CN114085293B

CN114085293B - Recombinant protein for preventing poultry ankara disease, construction method and application

Info

Publication number: CN114085293B
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: 2023-05-12
Anticipated expiration: 2041-11-14
Also published as: CN114085293A

Abstract

The invention relates to a recombinant protein for preventing fowl ankara disease, a construction method and application thereof, belonging to the technical field of genetic engineering biological medicine. Respectively constructing and chemically synthesizing encoding DNA of multi-epitope tandem proteins of four structural proteins of the 4-type avian adenovirus, connecting the encoding DNA of the four multi-epitope tandem proteins to obtain genes for encoding multi-fusion recombinant proteins, constructing recombinant expression plasmids for a prokaryotic expression system of escherichia coli, transferring competent cells of the escherichia coli to obtain genetically engineered escherichia coli expression bacteria, inducing expression and purifying the multi-fusion recombinant proteins in a large quantity, and mixing the multi-fusion recombinant proteins with an immunoadjuvant to prepare the multi-epitope subunit vaccine of the avian ankara. The subunit vaccine has strong immunogenicity, can stimulate organisms to produce protective antibodies, has long antibody maintenance time, can effectively prevent the onset of avian adenovirus type 4 infection, and has a preventive and protective effect on avian ankara.

Description

Recombinant protein for preventing poultry ankara disease, construction method and application

Technical Field

The invention belongs to the technical field of genetic engineering biological medicines, and particularly relates to a multi-fusion recombinant protein for preventing fowl ankara, a construction method and application thereof.

Background

Fowl ankara disease is a fowl epidemic disease caused by fowl ankara virus, and the disease has remarkable effect on 3-6 week-old broiler chickens, breeding hens and laying hens can also be ill at similar ages, and the death rate is as high as 20% -80%. The avian ankara virus is avian adenovirus type 4 virus (FAdV-4) of subgroup I avian adenovirus genus avian adenovirus type C. Avian adenoviruses are divided into three subgroups I, II and III, wherein the subgroup I has A, B, C, D, E, 12 serotypes (FAdV-1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10 and 11). The group of viruses are commonly existing in chickens, ducks and pigs, are frequently in recessive infection, act on poultry together as secondary pathogens, can be vertically transmitted, and pollute chicken embryos. The disease is found in the ankara area of Pakistan in 3 months of 1987, and in 2015, the disease is outbreak in areas such as Shandong, liaoning, jilin and Hebei in China, which causes huge economic loss for poultry industry in China. At present, the vaccine for effectively preventing the disease is less, the curative effect of antibiotics and antiviral drugs on the disease is not large, and no specific drug exists, so that the prevention and control of the disease are often unfavorable.

Ankara virus is DNA virus, and virus particles have no envelope and are spherical and have icosahedral symmetrical structures. The particle diameter is generally 70-90nm, and is composed of 252 capsid particles, of which 12 apical capsomers are Penton (Penton) and 240 non-apical capsomers are Hexon (Hexon) and Fiber protein (Fiber 1, fiber 2). Penton, hexon, and fiber proteins are viral capsid proteins and are immunogenic. Penton protein and fiber protein play a role in the process of adsorbing viruses and entering cells, and can effectively stimulate the organism to generate humoral immunity and induce the generation of neutralizing antibodies. The hexon protein contains specific antigenic determinants, is a neutralizing target of antibodies and has close relationship with pathogenicity.

Because of fast onset, high mortality and strong diffusion capability of the poultry ankara, the poultry ankara can be vertically and horizontally transmitted, and has caused great harm to the poultry industry. Therefore, to solve this problem, development of a novel effective vaccine is urgently required. At present, the disease is mainly prevented by inactivated vaccine and attenuated vaccine, but the two vaccines have a plurality of defects, and the problems of unstable protection effect, easy secondary infection, strong virulence return and the like exist.

Disclosure of Invention

The invention provides a recombinant protein for preventing fowl ankara disease, a construction method and application thereof, which are used for solving the problems of unstable protection effect, easy secondary infection, strong virulence return and the like of the existing inactivated vaccine and attenuated vaccine. The subunit vaccine for preventing the poultry ankara disease is prepared and applied to preventing the poultry ankara disease.

The technical scheme adopted by the invention is as follows:

the recombinant protein for preventing the poultry ankara disease is a multi-fusion recombinant protein FAdV4:F1-P-F2-H, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.

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

The invention discloses a method for constructing a coding gene of a recombinant protein for preventing fowl ankara, which comprises the following steps:

the coding genes FAdV4:F1, the coding DNA FAdV4:P of the multi-epitope tandem protein of the 4-type avian adenovirus Fiber2, the coding DNA FAdV4:F2 of the multi-epitope tandem protein of the 4-type avian adenovirus Fiber2 and the coding DNA FAdV4:H of the multi-epitope tandem protein of the 4-type avian adenovirus Fiber1 are connected in series by using a flexible Linker and restriction enzyme cleavage site connection method, so as to obtain the coding genes FAdV4:F1-P-F2-H.

The coding DNA of the multi-epitope tandem protein derived from the 4-type avian adenovirus Fiber1 comprises gene fragments of a plurality of epitopes of the 4-type avian adenovirus Fiber1, which are connected in series at flexible linker intervals, and the coding DNAFAdV4:F1 optimized by the codon 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 multi-epitope tandem protein derived from the type 4 avian adenovirus Penton comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Penton, the gene fragments are connected in series at flexible linker intervals, the coding DNAFAdV4:P optimized by the codon is chemically synthesized, the coding DNAFAdV4:P is shown as 382-714bp 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 multi-epitope tandem protein derived from the type 4 avian adenovirus Fiber2 comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Fiber2, which are connected in series at flexible linker intervals, and the code optimized coding DNAFAdV4:F2 is chemically synthesized, wherein the coding DNA is shown as 742-1077bp 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 multi-epitope tandem protein derived from the type 4 avian adenovirus Hexon comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Hexon, the gene fragments are connected in series at flexible linker intervals, the coding DNAFAdV4:H optimized by the codon is synthesized chemically, the coding DNAFAdV4:H is shown as 1099-1404bp of SEQ ID NO.2, and the corresponding amino acid sequence is shown as 367-468aa of SEQ ID NO. 1.

