CN114085293A

CN114085293A - Recombinant protein for preventing avian Ankara disease and construction method and application thereof

Info

Publication number: CN114085293A
Application number: CN202111344248.6A
Authority: CN
Inventors: 任洪林; 郭珣; 柳溪林; 胡盼; 卢士英; 柳增善; 李岩松; 张英; 祝万菊; 闫守庆
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2021-11-14
Filing date: 2021-11-14
Publication date: 2022-02-25
Anticipated expiration: 2041-11-14
Also published as: CN114085293B

Abstract

The invention relates to a recombinant protein for preventing avian Ankara disease and a construction method and application thereof, belonging to the technical field of gene engineering biomedicine. Respectively constructing and chemically synthesizing coding DNAs of multiple epitope tandem proteins of four structural proteins of the type 4 avian adenovirus, connecting the coding DNAs of the four multiple epitope tandem proteins to obtain a gene for coding a multiple fusion recombinant protein, constructing a recombinant expression plasmid for an escherichia coli prokaryotic expression system, transferring the recombinant expression plasmid into escherichia coli competent cells to obtain escherichia coli genetic engineering expression bacteria, performing induced expression and a large amount of purified multiple fusion recombinant protein, and mixing the multiple fusion recombinant protein with an immunologic adjuvant to prepare the avian ancavirus multiple epitope subunit vaccine. The subunit vaccine has strong immunogenicity, can stimulate an organism to generate protective antibodies, has long antibody maintenance time, can effectively prevent the infection and the morbidity of the avian adenovirus type 4, and has a prevention and protection effect on the avian Ankara disease.

Description

Recombinant protein for preventing avian Ankara disease and construction method and application thereof

Technical Field

The invention belongs to the technical field of gene engineering biomedicine, and particularly relates to a concatameric fusion recombinant protein for preventing avian Ankara disease, a construction method and application thereof.

Background

The avian Ankara disease is a poultry epidemic disease caused by avian Ankara virus, the disease has a remarkable effect on broilers of 3-6 weeks, breeding hens and laying hens can also have diseases in similar age groups, and the mortality rate is up to 20% -80%. The avian Ankara virus is avian adenovirus type 4 virus (FAdV-4) of avian adenovirus type C of subgroup I of avian adenoviruses of the family fowl adenoviridae. The avian adenovirus is divided into three subgroups I, II and III according to the different antigens of the specific groups, wherein the subgroup I comprises A, B, C, D, E five serotypes (FAdV-1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10 and 11). The group of viruses generally exists in chicken, duck and pig bodies, is often in recessive infection, acts on poultry together as secondary pathogen, can be vertically spread and pollutes chick embryos. The disease is discovered in Ankara area of Pakistan in 3 months of 1987, and outbreaks in areas of Shandong, Liaoning, Jilin, Hebei and the like in China since 2015 cause huge economic losses to poultry industry in China. At present, a few vaccines for effectively preventing the disease exist, the curative effect of antibiotics and antiviral drugs on the disease is not large, no specific drugs exist, and the disease is often prevented and controlled disadvantageously.

The Ankara virus is DNA virus, and the virus particle has no envelope and is spherical and in icosahedral symmetrical structure. The particle diameter is generally 70-90nm, and is composed of 252 capsomeres, wherein 12 apical capsomeres are Penton (Penton), and 240 non-apical capsomeres are Hexon (Hexon) and spike protein (Fiber1, Fiber 2). Penton, hexon and spike proteins are capsid proteins of viruses and have immunogenicity. The penton protein and the spike protein play a role in the processes of virus adsorption and cell entry, and can effectively stimulate an organism to generate humoral immunity and induce the generation of a neutralizing antibody. The hexon protein contains specific antigenic determinants, is a neutralization target of the antibody and is closely related to pathogenicity.

Because the Ankara disease of the poultry is fast in onset, high in death rate and strong in diffusion capability, the Ankara disease can be vertically and horizontally propagated, and great harm is caused to the poultry industry. Therefore, in order to solve this problem, the development of a novel effective vaccine is urgently required. At present, the prevention of the disease mainly depends on inactivated vaccine and attenuated vaccine, but the two vaccines have a plurality of defects, and have the problems of unstable protection effect, easy secondary infection, strong virulence return and the like.

Disclosure of Invention

The invention provides a recombinant protein for preventing avian Ankara disease, and a construction method and application thereof, and aims to solve the problems of unstable protection effect, easy secondary infection, strong toxicity and the like of the existing inactivated vaccine and attenuated vaccine. The avian Ankara disease subunit vaccine is prepared and applied to preventing avian Ankara disease.

The technical scheme adopted by the invention is as follows:

the recombinant protein for preventing the avian Ankara disease is concatameric fusion recombinant protein FAdV4: F1-P-F2-H, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 1.

The nucleotide sequence of the coding gene of the recombinant protein is shown in SEQ ID NO. 2.

