Chicken infectious rhinitis trivalent inactivated vaccine and preparation method thereof

Abstract

The invention discloses a chicken infectious rhinitis trivalent inactivated vaccine and a preparation method thereof, and relates to the technical field of bioengineering. The avibacterium paragallinarum immunoprotective antigen protein provided by the invention is formed by fusion of an immune enhancement sequence and an immunogenic protein HMTp210 of avibacterium paragallinarum. The invention further provides a chicken infectious rhinitis subunit vaccine. Immunoactive components of the chicken infectious rhinitis subunit vaccine comprise the A type avibacterium paragallinarum immunoprotective antigen protein, the B type avibacterium paragallinarum immunoprotective antigen protein and the C type avibacterium paragallinarum immunoprotective antigen protein. The vaccine can obviously enhance the resistance of chickens to three serotypes of avibacterium paragallinarum A, B and C, the safety and the protective effect of the vaccine are superior to those of a whole-cell inactivated vaccine, and the vaccine can be used for preventing chicken infectious rhinitis caused by different serotypes of avibacterium paragallinarum.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and particularly relates to a trivalent inactivated vaccine against infectious coryza in chickens and a preparation method thereof. Background Art

[0002] Avibacterium paragallinarum (Apg) is a short Gram-negative bacterium in the family Pasteurellaceae. Its basic characteristics are non-motility, pleomorphic cells, and capsulated virulent strains. Apg mainly causes infectious coryza in chickens, a disease mainly characterized by symptoms such as eyelid swelling, nasal discharge, and decreased egg production in chickens. With the development of intensive production methods and the promulgation and implementation of regulations restricting or banning the use of antibiotics in the breeding process, infectious coryza in chickens has attracted more and more attention.

[0003] To prevent infectious coryza in chickens, inactivated vaccines have been widely used at home and abroad. The inactivated vaccine is obtained by inactivating the whole bacterial cells of A-type, B-type, and C-type Avibacterium paragallinarum with formalin, etc. The inactivated vaccine has a good effect in preventing infection of the same serotype, but lacks cross-protection for different serotypes. In production practice, the side effects of the whole-bacterium inactivated vaccine are also problems that are frequently reported by chicken farmers.

[0004] In recent years, the research on protein-based subunit vaccines has become a hot topic. Some studies have shown that a subunit vaccine based on the outer membrane protein HMTp210 of Avibacterium paragallinarum can provide protection. Abascal EN et al. isolated and identified the outer membrane protein gene HMTp210 encoding A-type Avibacterium paragallinarum, and the recombinant protein obtained by expressing part of this gene in Escherichia coli can be used as a protective antigen against infectious coryza in chickens. It can be known from phylogenetic tree analysis that the homology of the region 2 gene of the outer membrane protein gene among strains of the same serotype of Avibacterium paragallinarum is relatively high, with the homology of A-type isolates being 99.5% - 100%, that of B-type isolates being 99.2% - 100%, and that of C-type isolates being 95.5% - 100%; while the homology of the region 2 gene of the outer membrane protein gene among the three different serotypes is relatively low, with the lowest being about 55%. Therefore, in this study, the HMTp210 outer membrane proteins of A-type, B-type, and C-type Avibacterium paragallinarum were expressed separately to prepare a trivalent inactivated vaccine against Avibacterium paragallinarum, so as to provide a subunit vaccine with low side effects and good immune efficacy. Summary of the Invention

[0005] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a trivalent inactivated vaccine against infectious coryza in chickens and a preparation method thereof. The vaccine composition prepared by the preparation method of the Avibacterium paragallinarum fusion protein can effectively prevent and treat the infection of Avibacterium paragallinarum.

[0006] The present invention provides a trivalent inactivated vaccine against infectious coryza of chickens. The immunologically active components of the trivalent inactivated vaccine include immunoprotective antigen proteins of Avibacterium paragallinarum of serotype A, serotype B, and serotype C. The amino acid sequence of the immunoprotective antigen protein of serotype A Avibacterium paragallinarum is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.2. The amino acid sequence of the immunoprotective antigen protein of serotype B Avibacterium paragallinarum is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.4. The amino acid sequence of the immunoprotective antigen protein of serotype C Avibacterium paragallinarum is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.6.

[0007] Furthermore, the amino acid sequences of the immunoprotective antigen proteins of serotype A, serotype B, and serotype C Avibacterium paragallinarum are respectively formed by fusing an immune enhancing sequence with the HMTp210 sequence of the corresponding serotype of Avibacterium paragallinarum.