The method for connecting restriction enzyme cutting sites of the coding gene comprises the steps of introducing five restriction enzyme cutting sites of Nco I, ndeI, hindIII, xhoI and Bam HI to four multi-antigen epitopes, and connecting an upstream primer and a downstream primer of coding DNA of protein in series, wherein the upstream primer and the downstream primer comprise:

f1 upstream primer of coding DNAFAdV4 of multi-epitope tandem protein derived from 4-type avian adenovirus Fiber1 contains NcoI restriction enzyme site as shown in SEQ ID NO.3, and downstream primer contains NdeI restriction enzyme site as shown in SEQ ID NO. 4;

the upstream primer of the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the 4-type avian adenovirus Penton sequentially contains NdeI, hindIII and XhoI restriction enzyme sites, and is shown as SEQ ID NO.5, and the downstream primer contains BamHI restriction enzyme sites, and is shown as SEQ ID NO. 6;

f2 upstream primer of coding DNAFAdV4 of multi-epitope tandem protein derived from 4-type avian adenovirus Fiber2 sequentially contains NdeI restriction enzyme sites, bamHI restriction enzyme sites and HindIII restriction enzyme sites, and is shown as SEQ ID NO.7, and downstream primer contains XhoI restriction enzyme sites, and is shown as SEQ ID NO. 8;

the upstream primer of coding DNAFAdV4:H of the multi-epitope tandem protein derived from the avian adenovirus No.4 contains NdeI, bamHI and XhoI restriction enzyme sites in sequence, as shown in SEQ ID NO.9, and the downstream primer contains HindIII restriction enzyme sites, as shown in SEQ ID NO. 10.

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

The invention expresses a recombinant expression vector for preventing fowl ankara disease of recombinant protein: the basic vector of the recombinant expression vector comprises pET-28a, a recombinant protein coding DNAFAdV4:F1-P-F2-H upstream primer introduces NcoI restriction enzyme site, as shown in SEQ ID NO.3, and a downstream primer introduces Eco RI restriction enzyme site, as shown in SEQ ID NO.11, and the recombinant expression plasmid pET28a-FAdV4:F1-P-F2-H is constructed by enzyme digestion and connection between NcoI inserted into pET-28a and Eco RI enzyme digestion site.

The invention relates to an induction expression method of recombinant protein for preventing fowl ankara, which comprises the following steps:

transferring a recombinant expression vector pET28a-FAdV4:F1-P-F2-H into competent cells of E.coli Rosetta (DE 3) to obtain escherichia coli genetic engineering expression bacteria Ec-RD-FAdV4:F1-P-F2-H capable of expressing the multi-linked fusion recombinant protein FAdV4:F1-P-F2-H, and purifying by IPTG induced expression and Ni column affinity chromatography to obtain the recombinant protein FAdV4:F1-P-F2-H.

The recombinant protein for preventing the poultry ankara disease is applied to the preparation of subunit vaccines of the poultry ankara disease.

A subunit vaccine for preventing fowl ankara disease, comprising recombinant protein for preventing fowl ankara disease and an immunologically acceptable vaccine adjuvant 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 four capsid protein antigen epitopes of 4-type avian adenovirus to construct a multi-fusion recombinant protein FAdV4:F1-P-F2-H, and the subunit vaccine of the avian ankara disease prepared by using the protein has more capsid protein antigen epitopes, stimulates organisms to generate more kinds and more amounts of protective antibodies, and obviously improves the protective capability on the avian ankara disease. The selected multi-antigen epitope and the codon of the encoding DNA of the multi-fusion recombinant protein FAdV4:F1-P-F2-H are optimized according to the codon preference of the escherichia coli, so that the protein quantity of the escherichia coli expression system for soluble expression of the multi-fusion recombinant protein FAdV4:F1-P-F2-H is obviously improved, and the sufficient multi-fusion recombinant protein FAdV4:F1-P-F2-H is ensured to be used for preparing the subunit vaccine of the poultry ankara disease. The multi-fusion recombinant protein FAdV4:F1-P-F2-H subunit vaccine prepared by the invention can generate complete immune protection only by one immunization, thereby obviously reducing the cost of immune epidemic prevention. In a word, the subunit vaccine prepared by using the multi-fusion recombinant protein FAdV4:F1-P-F2-H and capable of preventing the poultry ankara disease has the characteristics of high antigen expression, long antibody maintenance time, high purity, good safety, strong immunogenicity, strong protectiveness and the like, and can effectively prevent the poultry ankara disease.

Drawings

FIG. 1 is a diagram showing the result of PCR amplification of the encoding genes of the fusion recombinant proteins in six different arrangements obtained by enzyme digestion and connection of the encoding DNA FAdV4:F1, FAdV4: P, FAdV4:F2 and FAdV4:H of four multi-epitope tandem proteins in the step (1) of the embodiment 2; wherein:

lane M is DL 2000DNAMarker;

lanes 1-6 are the encoding genes of the recombinant protein for multiplex fusion, 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, respectively;

FIG. 2 is a diagram showing the result of agarose gel electrophoresis of the double enzyme-digested products after the encoding gene of the recombinant protein in step (2) of the present invention is identified by double enzyme digestion with restriction enzymes NcoI and Eco RI; wherein:

lane M is DL 2000DNAMarker; lanes 1-6 are respectively the recombinant protein 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 after enzyme digestion;

