A recombinant avian adenovirus type 11 fiber protein, subunit vaccine and application thereof

Abstract

This invention discloses a recombinant avian adenovirus type 11 fiber protein, a subunit vaccine, and their applications, belonging to the field of genetic engineering technology. The invention obtains the recombinant avian adenovirus type 11 fiber protein by removing amino acids 1-281 from the avian adenovirus type 11 fiber protein. This recombinant avian adenovirus type 11 fiber protein has the amino acid sequence shown in SEQ ID NO:3. Furthermore, this recombinant avian adenovirus type 11 fiber protein not only exhibits good immunogenicity but also demonstrates efficient and soluble expression in prokaryotic expression systems. When this recombinant avian adenovirus type 11 fiber protein is prepared into a subunit vaccine, it exhibits good protective efficacy against avian adenovirus type 11 infection in chickens.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant avian adenovirus type 11 fiber protein, subunit vaccine and its application. Background Technology

[0002] Based on antigenic differences, avian adenovirus (FAdV) can be divided into three subgroups (subgroups I, II, and III). Subgroup I avian adenoviruses are mainly isolated from the respiratory tracts of chickens, turkeys, and geese, and they all share a subgroup-specific antigen. Based on restriction endonuclease digestion analysis and cross-neutralization tests, subgroup I avian adenoviruses can be further divided into five species (subgroups A-E) and twelve serotypes (1-8a, 8b-11). Subgroup A includes serotype 1, subgroup B includes serotype 5, subgroup C includes serotypes 4 and 10, subgroup D includes serotypes 2, 3, 9, and 11, and subgroup E includes serotypes 6, 7, 8a, and 8b.

[0003] In my country, the predominant serological variant is FAdV-4, accounting for over 50%, followed by FAdV-11 and FAdV-8a / 8b. Infection with FAdV-4 in chickens leads to hepatitis-pericardial effusion syndrome (HHS), characterized by rapid onset, high pathogenicity, and high mortality. Infection with FAdV-11 and FAdV-8a / 8b results in inclusion body hepatitis (IBH), primarily manifested as hepatic swelling and hemorrhage, with hepatocytes exhibiting fatty degeneration, vacuolar degeneration, and nuclear lysis. Clinically, FAdV-11 can be isolated from chickens of all ages, indicating widespread infection; therefore, developing vaccines to control FAdV-11 has become a research hotspot. Currently, several vaccines against FAdV-4 have been developed in China, fundamentally improving FAdV-4 control. However, to date, there are no reports of commercially available, effective, and safe vaccines against FAdV-11, and vaccines against FAdV-4 do not offer cross-protection against FAdV-11. Therefore, the prevalence of FAdV-11 will gradually increase in the future, and China will face a severe situation in prevention and control.

[0004] There are two main technical approaches to FAdV-11 vaccines: whole-virus inactivated vaccines and genetically engineered subunit vaccines. Whole-virus inactivated vaccines involve costly cell culture, consume significant energy, and are environmentally unfriendly; the inactivation process may also lead to a decrease in antigenicity. Genetically engineered subunit vaccines represent a greener and more environmentally friendly approach, offering advantages such as lower cost, higher yield, and easier scale-up. However, in developing FAdV-11 vaccines using this approach, the avian adenovirus type 11 antigen protein expressed in the E. coli expression system exhibits problems such as poor solubility, low expression levels, and poor immunogenicity. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant avian adenovirus type 11 fiber protein and subunit vaccine and its application. This addresses the problems of poor solubility, low expression levels, and poor immunogenicity of the avian adenovirus type 11 antigen protein expressed in the *E. coli* expression system during the preparation of existing FAdV-11 vaccines.

[0006] In a first aspect, the present invention provides a recombinant avian adenovirus type 11 fiber protein, the recombinant avian adenovirus type 11 fiber protein being selected from any of the following: A1) having an amino acid sequence as shown in SEQ ID NO:3; A2) having an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence defined in A1); A3) having an amino acid sequence with more than 80% sequence identity compared to the amino acid sequence defined in A1) or A2); A4) an amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

[0007] The recombinant avian adenovirus type 11 fiber protein provided by the present invention can be a natural, recombinant or synthetic active polypeptide. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants) using recombinant technology.

[0008] In this invention (A4), the linkage can be achieved through direct peptide bond connection or through a linker, using methods conventional in the art. The tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Flag tag protein, LacZ tag protein, GFP (green fluorescent protein), sfGFP (hyperfolded green fluorescent protein), and HA tag (hemagglutinin tag). Those skilled in the art can select appropriate tag proteins according to actual needs. The use of tags does not alter the function of the target protein (recombinant avian adenovirus type 11 fiber protein); its purpose is for separation, purification, detection, or tracing. The tags can be separated from the target protein (recombinant avian adenovirus type 11 fiber protein) using chemical cleavage methods or enzymatic methods (such as introducing protease cleavage sites and using TEV protease to remove the tag) known in the art.

[0009] In this invention, the recombinant avian adenovirus type 11 fiber protein is obtained by removing amino acids 1-281 from the avian adenovirus type 11 fiber protein. The 282-355aa fragment in the recombinant avian adenovirus type 11 fiber protein plays a stabilizing role in its structure, the 361-569aa fragment is the core antigenic structure that induces a protective immune response in the recombinant avian adenovirus type 11 fiber protein, and the 356-360aa fragment is used to link the above fragments. This recombinant avian adenovirus type 11 fiber protein not only has good immunogenicity but also exhibits efficient and soluble expression in prokaryotic expression systems.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the aforementioned recombinant avian adenovirus type 11 Fiber protein.