[0008] Furthermore, the immune enhancing sequence includes chicken granulocyte-macrophage colony-stimulating factor sequence, interleukin-2 sequence, interleukin-1 sequence, Thelper sequence, and chicken β-defensin 1 sequence. The nucleotide sequence of the Thelper sequence is shown in SEQ ID NO.1.

[0009] Furthermore, the region 2 sequence of the HMTp210 protein that constitutes the immunoprotective antigen protein of serotype C Avibacterium paragallinarum is from the DC-C strain of serotype C Avibacterium paragallinarum. The DC-C strain was deposited at the China General Microbiological Culture Collection Center on December 26, 2024, with the deposit number: CGMCC NO.32727; the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The present invention also relates to a method for preparing an immunoprotective antigen protein of Avibacterium paragallinarum. The preparation method is to respectively express the nucleotide sequences formed by fusing the region 2 sequences of the HMTp210 proteins of serotype A, serotype B, and serotype C Avibacterium paragallinarum with the enhancing sequence through secretory expression in a prokaryotic expression system, expression of soluble proteins in a prokaryotic expression system, expression of soluble proteins in a eukaryotic expression system, or expression of soluble proteins in a yeast expression system, or use artificially synthesized biologically active proteins; or the active proteins obtained after denaturation and renaturation of the formed inclusion bodies.

[0011] Furthermore, the expression vectors used for the nucleotide sequences formed by fusing the region 2 sequences of the HMTp210 proteins of Avibacterium paragallinarum with the enhancing sequence are one or more of the pET, pQE, and pGEX series, and the host cells used are Escherichia coli DH5α competent cells and Escherichia coli BL21 competent cells.

[0012] Furthermore, the sequence of the immunoprotective antigen protein of Avibacterium paragallinarum serotype A is fused with the Thelper sequence and the HMTp210 sequence amplified from the clinical isolate DC-A strain. The sequence of the immunoprotective antigen protein of Avibacterium paragallinarum serotype B is fused with the Thelper sequence and the HMTp210 sequence amplified from the clinical isolate DC-B strain. The sequence of the immunoprotective antigen protein of Avibacterium paragallinarum serotype C is fused with the Thelper sequence and the HMTp210 sequence amplified from the clinical isolate DC-C strain. The DC-C strain was deposited at the China General Microbiological Culture Collection Center on December 26, 2024, with the deposit number: CGMCC NO.32727; the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] The present invention also relates to a preparation method of the above-mentioned trivalent inactivated vaccine against infectious coryza in chickens, comprising the following steps:

[0014] S1. Respectively obtain the immunoprotective antigen protein of Avibacterium paragallinarum serotype A, the immunoprotective antigen protein of Avibacterium paragallinarum serotype B, and the immunoprotective antigen protein of Avibacterium paragallinarum serotype C by means of expression / synthesis.

[0015] S2. Mix the three obtained recombinant proteins with an adjuvant in a ratio of 1:1:1 to obtain a genetic engineering subunit vaccine composition against infectious coryza in chickens.

[0016] Furthermore, the adjuvant is an oil adjuvant, an aluminum hydroxide adjuvant, a water adjuvant, or a composite adjuvant.

[0017] Furthermore, the oil adjuvant is a white oil adjuvant.

[0018] Compared with the prior art, the improvement of the trivalent inactivated vaccine against infectious coryza in chickens and its preparation method provided by the present invention lies in constructing an immunogenic protein in the form of a fusion of an immune-enhancing sequence and the outer membrane protein of infectious coryza in chickens, which significantly enhances humoral immunity and cellular immunity. Currently, the prevalence of Avibacterium paragallinarum serotype C in clinical practice is on the rise. Existing vaccines are traditional vaccine strains, with a large sequence difference from the existing strains, and cannot provide good protection against the prevalent strains. In this study, the outer membrane protein sequence of the prevalent strain of Avibacterium paragallinarum serotype C was used for the synthesis / expression of the immunogenic protein. The vaccine prepared by expressing the protein with this sequence can provide better protection against the prevalent strain compared with the vaccine prepared by expressing the sequence of the traditional strain. The vaccine prepared in this study has good safety and absorption compared with traditional vaccines, and significantly reduces immune side reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a Gram staining and colony morphology diagram of Avibacterium paragallinarum in Example 1.