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

in the figure, A is an SDS-PAGE electrophoresis detection result diagram of gene engineering expression bacteria Ec-RD-FAdV4:F1-P-F2-H IPTG induced expression total protein, wherein: lane M is protein Marker; lane 1 is Ec-RD-FAdV4: F1-P-F2-H uninduced whole cells; lane 2 is Ec-RD-FAdV4: F1-P-F2-H induced whole cells; lane 3 is the supernatant of the Ec-RD-FAdV4 after centrifugation of the disrupted cells of F1-P-F2-H; lane 4 is the inclusion bodies after centrifugation of the Ec-RD-FAdV4: F1-P-F2-H disrupted cells;

b, C, D, E, F in the figure are respectively SDS-PAGE electrophoresis detection result diagrams of induction expression proteins of other five fusion recombinant proteins FAdV4:F1-P-H-F2, FAdV4:F1-H-F2-P, FAdV4:F1-F2-P-H, FAdV4:F1-F2-H-P, FAdV4:F 1-H-P-F2;

FIG. 4 is a graph showing the result of electrophoresis 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 diagram of the induced expression purified protein of the genetically engineered expression bacterium Ec-RD-FAdV4:F1-P-F2-H, wherein: lane M is protein Marker; lane 1 is E.coli Rosetta (DE 3) control bacteria containing only pET-28a empty vector, lane 2 is supernatant of purified Ec-RD-FAdV4:F1-P-F2-H after induced expression disruption thallus centrifugation, and lane 3 is purified multi-fusion recombinant protein FAdV4:F1-P-F2-H;

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

FIG. 5 is a graph showing the level of change of chicken serum antibodies at different periods after protein immunization according to the immunogenicity test of 1) the recombinant protein in step (2) of the present invention;

FIG. 6 is a graph showing the rule of antibody growth after immunization in the 2) immune-toxicity-counteracting protective assay of step (2) in example 4 of the present invention;

FIG. 7 is a graph showing survival after one immune challenge in the 2) immune challenge protective experiment of step (2) of example 4 of the present invention.

Detailed Description

Example 1 preparation of multiple fusion recombinant protein FAdV4:F1-P-F2-H

(1) Downloading the gene nucleotide sequences of Penton, hexon, fiber and Fiber2 capsid proteins according to the gene sequence of avian adenovirus 4 type in NCBI GenBank (GenBank accession number KU 587519.1), selecting protein fragments with four capsid proteins positioned in the outer membrane region, good hydrophilicity and concentrated antigen epitopes according to the characteristics of antigen epitopes, and constructing the coding DNA connection of the protein fragments with the selected antigen epitopes concentrated by using the coding DNA nucleotide sequence of the protein linker GGGGS into four multi-antigen epitope tandem protein coding DNA FAdV4:F1, FAdV4: P, FAdV4:F2 and FAdV4:H. Four multi-antigen epitope tandem protein coding DNAs are synthesized directly by a chemical method after the optimization of the E.coli codons according to the codon usage preference of an E.coli expression system, and synthesized by Suzhou Jin Weizhi biotechnology limited company.

(2) Primers containing NcoI, ndeI, hindIII, xhoI and BamHI enzyme cutting sites are designed by using Premier 5, so that the coding DNA FAdV4:F1, FAdV4: P, FAdV4:F2 and FAdV4:H of the constructed four multi-epitope tandem proteins are used as templates, and PCR amplification is carried out by using the primers in the table 1 according to the PCR system and the conditions in the table 2, so that the coding DNA of the multi-epitope tandem proteins with different restriction enzyme cutting sites at the 5 'and 3' ends is obtained. The specific method comprises the following steps: performing PCR amplification by taking the encoding DNA FAdV4:F1 of the multi-epitope tandem protein as a template, and performing PCR amplification by taking an upstream primer Fiber1-F containing NcoI restriction enzyme sites (shown as SEQ ID NO. 3) and a downstream primer Fiber1-R containing NdeI restriction enzyme sites (shown as SEQ ID NO. 4) to obtain a DNA fragment Nc-FAdV4:F1-Nd with the length of 369 bp; p is taken as a template, and an upstream primer Penton-F (shown as SEQ ID NO. 5) containing NdeI, hindIII and XhoI restriction enzyme sites and a downstream primer Penton-R (shown as SEQ ID NO. 6) containing Bam HI restriction enzyme sites are used for PCR amplification to obtain a DNA fragment Nd-Hi-Xh-FAdV4:P-Ba with the length of 372 bp; f2 is used as a template, and an upstream primer Fiber2-F containing NdeI, bamHI and HindIII restriction enzyme sites (shown as SEQ ID NO. 7) and a downstream primer Fiber2-R containing XhoI restriction enzyme sites (shown as SEQ ID NO. 8) are used for PCR amplification to obtain a DNA fragment Nd-Ba-Hi-FAdV4 with the length of 375 bp; the encoding DNA FAdV4:H of the multi-epitope tandem protein is used as a template, and an upstream primer Hexon-F containing NdeI, bamHI and XhoI restriction enzyme sites (shown as SEQ ID NO. 9) and a downstream primer Hexon-R containing HindIII restriction enzyme sites (shown as SEQ ID NO. 10) are used for PCR amplification, so that a DNA fragment Nd-Ba-Xh-FAdV4:H-Hi with the length of 345bp is amplified. After the PCR reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis, and 4 kinds of DNA fragments of the multi-epitope tandem proteins with different restriction enzyme sites at the upstream and downstream ends, nc-FAdV4:F1-Nd, nd-Hi-Xh-FAdV4:P-Ba, nd-Ba-Hi-FAdV4:F2-Xh, nd-Ba-Xh-FAdV4:H-Xh, were subjected to gel recovery, and were respectively connected with pMD-18T vectors in a water bath at 16℃overnight using T vector connection kits, and the connection system was shown in Table 3.