[0011] The nucleic acid molecules provided by this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as mRNA or hnRNA; and these nucleic acid molecules can usually be obtained by PCR amplification or artificial synthesis.

[0012] In some embodiments, the nucleic acid molecule is selected from any of the following: B1) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:4; B2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in B1) under stringent conditions and encodes the above-mentioned recombinant avian adenovirus type 11 Fiber protein; B3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) or B2) and encodes the above-mentioned recombinant avian adenovirus type 11 Fiber protein.

[0013] As used herein, the term "hybridization under stringent conditions" refers to the hybridization of two nucleic acid fragments under standard hybridization conditions as described in the section "Expression of Cloned Genes in E. coli" of *Molecular Cloning: A Laboratory Manual* (1989) (Cold Spring Lane Laboratory, New York, USA). Such conditions include hybridization in 6.0 × SSC at 45 °C, followed by washing in 2 × SSC at 50 °C. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 × SSC at 50 °C for low stringency and 2.0 × SSC at 50 °C for high stringency. Additionally, the temperature in the washing step can be varied between room temperature (approximately 22 °C) for low stringency and 65 °C for high stringency.

[0014] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0015] In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecules.

[0016] The recombinant vectors in this invention include cloning vectors and expression vectors. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be the pET28a vector.

[0017] In a fourth aspect, the present invention provides a recombinant cell comprising the above-described nucleic acid molecules or the above-described recombinant vector.

[0018] In some implementations, the method for preparing recombinant cells includes the step of converting the recombinant vector into expression host cells.

[0019] In this invention, the expression host cell is a conventional host cell in the art, as long as the recombinant vector can stably replicate itself and the gene it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli or yeast. For example, Escherichia coli can be E. coli BL21(DE3).

[0020] In a fifth aspect, the present invention provides a method for preparing recombinant avian adenovirus type 11 Fiber protein, comprising the following steps: culturing the above-mentioned recombinant cells, inducing expression to obtain a culture; and isolating the above-mentioned recombinant avian adenovirus type 11 Fiber protein from the culture.

[0021] In this invention, there are no special requirements for the culture method and culture conditions; it is sufficient to ensure the normal growth of the recombinant cells. Furthermore, the methods for isolating the aforementioned recombinant avian adenovirus type 11 fiber protein from the culture are all conventional methods in the art.

[0022] In some embodiments, the culture medium used in the preparation of recombinant avian adenovirus type 11 fiber protein is a protein-expressing culture medium in the art, preferably LB medium.

[0023] In some implementations, the method also includes a step of purifying the recombinant avian adenovirus type 11 fiber protein, specifically including ammonium sulfate fractionation followed by affinity chromatography.

[0024] In some preferred embodiments, ammonium sulfate fractionation precipitation specifically includes: first precipitating impurities with 30% ammonium sulfate, and then collecting the target protein with 40% ammonium sulfate.

[0025] In some preferred embodiments, affinity chromatography specifically includes: first removing contaminating proteins using a washing buffer (0.01M PBS, 10mM imidazole, pH 7.4), and then eluting the target protein using an elution buffer (0.01M PBS, 50mM imidazole, pH 7.4).

[0026] In a sixth aspect, the present invention provides a recombinant avian adenovirus type 11 Fiber protein subunit vaccine, comprising the above-mentioned recombinant avian adenovirus type 11 Fiber protein and an adjuvant.

[0027] The recombinant avian adenovirus type 11 fiber protein subunit vaccine provided by this invention has good protective efficacy against avian adenovirus type 11 infection in chickens.

[0028] In a seventh aspect, the present invention provides a method for preparing the recombinant avian adenovirus type 11 Fiber protein subunit vaccine as described above, comprising the following steps: filtering the recombinant avian adenovirus type 11 Fiber protein and adding a surfactant to obtain an aqueous phase; mixing the aqueous phase with an adjuvant and emulsifying it to obtain the recombinant avian adenovirus type 11 Fiber protein subunit vaccine; wherein the surfactant includes Tween-80, the adjuvant includes white oil adjuvant, and the volume ratio of the aqueous phase to the adjuvant is 1:(2-4), preferably 1:3; the content of recombinant avian adenovirus type 11 Fiber protein in the recombinant avian adenovirus type 11 Fiber protein subunit vaccine is 40-60 μg / mL, preferably 50 μg / mL.

[0029] The recombinant avian adenovirus type 11 fiber protein subunit vaccine provided by this invention has a simple preparation method, uses inexpensive and readily available raw materials, and is suitable for large-scale industrial production.

[0030] It is understood that the surfactant and adjuvant can be selected from conventional surfactants and adjuvants in the prior art according to actual use needs. In this invention, the surfactant preferably includes Tween-80, and the adjuvant preferably includes white oil adjuvant.

[0031] In an eighth aspect, the present invention provides the use of the recombinant avian adenovirus type 11 fiber protein, any of the above-mentioned nucleic acid molecules, the above-mentioned recombinant vector, the above-mentioned recombinant cells, the recombinant avian adenovirus type 11 fiber protein prepared by the above-mentioned preparation method, any of the above-mentioned recombinant avian adenovirus type 11 fiber protein subunit vaccines, and the recombinant avian adenovirus type 11 fiber protein subunit vaccines prepared by the above-mentioned preparation method in the preparation of a medicament for the prevention and / or treatment of avian adenovirus type 11 infection in chickens.