The invention relates to a method for constructing a coding gene of a recombinant protein for preventing avian Ankara disease, which comprises the following steps:

by using a flexible Linker and a restriction endonuclease site connection method, a DNA FAdV4: F1 encoding the multiple epitope tandem protein derived from the avian adenovirus type 4 Fiber1, a DNA FAdV4: P encoding the multiple epitope tandem protein derived from the avian adenovirus type 4 Penton, a DNA FAdV4: F2 encoding the multiple epitope tandem protein derived from the avian adenovirus type 4 Fiber2 and a DNA FAdV4: H encoding the multiple epitope tandem protein derived from the avian adenovirus type 4 Hexon are connected in series to obtain the encoding gene FAdV4: F1-P-F2-H.

The coding DNA of the multiple epitope tandem protein derived from the avian adenovirus type 4 Fiber1 comprises gene segments of multiple epitopes of avian adenovirus type 4 Fiber1, which are connected in series at intervals by flexible linkers, and the codon-optimized coding DNAFAdV4: F1 is chemically synthesized, wherein the coding DNA is shown as 1-348bp of SEQ ID NO.2, and the corresponding amino acid sequence is shown as 1-116aa of SEQ ID NO. 1;

the coding DNA of the multiple epitope tandem protein derived from the Penton of the avian adenovirus type 4 comprises gene fragments of multiple epitopes of the Penton of the avian adenovirus type 4, and is connected in series at intervals by a flexible linker, and the codon-optimized coding DNA FAdV4: P is chemically synthesized, wherein the coding DNA FAdV4: P is shown as 382-238 aa of SEQ ID NO.2, and the corresponding amino acid sequence is shown as 128-238aa of SEQ ID NO. 1;

the coding DNA of the multiple epitope tandem protein derived from the avian adenovirus type 4 Fiber2 comprises gene segments of multiple epitopes of avian adenovirus type 4 Fiber2, which are connected in series at intervals by flexible linkers, and the codon-optimized coding DNAFAdV4: F2 is chemically synthesized, wherein the coding DNA is shown as 742-359 aa of SEQ ID NO.2, and the corresponding amino acid sequence is shown as 248-359aa of SEQ ID NO. 1;

the coding DNA of the multiple epitope tandem protein derived from the avian adenovirus type 4 Hexon comprises gene segments of multiple epitopes of the avian adenovirus type 4 Hexon, and the gene segments are connected in series at intervals by a flexible linker, and the codon optimized coding DNAFAdV4: H is chemically synthesized, as shown by 1099-1404bp of SEQ ID NO.2, and the corresponding amino acid sequence is shown by 367-468aa of SEQ ID NO. 1.

The coding gene construction method of the invention, the restriction enzyme cutting site connection method is to introduce five restriction enzyme cutting sites of Nco I, NdeI, Hind III, XhoI and Bam HI to four multi-antigen epitopes, and the upstream primer and the downstream primer of the coding DNA of tandem protein, including:

the upstream primer of DNA FAdV4: F1 derived from the multiple epitope tandem protein of avian adenovirus type 4 Fiber1 contains NcoI restriction endonuclease site as shown in SEQ ID NO.3, and the downstream primer contains NdeI restriction endonuclease site as shown in SEQ ID NO. 4;

p of an upstream primer of the coding DNA FAdV4 of the multiple epitope tandem protein derived from the Penton of the avian adenovirus type 4 sequentially contains NdeI, Hind III and XhoI restriction endonuclease sites as shown in SEQ ID NO.5, and a downstream primer contains Bam HI restriction endonuclease sites as shown in SEQ ID NO. 6;

the coding DNA FAdV4: F2 of the multiple epitope tandem protein derived from avian adenovirus type 4 Fiber2 has an upstream primer containing NdeI, Bam HI and Hind III restriction enzyme sites in sequence as shown in SEQ ID NO.7, and a downstream primer containing an XhoI restriction enzyme site as shown in SEQ ID NO. 8;

the upstream primer of the DNA FAdV4: H encoding the multiple epitope tandem protein derived from the avian adenovirus type 4 Hexon sequentially contains NdeI, Bam HI and XhoI restriction endonuclease sites as shown in SEQ ID NO.9, and the downstream primer contains Hind III restriction endonuclease site as shown in SEQ ID NO. 10.

The recombinant expression system comprises a eukaryotic expression system or a prokaryotic expression system.

The invention expresses the recombinant expression vector used for preventing poultry Ankara disease and has the following characteristics: the basic vector of the recombinant expression vector comprises pET-28a, wherein the recombinant protein coding DNAFAdV4: F1-P-F2-H upstream primer is introduced into an NcoI restriction endonuclease site, as shown in SEQ ID NO.3, the downstream primer is introduced into an Eco RI restriction endonuclease site, as shown in SEQ ID NO.11, and the upstream primer and the downstream primer are inserted between the NcoI restriction endonuclease site and the Eco RI restriction endonuclease site of the pET-28a through enzyme digestion connection, so that a recombinant expression plasmid pET28a-FAdV4: F1-P-F2-H of the concatameric recombinant protein FAdV4: F1-P-F2-H is constructed.

The invention relates to an inducible expression method of recombinant protein for preventing avian Ankara disease, which comprises the following steps:

transferring a recombinant expression vector pET28a-FAdV4: F1-P-F2-H into an escherichia coli E.coli Rosetta (DE3) competent cell to obtain an escherichia coli genetic engineering expression strain Ec-RD-FAdV4: F1-P-F2-H capable of expressing concatameric fusion recombinant protein FAdV4: F1-P-F2-H, and performing IPTG induced expression and Ni column affinity chromatography purification to obtain the recombinant protein FAdV4: F1-P-F2-H.