TABLE 1 all primer information

Table 2, example 1 PCR amplification System and conditions in step (2)

Note that: the PCR reaction procedure was 94℃for 5min;32 cycles (94 ℃ 40s;70 ℃ 30s;72 ℃ 90 s); 72 ℃ for 10min; preserving at 4 ℃.

Table 3, example 1 connection System in step (2)

Reagent(s)	Volume of
		Recovery of the product from the fragment of interest	4μl
pMD-18T	1μl
		solution I	5μl
Total volume of	10μl

EXAMPLE 2 construction of prokaryotic expression genetically engineered Strain of recombinant protein FAdV4:F1-P-F2-H

(1) Transferring the recombinant cloning plasmid containing the target DNA after the ligation in the step (2) of the example 1 into E.coli competent cells E.coli DH5α, picking up positive transformation bacteria, transferring to the vinca-coumarone biotechnology Co., ltd for sequencing, and extracting the recombinant cloning plasmid inserted with the correct DNA fragment.

Four recombinant cloning plasmids inserted into target DNA fragments are respectively extracted by using a plasmid extraction kit, and different encoding genes of the multi-fusion recombinant proteins are constructed by using a restriction enzyme cleavage site connection method according to different arrangement and combination modes. The specific method comprises the following steps: and (3) respectively carrying out double enzyme digestion by using NcoI, ndeI, hindIII, xhoI, bamHI, ecoRI, wherein a double enzyme digestion system is shown in Table 4, carrying out constant temperature enzyme digestion for 3-4 hours at 37 ℃, then, utilizing a DNA gel recovery kit to recover target DNA fragments, obtaining target DNA fragments with different enzyme digestion site sticky ends, and connecting by using T4DNA ligase to obtain the coding gene of the multi-linked fusion recombinant protein. The coding DNAFAdV4: F1 of the multi-epitope tandem protein is arranged at the first position, the coding DNA of the other three multi-epitope tandem proteins is constructed into six different connection modes of multi-fusion recombinant proteins 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, FAdV: F1-F2-H-P in a permutation and combination mode, the connection system is shown in Table 5, and the connection system is placed in a constant temperature water bath pot at 16 ℃ for overnight connection. The PCR amplification results of the encoding genes of the fusion recombinant proteins of six different connection modes obtained after enzyme digestion and connection are shown in figure 1, and then the connection products are transformed, sequenced and plasmids are extracted for standby. According to the experimental result of the immune toxicity attack protection rate of the prepared subunit vaccine, a preferable multi-fusion recombinant protein FAdV4:F1-P-F2-H is screened out, the invention specifically describes around the FAdV4:F1-P-F2-H, and other experimental operation methods of the multi-fusion recombinant protein with different connection modes are the same as the FAdV4:F1-P-F2-H.

(2) PCR amplification was performed using the encoding gene of the fusion recombinant protein FAdV4:F1-P-F2-H in step (1) as a template, using the upstream primer Fiber1-F containing the NcoI restriction enzyme site (shown as SEQ ID NO. 3) and the downstream primer FPBH-R containing the EcoRI restriction enzyme site (shown as SEQ ID NO. 11), and the PCR reaction system was the same as that in example 1. Introducing NcoI and EcoRI restriction sites into 5 'and 3' ends of a coding gene of a multi-linked fusion recombinant protein FAdV4:F1-P-F2-H, performing double restriction enzyme digestion by using two restriction enzymes of NcoI and EcoRI, wherein a double restriction enzyme digestion system is shown in Table 6, the double restriction enzyme digestion system is placed in a constant temperature water bath kettle at 37 ℃ to act for 3-4 hours, the agarose gel electrophoresis result of a double restriction enzyme digestion product is shown in FIG. 2, and then performing gel recovery on the double restriction enzyme digestion product. Recovering the double-enzyme-digested encoding gene DNA by using a DNA gel recovery kit, then connecting the recovered encoding gene DNA with NcoI/EcoRI double-enzyme tangential pET-28a plasmid by using T4DNA ligase, placing the connecting system in a constant temperature water bath at 16 ℃ for overnight connection, converting the connecting product, sequencing, extracting the plasmid for later use, and preparing the recombinant plasmid with the name of: pET28a-FAdV4: F1-P-F2-H. Then the recombinant plasmid is transformed into competent cells of escherichia coli Rosetta (DE 3) by a heat shock method, after PCR identification by using a bacterial liquid, the positive recombinant plasmid is delivered to vinca-kunmei biotechnology limited company for sequencing, and the nucleotide sequence of the coding gene of the 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 which can express the multiple fusion recombinant protein FAdV4:F1-P-F2-H in large quantity are obtained by screening at different temperatures and different inducer concentrations. The upstream primers of PCR amplification of the coding genes of the other 5 multi-fusion recombinant proteins are primers Fiber1-F containing NcoI restriction enzyme sites, and the downstream primers are different and all contain EcoRI restriction enzyme sites. Wherein, the downstream primer of PCR amplification of the encoding gene of the multiplex fusion recombinant protein FAdV4:F1-F2-P-H is the same as the downstream primer of FAdV4:F1-P-F2-H, and is a primer FPBH-R; the downstream primer of the PCR amplification of the encoding genes of the fusion recombinant proteins FAdV4:F1-P-H-F2 and FAdV4:F1-H-P-F2 is a primer FPHB-R, as shown in SEQ ID NO. 12; the downstream primer FHBP-R of the PCR amplification of the encoding genes of the recombinant proteins FAdV4:F1-H-F2-P and FAdV4:F1-F2-H-P is shown as SEQ ID NO. 13.