[0032] In a ninth aspect, the present invention provides a method for verifying the efficacy of the recombinant avian adenovirus type 11 fiber protein subunit vaccine as described above, comprising the following steps: immunizing 140-day-old chickens with the recombinant avian adenovirus type 11 fiber protein subunit vaccine to obtain immunized hens; incubating the eggs produced by the immunized hens to obtain test chickens containing maternal antibodies; simultaneously incubating the eggs produced by unimmunized hens to obtain challenge control chickens without maternal antibodies; intramuscularly injecting avian adenovirus type 11 strain into 1-day-old test chickens containing maternal antibodies and 1-day-old challenge control chickens without maternal antibodies, respectively; determining the survival and disease incidence of the test chickens and challenge control chickens, as well as the FAdV-11 strain load of the test chickens, challenge control chickens, and negative control chickens; if the test chickens survive without developing the disease, and the FAdV-11 strain load of the test chickens is less than the viral load of the challenge control chickens and similar to the viral load of the negative control chickens, it indicates that the recombinant avian adenovirus type 11 fiber protein subunit vaccine has protective efficacy against chickens infected with the FAdV-11 strain.

[0033] The verification method for the recombinant avian adenovirus type 11 fiber protein subunit vaccine established in this invention is simple and has good application prospects.

[0034] The beneficial effects of this invention are as follows: Unlike the prior art, this invention obtains recombinant avian adenovirus type 11 fiber protein by removing amino acids 1-281 from the avian adenovirus type 11 fiber protein. The 282-355aa fragment in this recombinant avian adenovirus type 11 fiber protein has a stabilizing effect on its structure, the 361-569aa fragment is the core antigen structure that induces a protective immune response in the recombinant avian adenovirus type 11 fiber protein, and the 356-360aa fragment is used to connect the above fragments. This recombinant avian adenovirus type 11 fiber protein not only has good immunogenicity but also exhibits efficient and soluble expression in prokaryotic expression systems. When this recombinant avian adenovirus type 11 fiber protein is prepared into a subunit vaccine, it has good protective efficacy against avian adenovirus type 11 infection in chickens. Attached Figure Description

[0035] Figure 1 The image shows the SDS-PAGE results of the FAdV-11 Fiber protein expressed in prokaryotes in Example 1 of this invention. Lane M: pre-stained protein standard molecular weight; Lane 1: negative control; Lane 2: total FAdV-11 Fiber protein expressed in prokaryotes; Lane 3: soluble FAdV-11 Fiber protein expressed in prokaryotes.

[0036] Figure 2 The FAdV-11 Fiber expressed in the prokaryote in Example 1 of this invention 282-569 SDS-PAGE results of the protein, where lane M: pre-stained protein standard molecular weight; lane 1: FAdV-11 Fiber. 282-569 Total protein expressed in prokaryotes; Lane 2: FAdV-11 Fiber 282-569 Prokaryotic expression of soluble protein; Lane 3: negative control;

[0037] Figure 3 The above are the SDS-PAGE results of the purified recombinant avian adenovirus type 11 fiber protein in Example 1 of this invention. Lane M represents the pre-stained protein standard molecular weight; lane 1 represents the purified FAdV-11 fiber. 282-569 protein;

[0038] Figure 4 The Western blot results for the purified recombinant avian adenovirus type 11 fiber protein in Example 1 of this invention are shown below. Lane M: pre-stained protein standard molecular weight; Lane 1: E. coli BL21(DE3) / pET28a supernatant; Lane 2: E. coli BL21(DE3) / pET28a-FAdV-11 fiber. 282-569 Supernatant; Lane 3: Liver tissue homogenate carrying FAdV-11;

[0039] Figure 5 The survival rate results of chickens in each group after being inoculated with the FAdV-11 strain in different ways in Example 2 of the present invention;

[0040] Figure 6 The results of FAdV-11 load in different tissues of chickens after inoculation with FAdV-11 strains in different ways in Example 2 of the present invention;

[0041] Figure 7 The results show the survival rates of SPF chickens of different ages after inoculating them with the FAdV-11 strain in Example 2 of this invention.

[0042] Figure 8 This is the result of the FADV-11 load in the liver of SPF chickens of different ages after inoculation with the FADV-11 strain in Example 2 of the present invention;

[0043] Figure 9 The results of liver weight ratio, spleen weight ratio, and bursa of Fabricius weight ratio of SPF chickens of different ages after inoculating them with the FAdV-11 strain in Example 2 of this invention.

[0044] Figure 10 This is the survival rate result of SPF chickens in different treatment groups after being inoculated with the FAdV-11 strain in Example 2 of the present invention;

[0045] Figure 11 This is the necropsy observation result of SPF chickens in different treatment groups after being inoculated with the FAdV-11 strain in Example 2 of the present invention;

[0046] Figure 12 The above are the HE staining results of SPF chickens in different treatment groups after inoculating them with the FAdV-11 strain in Example 2 of this invention.

[0047] Figure 13 The results of FADV-11 load in the livers of SPF chickens in different treatment groups after inoculation with the FADV-11 strain in Example 2 of this invention. Detailed Implementation

[0048] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0049] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0050] Example 1: Modification of the Fiber gene of avian adenovirus type 11 and construction of prokaryotic expression engineered bacteria

[0051] 1.1 Total DNA extraction from pathogen samples and cloning of the fiber gene