[0020] Figure 2It is the base sequence diagram of region 2 of the HMTp210 gene of Avibacterium paragallinarum strain DC-C.

[0021] Figure 3 It is the schematic diagram for the construction of recombinant plasmid.

[0022] Figure 4 It is the expression effect diagram of 3 kinds of recombinant outer membrane proteins of Avibacterium paragallinarum.

[0023] In the figure, M: protein molecular weight standard;

[0024] 1 - 2 are the precipitate and supernatant after induction of BL21-HMTpA strain; 3 - 4 are the precipitate and supernatant of the non-induced negative control;

[0025] 5 - 6 are the precipitate and supernatant after induction of BL21-HMTpB strain; 7 - 8 are the precipitate and supernatant of the non-induced negative control;

[0026] 9 - 10 are the precipitate and supernatant after induction of BL21-HMTpC strain; 11 - 12 are the precipitate and supernatant of the non-induced negative control. Detailed implementation mode

[0027] The present invention will be further described below in conjunction with the attached drawings and specific embodiments. The technical solutions in the embodiments of the present invention are clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work fall within the scope of protection of the present invention.

[0028] Example 1

[0029] 1. Collection and treatment of Avibacterium paragallinarum samples. After collecting suspected samples of infectious coryza from diseased chicken farms in Hebei, Shandong and Henan, they were isolated and identified by Beijing Dingchi Biotechnology Co., Ltd. The heads of diseased chickens were placed in a laminar flow hood, the outer surface of the infraorbital sinus was disinfected with alcohol, the infraorbital sinus was cut open with a sterile scalpel, the contents of the infraorbital sinus were picked up with a cotton swab and streaked on an Avibacterium paragallinarum agar plate. The plate was inverted and cultured at 37°C in 5% CO 2 for 24 hours, and suspected single colonies were picked for purification culture. After disinfecting the chicken head with alcohol and cutting open the infraorbital sinus, caseous secretions could be seen in the infraorbital sinus. After inoculating the caseous secretions into an Avibacterium paragallinarum agar medium, it was placed in an incubator at 37°C in 5% CO 2 and cultured for 24 hours to form dew-drop-shaped colonies with a diameter of 0.5 - 1 mm, round, slightly convex, smooth surface, grayish-white and translucent.

[0030] 2. Purification and passage of the isolates. Pick several typical colonies with strong fluorescence, streak them on the agar medium for Avibacterium paragallinarum, and place them in an incubator at 37°C with 5% CO 2 for 24 hours. Pick several typical colonies with strong fluorescence, suspend them in 1 ml of the liquid medium for Avibacterium paragallinarum, and then take 0.2 ml and inject it into the yolk sac of 6-day-old SPF chicken embryos. Incubate at 37°C continuously. If the chicken embryos die within 30 hours, harvest the infected yolk fluid, which is the purified strain, denoted as the E0 generation. A total of 3 suspected Avibacterium paragallinarum strains were isolated from the infraorbital sinuses, and the isolated bacteria were further identified.

[0031] 3. Morphological, biochemical and cultural characteristics of the E0 generation of the isolates. The isolated bacteria do not grow on ordinary agar medium and can only grow on the agar medium for Avibacterium paragallinarum containing chicken serum and NADH. After incubation at 37°C with 5% CO 2 for 24 hours, colonies with a diameter of 0.5 - 1 mm, round, slightly convex, smooth surface, grayish-white, translucent and dewdrop-like can be formed, as Figure 1 shown. The pure culture of the isolated strain was Gram-stained and examined under the microscope as Gram-negative short bacilli, and there were also a small number of long filamentous bacilli, showing obvious pleomorphism. It does not grow on ordinary broth and ordinary agar plates. In the liquid medium for Avibacterium paragallinarum containing chicken serum and NADH, after incubation at 37°C for 24 hours, it shows a slightly uniform turbidity. On the plate cross-inoculated with Staphylococcus aureus, satellite phenomenon is formed around the staphylococcal lawn, indicating that the isolated strain requires V factor for growth.

[0032] 4. Physiological and biochemical characteristics. Perform biochemical tests on the 3 isolated suspected Avibacterium paragallinarum strains. The strains can reduce nitrate to nitrite, ferment glucose but do not produce gas, have oxidase activity, alkaline phosphatase is present, do not produce indole, do not hydrolyze urea or do not liquefy gelatin. The results are shown in Table 1.