The recombinant protein for preventing the avian Ankara disease is applied to preparation of avian Ankara disease subunit vaccines.

The subunit vaccine for avian Ankara disease comprises recombinant protein for preventing avian Ankara disease and immunologically accepted vaccine adjuvant which are mixed in a volume ratio of 1:1, wherein the vaccine adjuvant comprises Freund's adjuvant or aluminum hydroxide sol adjuvant.

The invention has the advantages that:

the invention selects a plurality of epitopes of four capsid proteins of the avian adenovirus type 4 to construct a concatenated fusion recombinant protein FAdV4: F1-P-F2-H, and the subunit vaccine for the avian ancavirus prepared by using the protein has more capsid protein epitopes, stimulates organisms to generate more types and more quantities of protective antibodies, and obviously improves the protection power to the avian ancavirus. The selected multiple antigen epitopes and the codons of the encoding DNA of the concatameric fusion recombinant protein FAdV4: F1-P-F2-H are optimized according to the codon preference of escherichia coli, so that the protein amount of the soluble expression concatameric fusion recombinant protein FAdV4: F1-P-F2-H of an escherichia coli expression system is remarkably increased, and sufficient concatameric fusion recombinant protein FAdV4: F1-P-F2-H for preparing the avian ancara subunit vaccine is ensured. In addition, the concatenated fusion recombinant protein FAdV4: F1-P-F2-H subunit vaccine prepared by the invention can generate complete immune protection only by immunization once, and the cost of immune epidemic prevention is obviously reduced. In a word, the subunit vaccine which is prepared by using the concatenated fusion recombinant protein FAdV4: F1-P-F2-H and can prevent the avian ancara disease has the characteristics of high antigen expression amount, long antibody maintenance time, high purity, good safety, strong immunogenicity, strong protectiveness and the like, and can effectively prevent the avian ancara disease.

Drawings

FIG. 1 is a diagram showing the PCR amplification results of coding genes of concatameric fusion recombinant proteins obtained by enzyme digestion and ligation of four multi-antigen epitope tandem protein-encoding DNAs FAdV4: F1, FAdV4: P, FAdV4: F2 and FAdV4: H in step (1) of example 2 of the present invention in six different arrangement and ligation modes; wherein:

lane M is DL 2000DNA Marker;

lanes 1-6 are respectively encoding genes FAdV4: F1-P-H-F2, FAdV4: F1-P-F2-H, FAdV4: F1-H-P-F2, FAdV4: F1-H-F2-P, FAdV4: F1-F2-P-H, FAdV4: F1-F2-H-P of concatameric fusion recombinant protein;

FIG. 2 is a diagram of the result of agarose gel electrophoresis of a double digestion product after the coding gene of the concatameric fusion recombinant protein in step (2) of example 2 of the present invention is identified by double digestion with restriction enzymes NcoI and Eco RI; wherein:

lane M is DL 2000DNA Marker; lane 1-6 are the encoded gene FAdV4, F1-P-H-F2, FAdV4, F1-P-F2-H, FAdV4, F1-H-P-F2, FAdV4, F1-H-F2-P, FAdV4, F1-F2-P-H, FAdV4, F1-F2-H-P after restriction enzyme digestion;

FIG. 3 is a diagram showing the results of SDS-PAGE electrophoretic detection of each recombinant protein in step (1) of example 3 of the present invention;

in the figure, A is a SDS-PAGE electrophoresis detection result picture of IPTG induced expression total protein of gene engineering expression bacteria Ec-RD-FAdV4: F1-P-F2-H, wherein: lane M is protein Marker; lane 1 shows the whole cell body of Ec-RD-FAdV4, F1-P-F2-H, which was not induced; lane 2 shows Ec-RD-FAdV4, F1-P-F2-H-induced whole cells; lane 3 is the supernatant of the disrupted Ec-RD-FAdV4, F1-P-F2-H cells after centrifugation; lane 4 is the inclusion body of the disrupted Ec-RD-FAdV4, F1-P-F2-H cells after centrifugation;

in the figure, B, C, D, E, F is a SDS-PAGE electrophoresis detection result picture of induced expression proteins of other five concatenated fusion recombinant proteins FAdV4, F1-P-H-F2, FAdV4, F1-H-F2-P, FAdV4, F1-F2-P-H, FAdV4, F1-F2-H-P, FAdV4, F1-H-P-F2;

FIG. 4 is a graph showing the results of electrophoretic detection of each purified recombinant protein in step (2) of example 3 of the present invention;

in the figure, A is a SDS-PAGE electrophoresis detection result picture of the purified protein induced and expressed by the gene engineering expression bacterium Ec-RD-FAdV4: F1-P-F2-H, wherein: lane M is protein Marker; lane 1 is E.coli Rosetta (DE3) control containing pET-28a empty vector only, Lane 2 is the supernatant of the E.coli Rosetta (DE3) after the Ec-RD-FAdV4: F1-P-F2-H before purification is induced to express the broken thallus and centrifuged, Lane 3 is the purified concatameric fusion recombinant protein FAdV4: F1-P-F2-H;

b, C, D, E in the figure is a SDS-PAGE electrophoresis detection result picture of purified concatameric fusion recombinant proteins FAdV4, F1-P-H-F2, FAdV4, F1-H-F2-P, FAdV4, F1-F2-P-H, FAdV4, F1-F2-H-P respectively;