[0052] Chicken liver tissue infected with avian adenovirus type 11 (FAdV-11) strain was collected and phosphate buffer (pH 7.2) was added at a mass-to-volume ratio of 1:5. The mixture was then thoroughly ground using a high-throughput cryogenic grinder (conditions: frequency 50 Hz, grinding time 90 s, grinding temperature -30 ℃). The supernatant was then collected by centrifugation at 4000 rpm for 5 min. Take 500 μL of the liver homogenate supernatant, add an equal volume of Tris-saturated phenol, and vigorously vortex until milky white. Let stand at room temperature for about 10 min. Centrifuge at 12000 rpm for 10 min at 4℃, and transfer the supernatant to a new 1.5 mL EP tube. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix well, and let stand for 5 min. Centrifuge at 12000 rpm for 10 min at 4℃, and transfer the supernatant to a new 1.5 mL EP tube. Add an equal volume of isopropanol to the supernatant and precipitate at -20℃ for 1 h. Centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, and retain the precipitate. Wash the precipitate 1-2 times with 75% ethanol, centrifuge at 12000 rpm for 10 min, dry at room temperature, dissolve in 30 μL of ddH2O, and store at -20℃. This solution is the extracted total DNA sample containing the FAdV-11 genome.

[0053] Artificial primer FAdV-11 Fiber F: 5'- CCC GGATCC ATGGCGAAATCGACTCCTTTC-3' (SEQ ID NO:5, the italicized part is the introduced protective base, and the underlined part is the introduced protective base). Bam HI restriction enzyme site); FAdV-11 Fiber R: 5'- CCC CTCGAG TTAGGGTTGTGTTAATTTGTTG-3' (SEQ ID NO:6, the italicized part is the introduced protective base, the underlined part is the introduced protective base) Xho I (the restriction enzyme site).

[0054] Take 1 μL of the above total DNA as a PCR amplification template, and use FAdV-11 Fiber F and FAdV-11 Fiber R primers to amplify the gene sequence of FAdV-11 Fiber by PCR.

[0055] The amplification product was subjected to agarose gel electrophoresis, and the approximately 1800 bp band was purified by gel extraction. The purified fragment was named FAdV-11 Fiber. This DNA fragment was subcloned into the pTOPOT vector, transformed into DH5α competent cells, and the strain correctly identified by PCR was named E. coli DH5α / pTOPOT-FAdV-11 Fiber and sent to Shanghai Bioengineering Co., Ltd. for sequencing. The nucleotide sequence of FAdV-11 Fiber is shown in SEQ ID NO:2, and the amino acid sequence of FAdV-11 Fiber is shown in SEQ ID NO:1.

[0056] 1.2 FAdV-11 Fiber Homology Modeling and Structural Analysis

[0057] The amino acid sequence of FAdV-11 Fiber was homology-modeled and analyzed using the online modeling tool SWISS-MODEL. The results are shown in Table 1.

[0058] Table 1. Results of FAdV-11 Fiber Homology Modeling

[0059]

[0060] As shown in Table 1, FAdV-11 Fiber can form a homotrimeric structure. The C-terminal region (361-569 aa) matches well with the structure of FAdV-4 Fiber2, forming the "head" of the fiber, which is an important amino acid region for immune recognition and immune response. The N-terminus region (282-355 aa, 97-161 aa and 166-225 aa, 320-359 aa) shows varying degrees of similarity to the reovirus capsid outer attachment protein sigma1, forming the "tail-stalk" of the fiber, which is embedded on the surface of the FAdV-11 virus and stabilizes the fiber structure.

[0061] 1.3 Construction and expression of FAdV-11 Fiber prokaryotic expression vector

[0062] The plasmid pTOPOT-FAdV-11 Fiber was extracted from the E. coli DH5α / pTOPOT-FAdV-11 Fiber bacterial culture obtained in step 1.1, and then... Bam HI and XhopTOPOT-FAdV-11 Fiber and pET28a were digested with restriction endonucleases, respectively. The double digestion products were recovered by electrophoresis and ligated using T4 DNA ligase. The ligation products were transformed into E. coli BL21(DE3) competent cells and screened using kanamycin plates. Single clones were picked for PCR verification, and the correctly verified strain was named E. coli BL21(DE3) / pET28a-FAdV-11 Fiber.

[0063] Thaw the *E. coli* BL21(DE3) / pET28a-FAdV-11 Fiber strain and streak the bacterial culture onto solid LB medium containing kanamycin using an inoculation loop. Incubate overnight at 37°C. Pick a single colony and incubate in 30 mL of LB medium at 37°C and 200 rpm for approximately 10 h. Add the activated seed culture at a ratio of 3% to 300 mL of LB medium containing kanamycin in a culture flask and incubate at 37°C and 200 rpm for 3 h until OD (Organic Dysplasia) occurs. 600 The value was 0.8~1.0. α-lactose was added to a final concentration of 30 mmol / L, the temperature was adjusted to 32℃, and the cells were induced and cultured at 200 rpm for 12 h. The cells were then centrifuged at 4000g for 10 min, and the cells were collected. 0.01 mol / L PBS buffer (pH 7.4) was added to the cells, and after thorough resuspending, the cells were lysed by sonication. The cells were centrifuged at 12000g at 4℃ for 10 min, and the supernatant was collected; the supernatant contained soluble protein. Protein content was determined using the BCA method and detected using SDS-PAGE. The results are as follows: Figure 1 As shown.

[0064] from Figure 1 As can be seen, the molecular weight of FAdV-11 Fiber is 60kDa. In the prokaryotic system, it is mainly expressed as inclusion bodies, which do not have the correct conformation and cannot induce the production of protective antibodies.