Table 4, example 2 cleavage System in step (1)

Note that: a: ncoI-FAdV4, F1-NdeI; b: ndeI-FAdV 4P-BamHI; c: bamHI-FAdV 4F 2-XhoI; d: xhoI-FAdV 4H-HindIII; e: bamHI-FAdV 4H-HindIII; f: hindIII-FAdV 4: F2-XhoI; g: ndeI-FAdV 4H-HindIII; h: hindIII-FAdV 4: P-BamHI; i: bamHI-FAdV 4F 2-XhoI; j: hindIII-FAdV 4: F2-XhoI; k: xhoI-FAdV 4P-BamHI; l: ndeI-FAdV4, F2-XhoI; m: xhoI-FAdV 4P-BamHI; n: bamHI-FAdV 4H-HindIII; o: xhoI-FAdV 4H-HindIII; p: hindIII-FAdV 4: P-BamHI;

table 5, example 2 connection System in step (1)

Component of connection system	Volume (mul)
		10* T4 DNAbuffer	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 of	20

Table 6, example 2 cleavage System in step (2)

Table 7, example 2 connection System in step (2)

EXAMPLE 3 expression and purification of the recombinant protein FAdV4:F1-P-F2-H

(1) Inoculating the genetically engineered expression strain Ec-RD-FAdV4:F1-P-F2-H into 5ml liquid LB culture medium containing 100 mug/ml kanamycin, culturing for 2H at 37 ℃, adding IPTG to the final concentration of 0.1mmol/L, and inducing for 8H. And detecting the expression condition of the target protein through SDS-PAGE electrophoresis, wherein the FAdV4:F1-H-P-F2 of the multi-fusion recombinant protein cannot be expressed after induction, and the other 5 connecting modes of the multi-fusion recombinant protein are successfully expressed, so that the subsequent immunogenicity and toxicity attack protective experiment is operated based on the 5 multi-fusion recombinant proteins with successful expression. And (3) carrying out ultrasonic disruption on the induced expression strain, centrifuging the ultrasonic strain liquid at 8000rpm and 4 ℃ for 10min, collecting supernatant and precipitate, and detecting the expression quantity and the dissolution state of the target protein in the genetically engineered expression strain through SDS-PAGE electrophoresis.

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

(2) Purification of the recombinant protein FAdV4:F1-P-F2-H

After the gene engineering expression bacteria Ec-RD-FAdV4: F1-P-F2-H are induced to express by IPTG, the bacteria are broken by ultrasound, the bacteria liquid after ultrasound is centrifugated for 50min at 8000rpm and 4 ℃, the supernatant is collected, and the collected supernatant is purified by using an affinity chromatography medium Ni-NTA.

The SDS-PAGE detection result shows that the recombinant protein is expressed in 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℃was inoculated into 1L of sterilized LB, 100. Mu.g/ml of kanapecillin was added, and the mixture was subjected to shaking culture at 37℃and 160rpm for 2 hours, and further to IPTG was added to give a final concentration of 0.1mmol/L, and the mixture was subjected to shaking culture at 37℃and 160rpm for 8 hours to induce the same. After induction, the bacterial liquid was centrifuged at 8000rpm at 4℃for 10min, bacterial pellet was collected, resuspended in binding Buffer A (Tris 6.058g, glycerol 100ml, nacl29.25g, imidazole 0.68g to a constant volume of 1L and pH was adjusted to 7.2-7.4), the bacterial pellet was sonicated, the post-sonicated bacterial liquid was centrifuged at 8000rpm at 4℃for 50min, and then the supernatant was collected and purified using affinity chromatography medium Ni-NTA.

The method comprises the steps of suspending the collected thalli by using a Buffer A for ultrasonic crushing, and combining the supernatant after centrifugation with a nickel column; washing the column with elution Buffer B (Tris 6.058g, glycerol 100ml, naCl29.25g, imidazole 34.04g to 1L to adjust pH to 7.2-7.4) to remove the impurity protein; eluting proteins with an elution buffer containing 60mM imidazole (prepared by mixing 89.80ml buffer A with 0.20ml buffer B) and an elution buffer containing 80mM imidazole (prepared by mixing 85.71ml buffer A with 14.29ml buffer B), respectively; the target protein was eluted with an elution buffer containing 220mM imidazole (prepared by mixing 57.14ml buffer A with 42.86ml buffer B) and the protein eluate was collected. And (3) loading the collected protein eluent into a dialysis bag with 3500Dal molecular weight cut-off so as to remove 220mM imidazole in the protein eluent, immersing the dialysis bag with the protein eluent into PBS for 3-4 times, changing fresh PBS for 3 hours each time for dialysis, and concentrating with PEG20000 for 2 hours after the dialysis is finished, wherein the obtained concentrated liquid is purified target protein FAdV4:F1-P-F2-H. The purified target protein concentration was measured using BCA protein concentration measurement kit from bi yun. After the purification, the concentration of the recombinant protein FAdV4:F1-P-F2-H of the multi-fusion is 1-2mg/ml.

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

Example 4 preparation of avian Ankara disease genetically engineered subunit vaccine and immunoprotection analysis experiments

(1) Preparation of subunit vaccine

The 5 kinds of the fusion recombinant proteins purified in example 3 were diluted to 100. Mu.g/ml and mixed with Freund's adjuvant at a volume ratio of 1:1 to prepare subunit vaccine.

(2) Subunit vaccine immunogenicity analysis experiments

1) Immunogenicity analysis

The SPF sea blue white laying hens without specific pathogen at 14 days of age are divided into 6 groups, 6 chickens in each group are immunized by 5 subunit vaccines prepared by the multi-fusion recombinant proteins respectively, the 6 th group is a PBS control group, PBS with the same volume is injected, wherein the first immunization is emulsified by the Freund complete adjuvant with the same volume, the subsequent immunization is emulsified by the Freund incomplete adjuvant, the immunization period is 7 days, the immunization program is four times immunization, and the immunization mode is leg intramuscular injection inoculation. Immune chicken serum samples were collected weekly for antibody titer change level detection starting one week after the first immunization of the chicken flock by means of fin vein blood sampling, antibody titers in the serum were detected by an indirect ELISA method and P/N values were calculated. As shown in FIG. 5, the subunit vaccines prepared from 5 kinds of multi-fusion recombinant proteins can stimulate the organism to produce antibodies, and the antibodies have longer maintenance time, which indicates that the prepared 5 kinds of multi-fusion recombinant proteins have good immunogenicity and can stimulate the organism to produce antibodies with strong binding capacity and long maintenance time.