FIG. 5 is a graph of variation levels of chicken serum antibodies at different periods after protein immunization, which is obtained by analyzing immunogenicity of a concatameric fusion recombinant protein in step (2) of step 1) of immunogenicity experiment in example 4 of the present invention;

FIG. 6 is a graph showing the growth and development of the antibodies after immunization in the 2) immune toxicity-counteracting protective experiment in step (2) of example 4 of the present invention;

FIG. 7 is a survival chart of the single challenge in the 2) challenge protective experiment in step (2) of example 4 of the present invention.

Detailed Description

Example 1 preparation of concatameric fusion recombinant protein FAdV 4F 1-P-F2-H

(1) The gene nucleotide sequences of four capsid proteins of Penton, Hexon, Fiber1 and Fiber2 are downloaded according to an avian adenovirus type 4 gene sequence (GenBank accession number KU587519.1) in NCBI GenBank, protein fragments with good hydrophilicity and concentrated antigen epitopes of the four capsid proteins are selected according to the characteristics of the antigen epitopes, and coding DNA of the protein fragments in the selected antigen epitope set is connected by using a coding DNA nucleotide sequence of a protein linker GGGGGGS to construct four multi-antigen epitope tandem protein coding DNAs FAdV4: F1, FAdV4: P, FAdV4: F2 and FAdV4: H. Four kinds of multi-antigen epitope tandem protein coding DNA are synthesized by a chemical method directly after codon optimization of escherichia coli according to codon usage preference of an escherichia coli expression system and synthesized by Suzhou Jinzhi biological science and technology limited.

(2) Primers containing NcoI, NdeI, Hind III, XhoI and BamHI cleavage sites are designed by using Premier 5, and the constructed DNA coding for the multi-epitope tandem protein FAdV4: F1, FAdV4: P, FAdV4: F2 and FAdV4: H are used as templates, and PCR amplification is carried out by using the primers in the table 1 according to the PCR system and conditions in the table 2, so as to obtain the coding DNA of the multi-epitope tandem protein with different restriction sites at the 5 'and 3' ends. The specific method comprises the following steps: carrying out PCR amplification by using a DNA FAdV4: F1 coding the multi-epitope tandem protein as a template and an upstream primer Fiber1-F (shown as SEQ ID NO. 3) containing an NcoI restriction endonuclease site and a downstream primer Fiber1-R (shown as SEQ ID NO. 4) containing an NdeI restriction endonuclease site to amplify a DNA fragment Nc-FAdV4: F1-Nd with the length of 369 bp; carrying out PCR amplification by using a DNA FAdV4: P coded by a multi-epitope tandem protein as a template, an upstream primer Penton-F (shown as SEQ ID NO. 5) containing NdeI, Hind III and XhoI restriction endonuclease sites and a downstream primer Penton-R (shown as SEQ ID NO. 6) containing Bam HI restriction endonuclease sites to amplify a DNA fragment Nd-Hi-Xh-FAdV4: P-Ba with the length of 372 bp; carrying out PCR amplification by using DNA FAdV4: F2 encoding the multi-epitope tandem protein as a template, an upstream primer Fiber2-F (shown as SEQ ID NO. 7) containing NdeI, Bam HI and Hind III restriction enzyme sites and a downstream primer Fiber2-R (shown as SEQ ID NO. 8) containing XhoI restriction enzyme sites to amplify a DNA fragment Nd-Ba-Hi-FAdV4: F2-Xh with the length of 375 bp; DNA FAdV4: H encoded by the multi-epitope tandem protein is taken as a template, upstream primer Hexon-F (shown as SEQ ID NO. 9) containing NdeI, Bam HI and XhoI restriction enzyme sites and downstream primer Hexon-R (shown as SEQ ID NO. 10) containing Hind III restriction enzyme sites are subjected to PCR amplification, and DNA fragment Nd-Ba-Xh-FAdV4: H-Hi with the length of 345bp is amplified. After the PCR reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and 4DNA fragments encoding epitope-rich tandem proteins having different restriction sites at the upstream and downstream ends were subjected to gel recovery, such as Nc-FAdV4, F1-Nd, Nd-Hi-Xh-FAdV4, P-Ba, Nd-Ba-Hi-FAdV4, F2-Xh, and Nd-Ba-Xh-FAdV4, H-Xh, and were linked to pMD-18T vector in a 16 ℃ water bath overnight using a T-vector linking kit, respectively, in which the linking system is shown in Table 3.

TABLE 1 all primer information

TABLE 2 PCR amplification System and conditions in step (2) of example 1

Note: the PCR reaction program is 94 ℃ for 5 min; 32 cycles (94 ℃ for 40 s; 70 ℃ for 30 s; 72 ℃ C.)

90 s); 10min at 72 ℃; storing at 4 ℃.