[0065] 1.4 N-terminally truncated FAdV-11 Fiber 282-569 Construction, Induction, Expression, and Purification of Prokaryotic Expression Vectors

[0066] As shown in section 1.3, FAdV-11 Fiber is mainly expressed as inclusion bodies in the prokaryotic system, lacking the correct conformation and failing to induce protective antibodies. Furthermore, homology modeling and structural analysis of FAdV-11 Fiber in section 1.2 revealed that the C-terminal 361-569aa of FAdV-11 Fiber can assemble into a trimer structure, forming the "head" of the fiber and serving as the core region for inducing a protective immune response. Simultaneously, the N-terminus (e.g., 282-355aa) of FAdV-11 Fiber constitutes the "tail-stalk," playing a role in anchoring and stabilizing the fiber structure.

[0067] To enable soluble expression of the immunogenic fiber protein in a prokaryotic system, the fiber protein was truncated: the 361-569aa fragment of the FAdV-11 fiber "head" structure was selected as the immunodominant fragment, the 282-355aa fragment of the FAdV-11 fiber "stalk" structure was selected as the fragment for stabilizing the trimer structure, and the 356-360aa fragment between the two fragments was selected as the linker sequence, resulting in the recombinant avian adenovirus type 11 fiber protein, the amino acid sequence of which is shown in SEQ ID NO:3, and the nucleotide sequence of which is shown in SEQ ID NO:4.

[0068] Artificial primer FAdV-11 Fiber-282 F: 5'- CCC GGATCC TCTAGCAATGGGATAGCAGTAG-3' (SEQ ID NO:7, the italicized part is the introduced protective base, and the underlined part is the introduced protective base) Bam (HI restriction site). Using pTOPOT-FAdV-11 Fiber plasmid as a PCR template, and using FAdV-11 Fiber-282 F and FAdV-11 Fiber R primers, FAdV-11 Fiber was amplified by PCR. 282-569 The gene sequence was obtained. Approximately 900 bp of DNA was amplified, recovered, and purified, and named FAdV-11 Fiber. 282-569 .

[0069] Double digestion of FAdV-11 Fiber 282-569 The gene product and pET28a plasmid were collected, and the double-digested products were recovered by electrophoresis and ligated using T4 DNA ligase. The ligation product was transformed into *E. coli* BL21(DE3) competent cells, and selection was performed using kanamycin plates. Single clones were picked for PCR verification, and the correctly verified strain was named *E. coli* BL21(DE3) / pET28a-FAdV-11 Fiber. 282-569Lactose-induced expression, bacterial sonication disruption, and SDS-PAGE analysis were performed according to the method described in step 1.3. The results are as follows: Figure 2 As shown.

[0070] from Figure 2 As can be seen, the recombinant avian adenovirus type 11 fiber protein (FAdV-11 Fiber) 282-569 It can perform efficient soluble expression.

[0071] Next, the recombinant avian adenovirus type 11 fiber protein was purified. Specifically, the product induced by the above method was added to a 30% ammonium sulfate solution and mixed rapidly. After incubation at 4°C for 2 hours, the mixture was centrifuged at 12000g for 10 minutes, and the supernatant was retained. The supernatant was then added to a 40% ammonium sulfate solution and mixed rapidly. After incubation at 4°C for 2 hours, the mixture was centrifuged at 12000g for 1 minute, and the precipitate was retained. The precipitate was resuspended by adding 0.02M pH 7.4 PB solution at a mass-to-volume ratio of 1:5 to obtain the initially purified product.

[0072] The Ni-FF chromatography column and system were equilibrated sequentially with ddH2O and 0.01M pH 7.4 PBS solution. The pre-purified product was filtered through a 0.22μm microporous membrane and loaded at a flow rate of 1mL / min. After loading, the column was incubated at 4℃ for 30min. Unbound protein was washed with 0.01M pH 7.4 PBS solution, followed by washing with 5 column volumes of washing buffer (0.01M PBS, 10mM imidazole, pH 7.4) to remove contaminating proteins. The target protein was then eluted with elution buffer (0.01M PBS, 50mM imidazole, pH 7.4) and collected to obtain purified recombinant avian adenovirus type 11 fiber protein. Protein concentration was determined using the BCA method. The column was stored at 4℃ for later use, and SDS-PAGE analysis was performed simultaneously. The results are shown below. Figure 3 As shown.

[0073] from Figure 3 As can be seen, the purified recombinant avian adenovirus type 11 fiber protein has a high purity.

[0074] Furthermore, the purified recombinant avian adenovirus type 11 (AAV11) fiber protein was detected by Western blotting. The AAV11 fiber protein was subjected to SDS-PAGE electrophoresis. After electrophoresis, the gel was removed and transferred to a membrane. Chicken anti-FAdV-11 positive serum was added at a ratio of 1:500, and the membrane was incubated at 37°C for 2 hours. After washing the NC membrane, HRP-labeled goat anti-chicken antibody was added at a ratio of 1:10000, and the membrane was incubated at 37°C for 2 hours. After washing the NC membrane, chemiluminescence staining was performed using an ECL colorimetric kit. The results are shown below. Figure 3 As shown.

[0075] from Figure 4 As can be seen, the purified recombinant avian adenovirus type 11 fiber protein and FAdV-11 strain can both be specifically recognized by positive serum.

[0076] Example 2: Immunization challenge and evaluation of recombinant avian adenovirus type 11 fiber protein subunit vaccine

[0077] 2.1 Preparation of Recombinant Avian Adenovirus Type 11 Fiber Protein Subunit Vaccine

[0078] The purified recombinant avian adenovirus type 11 fiber protein from Example 1 was filtered through a 0.22 μm microporous membrane, diluted with sterile PBS solution, and then diluted with 2% Tween-80 to prepare an aqueous phase. The aqueous phase was mixed with white oil adjuvant at a volume ratio of 1:3, and sheared at 20,000 rpm for 3 minutes, paused for 2 minutes, and repeated 3 times. A small amount of the emulsified vaccine was dropped into clean, cold water; if all drops except the first one did not immediately disperse, emulsification was complete. 2 mL of the completely emulsified vaccine was centrifuged at 3000 rpm for 15 minutes; if no stratification was observed, the vaccine stability was good. The final concentration of the prepared vaccine was 50 μg / mL.