2) Animal toxicity-counteracting immunoprotection experiments

(1) Experimental chicken grouping and immunization

The SPF sea blue white laying hens of 14 days old are selected, 6 laying hens are in each group, the immune period is 14 days, and the immune program is divided into one immunization and two immunization, and the immune mode is intramuscular injection of legs.

Experimental group: the primary immunization group is immunized with the 5 subunit vaccines (100 mu of protein for the blood glucose meter/animal) at the age of 14 days of chicken, and the immunization is carried out for 14 days to attack toxin; the two immunization groups are immunized again after 14 days of one immunization, the immunization dose and the immunization mode are the same as those of the one immunization, and the two immunization groups are detoxified after 14 days of the two immunization groups;

negative control: a group which is not immunized and is not attacked by poison;

positive control (PBS group): and injecting PBS into the virus-fighting group.

(2) Post-immunization antibody titer detection

The results show that the P/N value of the serum antibody titer of the experimental chickens of each immune group is not significantly different, but most of the serum antibodies in the experimental chickens after 1 week of immunization are positive; there was a significant difference from the PBS group, and the results are shown in fig. 6.

(3) Toxicity attack protective experiment

Day 14 after the first immunization, the immune group and positive control group of chicken groups were intramuscular injected with FAdV-4 isolate SDBZ-1, with a viral titer of 10 ⁴ TCID ₅₀ According to 100TCID ₅₀ The dosage is used for counteracting toxic substances. One week after toxin expelling, observing clinical symptoms of chicken every day, and recording infected chickenAnd (3) carrying out section examination on dead chickens under the death condition, and carrying out pathological change observation. The results of the toxicity attack protection experiment are shown in table 8, and the survival curves are shown in fig. 7. The toxicity attack protection rate of the primary immune group indicates that the multi-fusion recombinant protein has different immunity protection induction capacities. Compared with subunit vaccines of other multi-fusion recombinant proteins (FAdV 4: F1-P-H-F2, FAdV4: F1-H-F2-P, FAdV: 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 immunization. After the second immunity is free from virus attack, the virus attack protection force among immune groups is not significantly different, and 5 subunit vaccines can provide protection rate close to 100%.

TABLE 8 results of primary immune toxicity-counteracting protective experiments

According to the result of the toxicity attack protection experiment, the subunit vaccine FAdV4:F1-P-F2-H can provide complete protection in an immune group, and the death rate of all immunized chickens can be completely protected after one immunization is carried out, so that the toxicity attack death rate is 0. And the chicken in the PBS positive control group only attacks the virus, most of the chicken is ill and dead, and the chicken in the negative control group is not ill and dead. The subunit vaccine of the preferred multi-fusion recombinant protein FAdV4:F1-P-F2-H can play a complete role in protection by only one immunization, so that the immunization cost is greatly reduced, and the vaccine prepared by the multi-fusion recombinant protein FAdV4:F1-P-F2-H is high-efficiency and economical.

The aluminum hydroxide sol adjuvant used in the invention is used for replacing Freund's adjuvant to prepare subunit vaccine, the method and the result of immunogenicity analysis and animal toxicity-attacking immune protective experiment are the same as those of Freund's adjuvant, the method and the result of animal toxicity-attacking immune protective experiment can stimulate the organism to produce the antibody with strong binding capacity and long maintenance time, and the toxicity-attacking protective rate reaches 100%. Freund's adjuvant and aluminum hydroxide sol can be used as immunological adjuvant of subunit vaccine in the invention, and can achieve ideal immune and toxicity-counteracting protection effects.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

SEQUENCE LISTING

<110> Jilin university

<120> recombinant protein for preventing fowl ankara disease, construction method and application thereof