TABLE 3 ligation System in step (2) of example 1

Reagent	Volume of
		Recovering the target fragment glue	4μl
pMD-18T	1μl
		solution I	5μl
Total volume	10μl

Example 2 construction of prokaryotic expression genetic engineering bacteria of concatameric fusion recombinant protein FAdV4: F1-P-F2-H

(1) The recombinant cloning plasmid containing the target DNA after ligation in step (2) of example 1 was transferred into E.coli competent cells E.coli DH5. alpha. and positive transformants were selected and sent to Changchun Kumai Biotech Ltd for sequencing and the recombinant cloning plasmid with the correct DNA fragment inserted was extracted.

And (3) respectively extracting four recombinant cloned plasmids inserted into the target DNA fragments by using a plasmid extraction kit, and constructing different encoding genes of the multi-linked fusion recombinant protein by using a restriction enzyme cutting site connection method according to different permutation and combination modes. The specific method comprises the following steps: performing double enzyme digestion by NcoI, NdeI, HindIII, XhoI, BamHI and EcoRI respectively, wherein the double enzyme digestion system is shown in Table 4, performing enzyme digestion at the constant temperature of 37 ℃ for 3-4h, then performing gel recovery on a target DNA fragment by using a DNA gel recovery kit to obtain target DNA fragments with sticky ends of different enzyme digestion sites, and performing ligation by using T4DNA ligase to obtain the coding gene of the multi-linked fusion recombinant protein. The coding DNA of the multi-antigen epitope tandem protein, DNAFADV4: F1, is fixed at the first position, the coding DNA of the other three multi-antigen epitope tandem proteins is combined in an arrangement mode to form six different connection modes of the concatameric fusion recombinant protein FAdV4: F1-P-H-F2, FAdV4: F1-P-F2-H, FAdV4: F1-H-P-F2, FAdV4: F1-H-F2-P, FAdV4: F1-F2-P-H, FAdV4: F1-F2-H-P coding genes, and the connection system is placed in a water bath kettle at the constant temperature of 16 ℃ to be connected overnight as shown in Table 5. The PCR amplification results of the coding genes of the concatameric fusion recombinant proteins obtained by enzyme digestion and ligation in six different ligation modes are shown in FIG. 1, and then the ligation products are transformed, sequenced and plasmids are extracted for later use. A preferable multiple fusion recombinant protein FAdV4: F1-P-F2-H is screened out according to the experimental result of the immune toxicity attack protection rate of the prepared subunit vaccine, the invention specifically describes the FAdV4: F1-P-F2-H, and the experimental operation methods of the multiple fusion recombinant protein in other different connection modes are the same as those of FAdV4: F1-P-F2-H.

(2) And (2) carrying out PCR amplification by using the coding gene of the concatameric fusion recombinant protein FAdV4: F1-P-F2-H in the step (1) as a template and an upstream primer Fiber1-F (shown as SEQ ID NO. 3) containing an NcoI restriction endonuclease site and a downstream primer FPBH-R (shown as SEQ ID NO. 11) containing an EcoRI restriction endonuclease site, wherein the PCR reaction system and the reaction procedure are the same as those in the example 1. Introducing enzyme cutting sites NcoI and EcoRI into 5 'ends and 3' ends of coding genes of concatameric fusion recombinant protein FAdV4: F1-P-F2-H, performing double enzyme cutting by using restriction enzymes NcoI and EcoRI, wherein the double enzyme cutting system is shown in table 6, the enzyme cutting system is placed in a constant-temperature water bath kettle at 37 ℃ for acting for 3-4H, the agarose gel electrophoresis result of the double enzyme cutting product is shown in figure 2, and then performing gel recovery on the double enzyme cutting product. The DNA gel recovery kit is used for recovering the coding gene DNA after double enzyme digestion, then the recovered coding gene DNA is connected with pET-28a plasmid linearized by double enzyme digestion of NcoI/EcoRI by using T4DNA ligase, the connection system is shown in table 7, the connection system is placed in a constant temperature water bath kettle at 16 ℃ for connection overnight, then the connection product is transformed, sequenced, extracted plasmid is used for standby, and the prepared recombinant plasmid is named as: pET28a-FAdV 4F 1-P-F2-H. Then the recombinant plasmid is transformed into competent cells of escherichia coli Rosetta (DE3) by a heat shock method, after the PCR identification of conventional bacterial liquid, the positive recombinant plasmid is delivered to Changchun Ku American biotechnology limited company for sequencing, and the nucleotide sequence of the coding gene of the concatameric fusion recombinant protein FAdV4: F1-P-F2-H is shown as SEQ ID NO.2, and the corresponding amino acid sequence is shown as SEQ ID NO. 1. The genetic engineering expression bacteria Ec-RD-FAdV4: F1-P-F2-H capable of expressing the concatameric fusion recombinant protein FAdV4: F1-P-F2-H in a large amount are obtained by searching different temperatures and different inducer concentrations for screening. The PCR amplification upstream primers of the coding genes of the other 5 concatameric fusion recombinant proteins are all primers Fiber1-F containing NcoI restriction endonuclease sites, and the downstream primers are different and all contain EcoRI restriction endonuclease sites. Wherein, the PCR amplification downstream primer of the encoding gene of the concatameric fusion recombinant protein FAdV4: F1-F2-P-H and the downstream primer of FAdV4: F1-P-F2-H are the same as primer FPBH-R; the downstream primer of the PCR amplification of the coding genes of the concatameric fusion recombinant proteins FAdV4: F1-P-H-F2 and FAdV4: F1-H-P-F2 is a primer FPHB-R, and is shown as SEQ ID NO. 12; the downstream primer FHBP-R of the coding gene PCR amplification of the concatameric fusion recombinant proteins FAdV4: F1-H-F2-P and FAdV4: F1-F2-H-P is shown in SEQ ID No. 13.