[0079] 2.2 Establishment of the FAdV-11 strain challenge model

[0080] 2.2.1 Effects of FAdV-11 strain inoculation via different methods on 1-day-old SPF chickens

[0081] 1) Animal challenge program

[0082] The purchased SPF eggs were incubated in a dedicated incubator. Sixty one-day-old chicks that hatched successfully were randomly divided into three groups: three challenge groups (groups A-C) with 10 chicks per group, and three negative control groups (groups D-F) with 10 chicks per group. Groups A-C were challenged via intramuscular injection, 1 / 2 eye drops + 1 / 2 nasal drops, and 1 / 5 eye drops + 4 / 5 nasal drops, respectively. The challenge dose was 200 μL (10... 7 TCID 50 (each chicken) was infected with the FAdV-11 strain. Groups D through F were injected with 200 μL of PBS buffer as controls. Chickens in each group were observed for one week after challenge, their weight was measured, and their mental state was recorded. Any dead chickens were immediately necropsed. All chickens were necropsed on day 7 post-challenge. The specific challenge experiment design is shown in Table 2, and the results are as follows: Figure 5 As shown.

[0083] Table 2. Infection protocols for 1-day-old SPF chickens

[0084]

[0085] from Figure 5 As can be seen, only on the 4th day after intramuscular injection of the FAdV-11 strain did mortality begin and reach its peak, with a total of 8 chickens dying. On the 5th day, all chickens in the group died, with a survival rate of 0% (0 / 10). No chickens died in the other groups, and the survival rate was 100% (10 / 10).

[0086] 2) Absolute fluorescence quantitative PCR detection of viral load

[0087] A pair of real-time PCR primers were synthesized based on the FAdV-11 sequence. The forward primer sequence was FAdV-11 RT F1 (5'-GGTTACAGACAAGCATTCAGG-3'(SEQ ID NO:8)), and the reverse primer sequence was FAdV-11 RT R1 (5'-TCAGAGACAGACCGTTAGATGA-3'(SEQ ID NO:9)). Using the total DNA sample containing the FAdV-11 genome extracted in Example 1 as a template, a DNA fragment of approximately 150 bp was amplified using FAdV-11 RT F1 / FAdV-11 RT R1 as a template. After gel purification, the fragment was subcloned into the pTOPOT vector and transformed into DH5α cells. PCR was used to identify the correct clone, and the plasmid was extracted. Its concentration was determined using an ultra-micro spectrophotometer. This plasmid served as a standard plasmid for absolute quantification. The copy number of the extracted plasmid was calculated using the following formula: Copy number (copy / μL) = Avogadro's constant (copy / mol) × plasmid concentration (ng / μL) × 10⁻⁶ -9 / [vector fragment length (bp) + fragment size (bp)] × 660, Avogadro's constant is 6.02 × 10 23 .

[0088] The standard quality grain copy number was diluted in a 10-fold gradient to obtain 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 The standard series of copies / μL were used, with 3 replicates for each standard. The optimized reaction system (Table 3) and program were used for absolute real-time PCR. The standard curve was plotted with the corresponding Ct value as the ordinate and the logarithm of the gradient standard concentrations as the abscissa.

[0089] Table 3 Absolute Real-Time PCR Amplification Reaction System

[0090]

[0091] The reaction procedure is as follows: Step 1, pre-denaturation at 95℃ for 1 min; Step 2, denaturation at 95℃ for 10 s, annealing at 52℃ for 20 s, extension at 72℃ for 30 s, 40 cycles; Step 3, melting curve analysis, 95℃ for 10 s, 70℃-95℃, 0.5℃ / cycle.

[0092] After the challenge, the viral load in multiple organs and tissues of 1-day-old chickens that died after intramuscular injection of the FAdV-11 strain was detected using the absolute fluorescence quantitative PCR method described above. The results are as follows: Figure 6 As shown.

[0093] from Figure 6 The results show that the average viral load in the livers of all the dead chickens was 10. 10.07 Copies / mg, significantly higher than thymus (10) 6.62 Copies / mg), kidney (10) 7.30 Copies / mg) and bursa of Fabricius (10 7.06 Copies / mg) P <0.001), which was also significantly higher than that of the spleen (10 8.09 Copies / mg) and intestines (10 8.19 Copies / mg) P <0.05). The above results indicate that the liver is the main organ infected by FAdV-11, and the virus can infect most tissues and organs in chickens.

[0094] 2.2.2 Challenge test of FAdV-11 strain on SPF chickens of different ages

[0095] 1) Animal challenge program

[0096] The purchased SPF eggs were incubated in a dedicated incubator. Eighty one-day-old chicks that hatched successfully were randomly divided into four challenge groups (groups A-D) of 10 chicks each, and one negative control group of 40 chicks. Groups A-D were intramuscularly injected with the FAdV-11 strain at 1, 7, 14, and 21 days of age, at a dose of 200 μL (10... 7 TCID 50 / bird). Ten chickens were randomly selected from each negative control group at 1, 7, 14, and 21 days of age and injected with phosphate-buffered saline. Chickens in each group were observed for 7 consecutive days after challenge, their weight was measured, and their mental state was recorded. Any dead chickens were immediately necropsied. All chickens were necropsized on day 7 after challenge. The challenge experiment design is shown in Table 4, and the results are as follows: Figure 7 As shown.