<130> jlu-rhl2021p

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 468

<212> PRT

<213> Synthesis

<400> 1

Met Asp His His His His His His Arg Ala Leu Ser Glu Pro Ser Arg

1 5 10 15

Tyr Leu Ser Glu Gly Asp Glu Arg Arg Lys Pro Lys Arg Ala Arg Pro

20 25 30

Ala Thr Arg Ala Asn Gly Gly Gly Gly Gly Ser Asn Gly Asn Val Lys

35 40 45

Ser Lys Gly Leu Gln Asn Trp Ser Gly Gly Gly Gly Ser Val Pro Pro

50 55 60

Thr Val Ser Pro Thr Asn Gln Asn Gly Gly Gly Gly Ser Pro Asn Ser

65 70 75 80

Asn Gln Ser Asp Val Gly Tyr Leu Gly Leu Pro Pro His Thr Arg Asp

85 90 95

Asn Trp Tyr Gly Gly Gly Gly Ser Gln Thr Ser Gly Ser Asn Trp Phe

100 105 110

Asp Gln Asn Ala Gly Gly Gly Gly Ser His Met Lys Leu Leu Glu Pro

115 120 125

Pro Pro Pro Thr Glu Leu Thr Pro Ser Thr Gly Gly Gly Gly Ser Ala

130 135 140

Pro Thr Gly Gly Arg Asn Ser Ile Lys Tyr Arg Asp Tyr Thr Pro Cys

145 150 155 160

Arg Asn Gly Gly Gly Gly Ser Lys Ala Ser Asp Ile Asp Thr Tyr Asn

165 170 175

Lys Asp Ala Asn His Ser Asn Phe Gly Gly Gly Gly Ser Arg Gln Asn

180 185 190

Asn Val Gln Lys Ser Asp Ile Gly Gly Gly Gly Ser Gly Ile Gly Lys

195 200 205

Arg Glu Pro Tyr Ser Lys Gly Phe Val Ile Gly Gly Gly Gly Ser Phe

210 215 220

Ile Ala Pro Thr Gly Phe Lys Glu Asp Asn Thr Thr Asn Leu Gly Gly

225 230 235 240

Gly Gly Ser Gly Ser Lys Leu Gly Lys Pro Glu Thr Glu Ala Gly Pro

245 250 255

Ser Pro Ala Gly Gly Gly Gly Ser Ile Lys Asn Arg Ser Val Asp Leu

260 265 270

Ala His Asp Pro Ser Leu Asp Gly Gly Gly Gly Ser Val Ser Val Asp

275 280 285

Glu Ser Leu Gln Ile Val Asn Asn Thr Leu Glu Val Lys Pro Asp Pro

290 295 300

Ser Gly Pro Gly Gly Gly Gly Ser Ser Ala Thr Met Gly Asn Arg Pro

305 310 315 320

Gly Asp Leu Asn Ser Ala Asn Ala Lys Gly Gly Gly Gly Ser Pro Met

325 330 335

Ala Asn Arg Ser Val Thr Ser Pro Trp Thr Tyr Ser Ala Asn Gly Tyr

340 345 350

Tyr Glu Pro Ser Ile Gly Glu Gly Gly Gly Gly Ser Leu Glu Asn Val

355 360 365

Thr Thr Glu Lys Gly Gly Gly Gly Ser Ile Gln Thr Asp Asp Thr Ser

370 375 380

Thr Gly Gly Gly Gly Gly Ser Leu Asp Arg Gly Pro Ser Phe Lys Pro

385 390 395 400

Tyr Cys Gly Thr Gly Gly Gly Gly Ser Ser Met Phe Asn Asn Trp Ser

405 410 415

Glu Thr Ala Pro Gly Gln Asn Gly Gly Gly Gly Ser Phe Pro Asn Pro

420 425 430

Asn Gln Gly Pro Gly Arg Asn Pro Leu Arg Arg Val Gln Asn Ala Asn

435 440 445

Thr Gly Val Gly Gly Gly Gly Ser Glu Asp Tyr Asp Asp Tyr Asn Ile

450 455 460

Gly Thr Thr Arg

465

<210> 2

<211> 1413

<212> DNA

<213> Synthesis

<400> 2

atggatcatc atcaccatca ccatcgggcg ctgagcgaac cgagccgcta tctgagtgag 60

ggtgacgagc gacgtaagcc taagagggct agacctgcca cacgtgctaa tggcgggggc 120

gggggcagta atgggaacgt gaagagcaaa ggactccaga actggtcggg cggtggtgga 180

tcagttccac ctactgtatc tcctactaat cagaatggtg gcggcgggag cccgaacagc 240

aaccagagcg atgtgggcta tctgggcctg ccgccgcata cccgcgataa ctggtatggt 300

ggcggaggct cacagactag cggcagcaac tggtttgatc agaacgcggg cggaggcgga 360

agccatatga agcttctcga gccaccacct cctaccgaat taacgccgag cactggtgga 420

ggcgggtcgg caccgaccgg gggtcggaac agcattaaat atcgcgatta tacgccatgt 480

cgcaacggag gaggtggtag caaagctagc gatattgata cctataacaa agatgcgaac 540

catagcaact tcggcggtgg agggtcgcgt cagaataatg tgcagaagag tgacatagga 600

ggcggagggt ccggaatcgg caaacgcgaa ccgtatagca aaggctttgt gattggtggg 660

ggtgggtctt tcattgctcc aacaggtttc aaggaagata acaccaccaa cctgggcgga 720

ggcggaagcg gatccaagct tgggaaaccg gaaaccgaag cgggcccgag cccggccgga 780

ggtggcggat cgataaagaa ccggtccgtt gacttggcgc atgatccgag cctggatgga 840

ggtggcggta gcgtctcggt ggatgaaagc ctgcagattg tgaacaatac gttagaggtt 900

aagcccgacc cttctggacc cggaggcggt ggctctagcg cgacgatggg caatagacca 960

ggagacctca atagtgccaa tgccaagggc ggaggcggga gcccgatggc gaatcgatct 1020

gtaacaagtc cgtggaccta tagcgcgaac ggctattatg aaccgagcat tggcgaaggc 1080

ggaggcggaa gcctcgagaa cgtgaccacc gagaagggtg ggggaggaag tatccagacc 1140

gatgatacct ctacaggagg tggtggcgga agccttgatc gcggcccgag ctttaaaccg 1200

tattgcggta caggtggcgg agggtcctcc atgtttaata attggtccga gacggctcca 1260

ggccagaacg gagggggtgg atcgtttcca aatcctaatc aaggtccagg aaggaatccc 1320

ttacggagag tacagaacgc gaataccggc gtgggtggtg ggggcagcga ggactatgat 1380

gattataaca ttggcaccac ccgctaagaa ttc 1413

<210> 3

<211> 46

<212> DNA

<213> Synthesis

<400> 3

catgccatgg atcatcatca ccatcaccat cgggcgctga gcgaac 46

<210> 4

<211> 51

<212> DNA

<213> Synthesis

<400> 4

ggaattccat atggcttccg cctccgcccg cgttctgatc aaaccagttg c 51

<210> 5

<211> 48

<212> DNA

<213> Synthesis

<400> 5

ggaattccat atgaagcttc tcgagccacc acctcctacc gaattaac 48

<210> 6

<211> 44

<212> DNA

<213> Synthesis

<400> 6

cgggatccgc ttccgcctcc gcccaggttg gtggtgttat cttc 44

<210> 7

<211> 49

<212> DNA

<213> Synthesis

<400> 7

ggaattccat atgggatcca agcttgggaa accggaaacc gaagcgggc 49

<210> 8

<211> 41

<212> DNA

<213> Synthesis

<400> 8

ccctcgaggc ttccgcctcc gccttcgcca atgctcggtt c 41

<210> 9

<211> 50

<212> DNA

<213> Synthesis

<400> 9

ggaattccat atgggatccc tcgagaacgt gaccaccgag aagggtgggg 50

<210> 10

<211> 49

<212> DNA

<213> Synthesis

<400> 10

cccaagcttg cttccgcctc cgccgcgggt ggtgccaatg ttataatcat c 51

<210> 11

<211> 50

<212> DNA

<213> Synthesis

<400> 11

ggaattctta gcgggtggtg ccaatgttat aatcatcata gtcctcgctg 50

<210> 12

<211> 50

<212> DNA

<213> Synthesis

<400> 12

ggaattctta ttcgccaatg ctcggttcat aatagccgtt cgcgctatag 50

<210> 13

<211> 53

<212> DNA

<213> Synthesis

<400> 13

ggaattctta caggttggtg gtgttatctt ccttgaaacc tgttggagca atg 53

Claims

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