TABLE 4 enzyme digestion System in step (1) of example 2

Note: a: NcoI-FAdV 4F 1-NdeI; b: NdeI-FAdV 4P-BamHI; c: BamHI-FAdV4: F2-XhoI; d: XhoI-FAdV 4H-Hind III; e: BamHI-FAdV 4H-Hind III; f: hind III-FAdV 4: F2-XhoI; g: NdeI-FAdV 4H-Hind III; h: hind III-FAdV 4: P-BamHI; i: BamHI-FAdV4: F2-XhoI; j: hind III-FAdV 4: F2-XhoI; k: XhoI-FAdV 4P-BamHI; l: NdeI-FAdV 4F 2-XhoI; m: XhoI-FAdV 4P-BamHI; n: BamHI-FAdV 4H-Hind III; o: XhoI-FAdV 4H-Hind III; p: hind III-FAdV 4: P-BamHI;

TABLE 5 ligation System in step (1) of example 2

Connecting system component	Volume (μ l)
		10* T4 DNA buffer	2
T4 DNA ligase	2
		NcoI-FAdV4:F1-NdeI	4
NdeI-FAdV4:P-BamHI	4
		BamHI-FAdV4:F2-XhoI	4
XhoI-FAdV4:H-HindШ	4
		Total volume	20

TABLE 6 enzyme digestion System in step (2) of example 2

TABLE 7 ligation System in step (2) of example 2

Example 3 expression and purification of concatameric fusion recombinant protein FAdV 4F 1-P-F2-H

(1) The genetically engineered expression bacterium Ec-RD-FAdV4: F1-P-F2-H was inoculated into 5ml of liquid LB medium containing 100. mu.g/ml kanamycin, cultured at 37 ℃ for 2 hours, and then IPTG was added to a final concentration of 0.1mmol/L, and induced for 8 hours. The expression condition of the target protein is detected through SDS-PAGE electrophoresis, wherein the concatameric fusion recombinant protein FAdV4: F1-H-P-F2 cannot be expressed after induction, and the rest 5 concatameric fusion recombinant proteins are successfully expressed, so that the subsequent immunogenicity and toxicity counteracting protective experiments are operated based on the successfully expressed 5 concatameric fusion recombinant proteins. And (3) carrying out ultrasonic cell disruption on the induced expression strain, centrifuging the ultrasonic bacteria liquid at 8000rpm and 4 ℃ for 10min, collecting supernatant and precipitate, and detecting the expression amount and the dissolution state of the target protein in the gene engineering expression strain by SDS-PAGE electrophoresis.

The SDS-PAGE electrophoresis detection result of the gene engineering expression bacteria Ec-RD-FAdV4: F1-P-F2-H is shown in figure 3A, the SDS-PAGE electrophoresis detection result of other concatameric fusion recombinant proteins is shown in figure 3B-F, and the detection result is shown in figure 3F, so that the concatameric fusion recombinant proteins FAdV4: F1-H-P-F2 can not be successfully induced to express.

(2) Purification of concatameric fusion recombinant protein FAdV 4F 1-P-F2-H

After the genetic engineering expression bacteria Ec-RD-FAdV 4F 1-P-F2-H is induced and expressed by IPTG, the bacteria are crushed by ultrasonic, the bacteria liquid after ultrasonic is centrifuged for 50min at 8000rpm and 4 ℃, the supernatant is collected, and the collected supernatant is purified by using an affinity chromatography medium Ni-NTA.

SDS-PAGE detection results show that the recombinant protein is expressed in both the supernatant and the inclusion body, and the expression amount of the supernatant is far greater than that of the inclusion body, so that the recombinant protein is purified by adopting a supernatant purification mode. 5ml of Ec-RD-FAdV4: F1-P-F2-H bacterial liquid cultured overnight at 37 ℃ is inoculated into 1L of sterilized LB, 100 mu g/ml kanamycin is added, shaking table culture is carried out at 37 ℃ and 160rpm for 2H, IPTG is added to enable the final concentration to be 0.1mmol/L, shaking table culture is carried out at 37 ℃ and induction is carried out for 8H. Centrifuging the induced bacterial liquid at 8000rpm at 4 ℃ for 10min, collecting thallus precipitates, re-suspending with binding Buffer solution Buffer A (Tris 6.058g, glycerol 100ml, NaCl29.25g and imidazole 0.68g, fixing the volume to 1L, adjusting the pH to 7.2-7.4), ultrasonically crushing thallus, centrifuging the ultrasonically-treated bacterial liquid at 8000rpm at 4 ℃ for 50min, collecting supernatant, and purifying the collected supernatant by using an affinity chromatography medium Ni-NTA to obtain target protein.