[0097] Table 4. Infection protocols for SPF chickens of different ages

[0098]

[0099] from Figure 7 As can be seen, only 1-day-old SPF chickens showed lethargy, ruffled feathers, curling up and drowsiness after being injected with the FAdV-11 strain; 3 chickens died on the 5th, 6th and 7th days, with a survival rate of 10% (1 / 10) within 7 days, and the only surviving chicken also showed severe clinical symptoms; chickens in the other age groups and the negative control group did not show any deaths or clinical symptoms, and the survival rate was 100% (10 / 10).

[0100] 2) Absolute fluorescence quantitative PCR detection of viral load

[0101] After the challenge, the viral load in the liver tissue of chickens of different ages was detected using the absolute quantitative PCR method described above. The results are as follows: Figure 8 As shown.

[0102] from Figure 8 As can be seen, the viral load in the liver tissue of chickens in the four different age groups was significantly higher than that in the negative control group. P <0.001). The viral load in the liver tissue of group A (1-day-old infants challenged with the virus) was 10. 10.05 Copies / mg were significantly higher in group B (7-day-old challenge, 10 4.08 Copies / mg), Group C (14-day-old infants challenged, 10 3.01 Copies / mg), Group D (21-day-old challenge, 10 3.19 Copies / mg) and control group (10 1.64 Copies / mg) P <0.001). There was no significant difference between group C and group D. P >0.05), the viral load in the liver tissue of group B was significantly higher than that in groups C and D. P <0.01).

[0103] 3) Chicken organ index determination

[0104] In the challenge experiment, the body weight, liver, spleen, and bursa of Fabricius of the chickens were weighed after necropsy. The ratios of liver, spleen, and bursa of Fabricius to body weight were calculated, where: liver-to-body weight ratio = liver weight (g) / body weight (g) × 1000; spleen-to-body weight ratio = spleen weight (g) / body weight (g) × 1000; bursa of Fabricius-to-body weight ratio = bursa of Fabricius weight (g) / body weight (g) × 1000. The results are as follows: Figure 9 As shown.

[0105] from Figure 9 It can be seen that the liver-to-body weight ratio and spleen-to-body weight ratio of chickens in the 1-day-old and 7-day-old challenge groups were significantly higher than those in the control group. P <0.001, but the ratio of bursa of Fabricius body weight was not significantly different from the control group ( P >0.05); however, there were no significant differences in the liver weight ratio, spleen weight ratio, and bursa of Fabricius weight ratio between the 14-day-old and 21-day-old challenge groups and the control group. P >0.05), indicating swelling of the liver and kidneys in chickens when the disease occurs.

[0106] The above results indicate that intramuscular injection of the FAdV-11 strain into 1-day-old chickens can establish a good challenge model, which is beneficial for verifying the protective efficacy of the recombinant avian adenovirus type 11 fiber protein subunit vaccine.

[0107] 2.3 Evaluation of Immunization Challenge with Recombinant Avian Adenovirus Type 11 Fiber Subunit Vaccine

[0108] 1) Animal challenge program

[0109] Purchased SPF eggs were incubated in a dedicated incubator. Successfully hatched chicks were transferred to SPF-grade breeding isolators, with one rooster for every three hens. At 140 days of age, SPF hens were immunized via intramuscular injection of 50 μg per chick, followed by a booster immunization two weeks later. Eggs were collected two weeks after the booster immunization. Eggs from immunized hens were incubated (hatched chicks containing maternal antibodies against recombinant avian adenovirus type 11 fiber protein), while eggs from unimmunized hens were also incubated simultaneously (hatched chicks without maternal antibodies against recombinant avian adenovirus type 11 fiber protein), serving as a negative control.

[0110] Twenty one-day-old chicks containing maternal antibodies were randomly divided into two groups (Group A and Group B). Group A received an intramuscular injection of 200 μL of FAdV-11 strain (10... 7 TCID50 / bird), Group B was injected with PBS buffer. Twenty 1-day-old SPF chickens without maternal antibodies were randomly divided into two groups (Group C and Group D). Group C received an intramuscular injection of 200 μL (10 TCID50 / bird) of FAdV-11 strain. 7 Chickens were tested at TCID50 / bird, with group D injected with PBS buffer. Chickens in each group were observed for one week after challenge, their weight was measured, and their mental state was recorded. Any dead chickens were immediately necropsed. All chickens were necropsed on day 7 after challenge. The specific design of the challenge experiment is shown in Table 5, and the results are as follows: Figure 10 As shown.

[0111] Table 5. Challenge Test Protocol

[0112]

[0113] from Figure 10 As can be seen, the 1-day-old chicks in the challenge control group (Group C) began to die on the 4th day after challenge and reached the peak, with all of them dying within the 5th day, resulting in a survival rate of 0% (0 / 10). No deaths occurred in the maternal antibody chick challenge group (Group A), the maternal antibody chick non-challenge group (Group B), and the negative control group (Group D), with a survival rate of 100% (10 / 10).

[0114] 2) Animal necropsy and HE staining

[0115] After the challenge was completed, a necropsy was performed on the animal, and the results were as follows: Figure 11 As shown.

[0116] from Figure 11 As can be seen, in the challenge control group (Group C), the liver was yellow, swollen, hemorrhaged, and fragile, and the kidneys were yellow, swollen, with obvious granular morphology and accompanied by a small amount of bleeding. No lesions were found in the liver and kidneys of the maternal antibody chick challenge group (Group A), the maternal antibody chick non-challenge group (Group B), and the negative control group (Group D).