[0033] Table 1 Biochemical tests of the isolated strains

[0034]

[0035] 5. Serotype identification. Use the hemagglutination inhibition test to identify the serotypes of the isolated strains. The 3 strains are serotype A, B and C respectively, and are named DC-A, DC-B and DC-C respectively. The results are shown in Table 2.

[0036] Table 2 Results of strain typing identification

[0037]

[0038]

[0039] Note: “-” indicates complete inhibition of agglutination, which means that the red blood cells are spread flat on the bottom of the well or there are red blood cells concentrated in the shape of dots in the center of the bottom of the well; “+” indicates complete or partial agglutination of red blood cells, which means that the red blood cells are spread flat on the bottom of the well or there are red blood cells concentrated in the shape of dots in the center of the bottom of the well, with red blood cells spread flat on the bottom of the well around them.

[0040] 6. The outer membrane protein HMTp210 of A. paragallinarum is the main immunogenic protein of A. paragallinarum. Primers were designed to amplify the region 2 of the HMTp210 gene of the A. paragallinarum DC-C strain. By comparing the DC-C strain with the C-type classical strain, it was found that the variant region of the HMTp210 gene of the newly isolated DC-C strain had base insertions in multiple places, with the longest insertion fragment being 138 bases. This proves that the isolated strain is very different from the classical strain in base sequence, protein amino acid sequence and immunogenicity, and is a new variant strain. The results are as follows Figure 2 The isolated Avibacterium paragallinarum DC-C strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit date being December 26, 2024, and the deposit number being CGMCC No.32727.

[0041] 7. Animal regression test. After the original strain was revived and inoculated into the A. paragallinarum agar medium, a single colony was picked and inoculated into the A. paragallinarum liquid medium to prepare a bacterial suspension as a sample. The above sample was appropriately diluted to about 2.5×10 7 CFU / ml, the infraorbital sinus of 10 60-day-old SPF chickens were inoculated (0.2ml / bird) and clinical observation was carried out every day after inoculation. After 7 days of observation, all the infected chickens showed symptoms of the disease, with clear to serous viscous secretions flowing out of the nostrils; or swelling of the eyelids (infraorbital sinus).

[0042] Example 2 Construction of Avibacterium paragallinarum recombinant plasmid

[0043] 1. Primer design Based on the gene sequences of the hypervariable regions of the outer membrane protein HMTp210 of three representative isolates of Avibacterium paragallinarum, specific primers were designed in the conserved regions upstream and downstream of the hypervariable regions in this experiment to amplify the genes covering the hypervariable regions. Primers HMT-EcoRI-F and HMT-NotI-R were designed to amplify the gene sequences of serotype A, B, and C of Avibacterium paragallinarum fused with the enhancer sequence Thelper, and the lengths of the amplified fragments were 1857 bp, 1842 bp, and 1980 bp, respectively. The nucleotide sequence of the enhancer sequence Thelper is as shown in SEQ ID NO.1, the base sequence of the immunoprotective antigen protein of serotype A Avibacterium paragallinarum is as shown in SEQ ID NO.2, the base sequence of the immunoprotective antigen protein of serotype B Avibacterium paragallinarum is as shown in SEQ ID NO.4, and the base sequence of the immunoprotective antigen protein of serotype C Avibacterium paragallinarum is as shown in SEQ ID NO.6, all of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd. EcoRI and NotI restriction enzyme sites (underlined) were introduced into the upstream and downstream primers respectively, and the primer sequences are as follows:

[0044] Table 3 PCR primers for hypervariable region region 2

[0045]

[0046] PCR reaction procedure: After reacting at 98°C for 1 minute, 30 cycles of "denaturation (95°C, 10 seconds), annealing (56°C, 30 seconds), and extension reaction (72°C, 120 seconds)" were carried out, and then the repair extension reaction (72°C, 7 minutes) was performed.

[0047] 2. Construction of recombinant expression plasmids The above PCR products were digested with EcoRI and NotI. The enzyme digestion system of the PCR product fragment (50 μl): The above system was placed in a 37°C constant temperature water bath for 2 hours. After separating all the enzyme digestion products by 0.8% agarose gel electrophoresis, the common DNA gel recovery kit of Beijing Tiangen Biochemical Technology Co., Ltd. was used to recover the DNA fragments according to the steps of the kit instructions. The recovered target DNA fragments can be used immediately or stored at -15°C for later use. The expression vector pET28a was digested with EcoRI and NotI and the enzyme digestion products were recovered. The recovered and purified target DNA fragments and the recovered and purified expression vector pET28a were ligated to obtain recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli DH5α competent cells, and single colonies grown on the plate were picked and inoculated into LB liquid medium containing 50 μg / ml kanamycin sulfate respectively for the identification of recombinant plasmids. And the positive recombinant plasmids were extracted using a common plasmid extraction kit.