Suspending the collected thallus by Buffer A for ultrasonic crushing, and combining the centrifuged supernatant with a nickel column; washing the column with elution Buffer B (Tris 6.058g, glycerol 100ml, NaCl29.25g, imidazole 34.04g to 1L, adjusting pH to 7.2-7.4) to remove hetero-protein; washing off the hybrid protein with an elution buffer containing 60mM imidazole (prepared by mixing 89.80ml of buffer A and 0.20ml of buffer B) and an elution buffer containing 80mM imidazole (prepared by mixing 85.71ml of buffer A and 14.29ml of buffer B), respectively; the target protein was eluted with an elution buffer containing 220mM imidazole (prepared by mixing 57.14ml buffer A and 42.86ml buffer B together), and the protein eluate was collected. Filling the collected protein eluent into a dialysis bag with molecular weight cutoff of 3500Dal to remove 220mM imidazole in the protein eluent, immersing the dialysis bag with the protein eluent into PBS for dialysis for 3-4 times, replacing new PBS for dialysis every 3 hours, and after dialysis, concentrating with PEG20000 for 2 hours to obtain concentrated liquid, namely the purified target protein FAdV4: F1-P-F2-H. The purified target protein concentration was measured using BCA protein concentration measurement kit from pecan corporation. Through determination, the concentration of the purified concatameric fusion recombinant protein FAdV4, F1-P-F2-H protein is 1-2 mg/ml.

The SDS-PAGE electrophoresis detection result of the purified concatameric fusion recombinant protein FAdV4: F1-P-F2-H is shown in figure 4A, and the detection results of other concatameric fusion recombinant proteins are shown in figures 4B-E.

Example 4 preparation of avian Ankara disease genetic engineering subunit vaccine and immunoprotection analysis experiment

(1) Preparation of subunit vaccines

The 5 concatameric fusion recombinant proteins purified in the example 3 are diluted to 100 mu g/ml and mixed with Freund's adjuvant according to the volume ratio of 1:1 to prepare the subunit vaccine.

(2) Subunit vaccine immunogenicity assay

1) Immunogenicity assays

The 14-day-old specific pathogen-free SPF (specific pathogen free) Hailan white laying hens are divided into 6 groups, each group comprises 6 chickens, the first 5 groups are respectively immunized by 5 kinds of subunit vaccines prepared by multi-union fusion recombinant proteins and are used as an immunization group, the 6 th group is a PBS control group, PBS with the same volume is injected, wherein the first immunization is emulsified by equivalent volume of Freund's complete adjuvant, the subsequent immunization is emulsified by equivalent volume of Freund's incomplete adjuvant, the immunization period is 7 days, the immunization program is four times of immunization, and the immunization mode is leg intramuscular injection inoculation. Immune chicken serum samples were collected by collecting blood from the wing vein every week starting one week after the first immunization of the chicken flock for detection of the level of change in antibody titer, and the antibody titer in the serum was detected by an indirect ELISA method and the P/N value was calculated. The results are shown in fig. 5, and it is known from the results that all subunit vaccines prepared from 5 concatameric fusion recombinant proteins can stimulate the organism to produce antibodies, and the antibody maintenance time is long, which indicates that the prepared 5 concatameric fusion recombinant proteins have good immunogenicity, and can stimulate the organism to produce antibodies with strong binding ability and long maintenance time.

2) Animal challenge immune protective experiment

Grouping and immunizing experimental chickens

Selecting 14-day-old SPF (specific pathogen free) Hailan white laying hens, wherein each group comprises 6 laying hens, the immune cycle is 14 days, the immune process comprises one-time immunity and two-time immunity, and the immune mode is leg intramuscular injection.

Experimental groups: the primary immunization group immunizes the 5 subunit vaccines (100 mu g of protein/chicken) at the age of 14 days, and the immunization is carried out for 14 days and then the virus is attacked; the two immunization groups are immunized again after the first immunization for 14 days, the immunization dose and the immunization mode are the same as those of the first immunization, and the second immunization is performed for 14 days before the virus attack;

negative control: non-immune and non-toxic group;

positive control (PBS group): challenge group injected with PBS.

② detection of antibody titer after immunization

The result shows that the P/N value of the serum antibody titer of each immune group experiment chicken has no significant difference, but the serum antibody in most experiment chicken is shown to be positive in 1 week after the immunization; there were significant differences from the PBS group, and the results are shown in figure 6.

Toxic attack protective experiment

14 days after the first immunization, the chickens of the immune group and the positive control group are inoculated with the FAdV-4 isolate SDBZ-1 through intramuscular injection, and the virus titer is 10⁴TCID₅₀According to 100TCID₅₀The dosage is used for counteracting toxic substances. One week after challenge, the chickens were observed for clinical symptoms every day, the death condition of infected chickens was recorded, and the dead chickens were subjected to autopsy and lesion observation. The toxicity challenge protective experiment results are shown in table 8, and the survival curves are shown in fig. 7. The virus attacking protection rate of the primary immune group indicates that the multi-concatemeric fusion recombinant protein has the capacity of differently inducing immune protection. Compared with subunit vaccines of other concatameric fusion recombinant proteins (FAdV4: F1-P-H-F2, FAdV4: F1-H-F2-P, FAdV4: F1-F2-P-H and FAdV4: F1-F2-H-P), the subunit vaccine FAdV4: F1-P-F2-H has the best immune protection, and can generate 100% immune protection rate only by one-time immunization. After secondary immunization, the challenge protective force of each immune group has no significant difference, and 5 subunit vaccines can provide nearly 100% of protective rate.