[0117] After the challenge, liver tissue from the experimental animals was taken for formaldehyde fixation, paraffin embedding, tissue sectioning, and HE staining. The results are as follows: Figure 12 As shown.

[0118] from Figure 12 As can be seen, the histopathological changes in the liver specimens of the challenged control group (Group C) included multifocal severe necrosis of hepatocytes and basophilic intranuclear inclusions, severe mixed inflammatory cell infiltration in the portal space, and fat droplets on hepatocytes; the hepatocytes of the maternally antibody-challenged chicks (Group A), the maternally antibody-unchallenged chicks (Group B), and the negative control group (Group D) showed good growth, normal morphology, and no pathological conditions.

[0119] 3) Absolute fluorescence quantitative PCR detection of viral load

[0120] After the challenge, the viral load in the liver tissue of the challenged chickens was detected using the absolute quantitative PCR method described above. The results are as follows: Figure 13 As shown.

[0121] from Figure 13As can be seen, there were no significant differences in the detection results among the maternal antibody chick challenge group (Group A), the maternal antibody non-challenge chick group (Group B), and the negative control group (Group D), indicating that FAdV-11 virus was not detected in the maternal antibody chick challenge group (Group A). ​​The challenge control group (Group C) showed obvious FAdV-11 virus, and the viral load was significantly higher than that in the maternal antibody chick challenge group (Group A), the maternal antibody non-challenge chick group (Group B), and the negative control group (Group D). This further indicates that the challenge model (laying hen immunization-chick hatching-chick challenge) was successful, and chicks containing maternal antibodies against recombinant avian adenovirus type 11 fiber protein have the effect of resisting FAdV-11 strain infection.

[0122] In summary, the recombinant avian adenovirus type 11 fiber protein provided by this invention not only has good immunogenicity, but also exhibits efficient and soluble expression in prokaryotic expression systems. When this recombinant avian adenovirus type 11 fiber protein is prepared into a subunit vaccine, it has good protective efficacy against chickens infected with avian adenovirus type 11.

[0123] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A recombinant avian adenovirus type 11 fiber protein, characterized in that, The amino acid sequence of the recombinant avian adenovirus type 11 fiber protein is selected from any of the following: A1) The amino acid sequence is shown in SEQ ID NO:3; A2) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1).

2. A nucleic acid molecule encoding the recombinant avian adenovirus type 11 fiber protein as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleic acid molecule is: B1) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO:

4.

4. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 2 or 3.

5. A recombinant cell, characterized in that, It comprises the nucleic acid molecule of claim 2 or 3 or the recombinant vector of claim 4.

6. A method for preparing recombinant avian adenovirus type 11 fiber protein, characterized in that, Includes the following steps: The recombinant cells described in claim 5 were cultured and induced to express the desired expression to obtain a culture. The recombinant avian adenovirus type 11 fiber protein of claim 1 was isolated from the culture.

7. A recombinant avian adenovirus type 11 fiber protein subunit vaccine, characterized in that, Includes the recombinant avian adenovirus type 11 fiber protein and adjuvant as described in claim 1.

8. A method for preparing a recombinant avian adenovirus type 11 Fiber protein subunit vaccine as described in claim 7, characterized in that, Includes the following steps: The recombinant avian adenovirus type 11 fiber protein was filtered, and a surfactant was added to obtain an aqueous phase. The aqueous phase was mixed with an adjuvant and emulsified to obtain a recombinant avian adenovirus type 11 Fiber protein subunit vaccine. The surfactant includes Tween-80, the adjuvant includes white oil adjuvant, and the volume ratio of the aqueous phase to the adjuvant is 1:(2-4). The recombinant avian adenovirus type 11 fiber protein subunit vaccine contains 40-60 μg / mL of recombinant avian adenovirus type 11 fiber protein.

9. The use of the recombinant avian adenovirus type 11 fiber protein as described in claim 1, the nucleic acid molecule as described in any one of claims 2-3, the recombinant vector as described in claim 4, the recombinant cell as described in claim 5, the recombinant avian adenovirus type 11 fiber protein prepared by the preparation method as described in claim 6, the recombinant avian adenovirus type 11 fiber protein subunit vaccine as described in claim 7, and the recombinant avian adenovirus type 11 fiber protein subunit vaccine prepared by the preparation method as described in claim 8 in the preparation of a drug for the prevention and / or treatment of avian adenovirus type 11 infection in chickens.

10. A method for verifying the efficacy of the recombinant avian adenovirus type 11 Fiber protein subunit vaccine as described in claim 7, characterized in that, Includes the following steps: Immunized hens were obtained by immunizing 140-day-old chickens with recombinant avian adenovirus type 11 fiber protein subunit vaccine; Eggs produced by the immunized hens were incubated to obtain test chickens containing maternal antibodies; at the same time, eggs produced by unimmunized hens were incubated to obtain challenge control chickens that did not contain maternal antibodies. Avian adenovirus type 11 strain was injected intramuscularly into 1-day-old experimental chickens containing maternal antibodies and 1-day-old challenged control chickens without maternal antibodies. The survival and morbidity of the experimental chickens, challenged control chickens, and negative control chickens, as well as the FADV-11 strain load in the three groups of chickens, were measured. If the experimental chickens survive without developing the disease, and the viral load of the FAdV-11 strain in the experimental chickens is less than that in the challenged control chickens and similar to that in the negative control chickens, then the recombinant avian adenovirus type 11 fiber protein subunit vaccine is effective in protecting chickens infected with the FAdV-11 strain.

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