2. A gene encoding a recombinant protein for use in the prevention of avian ankara disease according to claim 1, characterized in that: the nucleotide sequence of the coding gene of the recombinant protein is shown as SEQ ID NO. 2.

3. A method of constructing a gene encoding a recombinant protein for preventing an avian ankara disease according to claim 2, comprising the steps of:

the method comprises the steps of connecting a flexible Linker with restriction enzyme cleavage sites, wherein the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the type 4 avian adenovirus Fiber1, the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the type 4 avian adenovirus Penton, the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the type 4 avian adenovirus Fiber2, the encoding DNA FAdV4 of the F2 and the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the type 4 avian adenovirus Hexon are connected in series to obtain the encoding gene FAdV4 of F1-P-F2-H.

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

the coding DNA of the multi-epitope tandem protein derived from the type 4 avian adenovirus Fiber1 comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Fiber1, which are connected in series at flexible linker intervals, and the coding DNA FAdV4:F1 optimized by the codon is synthesized chemically, 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 multi-epitope tandem protein derived from the type 4 avian adenovirus Penton comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Penton, the gene fragments are connected in series at flexible linker intervals, the coding DNA FAdV4 with optimized codons is synthesized chemically, the P is shown as 382-714bp 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 multi-epitope tandem protein derived from the type 4 avian adenovirus Fiber2 comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Fiber2, which are connected in series at flexible linker intervals, and the coding DNA FAdV4:F2 optimized by the codon is synthesized chemically, wherein the coding DNA is shown as 742-1077bp 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 multi-epitope tandem protein derived from the type 4 avian adenovirus Hexon comprises gene fragments of a plurality of epitopes of the type 4 avian adenovirus Hexon, the gene fragments are connected in series at flexible linker intervals, the coding DNA FAdV4:H optimized by the codon is synthesized chemically, the coding DNA is shown as 1099-1404bp of SEQ ID NO.2, and the corresponding amino acid sequence is shown as 367-468aa of SEQ ID NO. 1.

5. The method for constructing a coding gene according to claim 3, wherein: the restriction enzyme site connection method is to introduce five restriction enzyme sites of Nco I, ndeI, hindIII, xhoI and BamHI into an upstream primer and a downstream primer of encoding DNA of four multi-epitope tandem proteins, and the method comprises the following steps:

f1 upstream primer of the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the 4-type avian adenovirus Fiber1 contains NcoI restriction enzyme site as shown in SEQ ID NO.3, and downstream primer contains NdeI restriction enzyme site as shown in SEQ ID NO. 4;

f2 upstream primer of the encoding DNA FAdV4 of the multi-epitope tandem protein derived from the 4-type avian adenovirus Fiber2 sequentially contains NdeI restriction enzyme sites, bamHI restriction enzyme sites and HindIII restriction enzyme sites, and is shown as SEQ ID NO.7, and downstream primer contains XhoI restriction enzyme sites and is shown as SEQ ID NO. 8;

the upstream primer of the encoding DNA FAdV4:H of the multi-epitope tandem protein derived from the 4-avian adenovirus Hexon sequentially contains NdeI, bamHI and XhoI restriction enzyme sites, and is shown as SEQ ID NO.9, and the downstream primer contains HindIII restriction enzyme sites, and is shown as SEQ ID NO. 10.

6. Recombinant expression system of a recombinant protein for the prevention of avian ankara according to claim 1, characterized in that: the recombinant expression system comprises a eukaryotic expression system or a prokaryotic expression system.

7. Recombinant expression vector for the expression of a recombinant protein for the prevention of avian ankara according to claim 1, characterized in that: the basic vector of the recombinant expression vector comprises pET-28a, a recombinant protein encoding DNA FAdV4:F1-P-F2-H, an upstream primer of which is introduced with an NcoI restriction enzyme site, as shown in SEQ ID NO.3, and a downstream primer of which is introduced with an Eco RI restriction enzyme site, as shown in SEQ ID NO.11, and the recombinant expression plasmid pET28a-FAdV4:F1-P-F2-H of which is inserted between the NcoI and Eco RI restriction enzyme sites of pET-28a is constructed by enzyme digestion connection.

8. A method for the inducible expression of recombinant proteins for the prevention of avian ankara according to claim 1, comprising the steps of:

transferring the recombinant expression plasmid pET28a-FAdV4:F1-P-F2-H of claim 7 into competent cells of E.coli Rosetta (DE 3) to obtain escherichia coli genetic engineering expression bacteria Ec-RD-FAdV4:F1-P-F2-H of the expressible multi-fusion recombinant protein FAdV4:F1-P-F2-H, and purifying by IPTG induction expression and Ni column affinity chromatography to obtain the recombinant protein FAdV4:F1-P-F2-H.

9. Use of a recombinant protein for preventing an avian ankara according to claim 1 for the preparation of an avian ankara subunit vaccine.

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