TABLE 8 protective test results of primary immune challenge

According to the result of the challenge protective experiment, the subunit vaccine FAdV4, F1-P-F2-H can provide complete protection in an immune group, all immunized chickens can be completely protected by one-time immunization, and the death rate after challenge is 0. Most of the chickens in the PBS positive control group are attacked and killed after the challenge, and no chickens in the PBS positive control group are attacked and killed. The preferable subunit vaccine of the concatenated fusion recombinant protein FAdV4: F1-P-F2-H can play a complete protection role only by one-time immunization, so that the immunization cost is greatly reduced, and the vaccine prepared from the concatenated fusion recombinant protein FAdV4: F1-P-F2-H is efficient and economical.

The aluminium hydroxide sol adjuvant used in the invention replaces Freund's adjuvant to prepare subunit vaccine, the method and the result of immunogenicity analysis and animal challenge immunoprotection experiment are the same as those of using Freund's adjuvant, the aluminium hydroxide sol adjuvant can stimulate organism to generate antibody with strong binding ability and long maintenance time, and the challenge protection rate reaches 100%. Freund's adjuvant and aluminium hydroxide sol can be used as the immunological adjuvant of the subunit vaccine of the invention, and can achieve ideal immune and challenge protection effects.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

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Claims

1. A recombinant protein for preventing avian ancavirus, characterized in that: the recombinant protein is a concatameric fusion recombinant protein FAdV4: F1-P-F2-H, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 1.

2. The recombinant protein for preventing avian ancara according to claim 1, characterized in that: the nucleotide sequence of the coding gene of the recombinant protein is shown in SEQ ID NO. 2.

3. The method for constructing the gene coding for the recombinant protein for preventing the avian ancavirus according to claim 1 or 2, characterized by comprising the following steps:

by using a flexible Linker and a restriction endonuclease site connection method, a coding DNA FAdV4: F1 of a multiple epitope tandem protein derived from avian adenovirus type 4 Fiber1, a coding DNA FAdV4: P of a multiple epitope tandem protein derived from avian adenovirus type 4 Penton, a coding DNA FAdV4: F2 of a multiple epitope tandem protein derived from avian adenovirus type 4 Fiber2 and a coding DNA FAdV4: H of a multiple epitope tandem protein derived from avian adenovirus type 4 Hexon are connected in series to obtain a coding gene FAdV4: F1-P-F2-H.

4. The method of constructing a coding gene according to claim 3, wherein:

5. The method of constructing a coding gene according to claim 3, wherein: the restriction enzyme cutting site connection method is characterized in that five restriction enzyme cutting sites including Nco I, NdeI, Hind III, XhoI and Bam HI are introduced to four multiple antigen epitopes, and an upstream primer and a downstream primer of the coding DNA of tandem protein comprise:

the upstream primer of the DNA FAdV4: H encoding the multiple epitope tandem protein derived from the avian adenovirus type 4 Hexon sequentially contains NdeI, Bam HI and XhoI restriction endonuclease sites as shown in SEQ ID NO.9, and the downstream primer contains HindIII restriction endonuclease site as shown in SEQ ID NO. 10.

6. The recombinant expression system of recombinant proteins for preventing avian ancara as claimed in claim 1 or 2, wherein: the recombinant expression system comprises a eukaryotic expression system or a prokaryotic expression system.

7. Recombinant expression vector expressing the recombinant protein according to claim 1 or 2 for the prevention of avian ancara, characterized in that: the basic vector of the recombinant expression vector comprises pET-28a, wherein the recombinant protein coding DNAFAdV4: F1-P-F2-H upstream primer is introduced into an NcoI restriction endonuclease site, as shown in SEQ ID NO.3, the downstream primer is introduced into an Eco RI restriction endonuclease site, as shown in SEQ ID NO.11, and the upstream primer and the downstream primer are inserted between the NcoI restriction endonuclease site and the Eco RI restriction endonuclease site of the pET-28a through enzyme digestion connection, so that a recombinant expression plasmid pET28a-FAdV4: F1-P-F2-H of the concatameric recombinant protein FAdV4: F1-P-F2-H is constructed.

8. The method for inducing expression of recombinant proteins for preventing avian ancavirus according to claim 1 or 2, characterized in that it comprises the following steps:

9. Use of the recombinant protein for preventing avian ancara as claimed in claim 1 or 2 in the preparation of subunit vaccine for avian ancara.

10. An avian ancavirus subunit vaccine comprising the recombinant protein for the prevention of avian ancavirus according to claim 1 or 2, characterized in that: the recombinant protein for preventing the avian Ankara disease is mixed with an immunologically accepted vaccine adjuvant in a volume ratio of 1:1, and the vaccine adjuvant comprises Freund's adjuvant or aluminum hydroxide sol adjuvant.