 μL of 1M pre-cooled sorbitol solution is added immediately and evenly mixed, the resulting mixed solution is transferred into a 1.5 mL centrifuge tube, and cultured for 2 h in a constant temperature shaker at 30° C. at 180 rpm; the resuscitated bacterial solution was centrifuged at a low speed for 5 min and 150-200 μL of suspended thallus is retained and coated on an YPD plate containing 1 M sorbitol and cultured for 48 h in a constant temperature incubator at 30° C.

In the above method for preparing the subunit vaccine, the step 4 is specifically as follows: the thallus on the YPD plate in step 3 are collected and diluted into corresponding folds, 150-200 μL of bacterial solution is sucked to the YPD culture medium containing 0.4 g/ml G418 antibiotics, and statically cultured in a 30° C. constant temperature incubator for 2-4 days, transformants capable of normally growing on the resistance plate are picked to extract the transformant genome, and PCR amplification is performed on the target fragment.

In the above method for preparing the subunit vaccine, the step 5 is specifically as follows: the verified transformants are picked and inoculated in a BMGY culture solution to be subjected to shaking culture for 48 h at 30° C., then the cultured product is centrifuged under a low temperature at 4000 rpm for 10 min to remove the supernatant, then the BMMY culture solution are added to suspend the thallus, then the thallus is induced for 72 h with addition of methanol every 24 h; the culture solution was centrifuged under a low temperature at 4000 rpm for 10 min, and the supernatant is collected to obtain a protein solution, and SDS-PAGE identification is performed on the protein solution;

the step 6 is specifically as follows: absolute ethanol whose volume is as twice as that of protein solution is added overnight for settlement, the solution after settlement is centrifuged under a low temperature at 8000 rpm/min for 5 min to remove the supernatant, and the avian influenza H9 subtype HA and adenovirus type 4 fiber2 fusion antigen is collected and precipitated.

In the above method for preparing the subunit vaccine, the emulsifier used in the oil phase is span-80 and the solvent is white oil;

the emulsifier used in the water phase is Tween 80 and the solvent is water.

The disclosure has the beneficial effects:

The vaccine of the disclosure adopts the fusion antigen, which has the advantages:

1. The fusion antigen can induce poultries to produce high-level specific antibodies and protect poultry from avian influenza and fowl adenovirus infection;

2. The specific linker peptide is added between avian influenza virus HA and fowl adenovirus fiber2 to assist in the fusion expression of avian influenza virus HA and fowl adenovirus fiber2 and optimize the folding of fusion protein molecules;

3. The avian influenza virus HA protein and fowl adenovirus fiber2 protein are fused in series and expressed for the first time, so as to reduce the repetitive works in the actual production and increase labor efficiency.

The method for preparing the fusion antigen in the vaccine of the disclosure is genetic engineering fermentation. The method has the advantages of low cost, high antigen purity, good immunogenicity and high safety.