[0048] Example 3 Construction of recombinant expression strains

[0049] 1. Transformation of Recombinant Plasmids into E. coli BL-21 Competent Cells The extracted plasmids were separately transformed into E. coli BL21(DE3) competent cells. The method was the same as that for transforming E. coli DH5α competent cells. After transformation, single colonies grown on the plate were picked and identified as positive colonies using the T7 primer: 5’-TAATACGACTCACTATAGGG-3’ and the T7t primer: 5’-TGCTAGTTATTGCTCAGCGG-3’. The engineered bacteria identified could amplify bands of approximately 2130 bp, 2115 bp, and 2253 bp respectively, which were consistent with the target band sizes. The PCR products were separately sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing, and the sequencing results were consistent with the target sequences.

[0050] 2. Enzyme Digestion Identification of Recombinant Plasmids Single colonies grown on the plate were separately inoculated into LB liquid medium containing 50 μg / ml kanamycin sulfate. The bacterial cells were collected, and recombinant plasmids were extracted using a common plasmid extraction kit. The specific operation steps were carried out according to the kit instructions. The recombinant plasmids were identified by digestion with EcoRI and NotI. After digestion, foreign fragments of the expected sizes of 1841 bp, 1826 bp, and 1964 bp and a vector fragment of 5369 bp appeared, indicating correct recombinant plasmids. The schematic diagram of the constructed expression sequence is as Figure 3 shown.

[0051] 3. Purification and Propagation of Strains The identified correct strains were inoculated on LB solid medium containing 50 μg / ml kanamycin sulfate and cultured for 16 - 24 hours. Single colonies were picked and inoculated on a new LB solid medium containing 50 μg / ml kanamycin sulfate, and purified for three generations according to the above method. The obtained monoclonal strains were propagated in LB liquid medium containing 50 μg / ml kanamycin sulfate, and the propagated strains were designated as the E0 generation. 100 tubes of each strain were cryopreserved and stored at -70°C or below.

[0052] 4. Prokaryotic Expression of Outer Membrane Proteins The positive clones were separately inoculated into 5 ml of LB liquid medium containing 50 μg / ml kanamycin sulfate and cultured with shaking at 37°C. 1 ml of the cultured bacterial liquid was taken and inoculated into 100 ml of fresh LB liquid medium containing 50 μg / ml kanamycin sulfate, and cultured with shaking at 37°C for about 4 hours until OD 600When it reaches 0.6 - 1.0, add IPTG to a final concentration of 0.5 mmol / L, continue culturing for 5 hours, then collect the bacterial cells. Resuspend the bacterial cells with 50 mmol / L Tris-HCl solution, disrupt them twice under 800 bar high pressure, and analyze the expression of the target protein by SDS-PAGE. After SDS-PAGE detection, protein target bands appeared at the expected positions of 69 kD, 67 kD, and 71 kD respectively, and the expression results showed that the recombinant Escherichia coli BL21-HMTpA existed in the form of inclusion bodies; most of the expressed proteins of BL21-HMTpB and BL21-HMTpC strains were soluble proteins, and a small part existed in the inclusion bodies. The results are as Figure 4 shown.

[0053] 5. Western Blot Detection of Recombinant Antigen Proteins Western Blot detection was performed on the recombinant antigen proteins HMTpA, HMTpB, and HMTpC respectively with the positive sera of Avibacterium paragallinarum of serotype A, serotype B, and serotype C. The recombinant antigen proteins HMTpA, HMTpB, and HMTpC could react with the positive chicken sera of Avibacterium paragallinarum of serotype A, serotype B, and serotype C respectively, which was consistent with the expected results.

[0054] Example 4 Preparation of Trivalent Inactivated Vaccine against Infectious Coryza in Chickens

[0055] 1. Preparation of Vaccine-Used Proteins The BL21-HMTpA strain, BL21-HMTpB strain, and BL21-HMTpC strain were respectively subjected to protein expression, bacterial cell disruption, protein extraction, and filtration sterilization according to the method of Example 3 to obtain the recombinant antigen proteins HMTpA, HMTpB, and HMTpC respectively. The obtained proteins were quantitatively analyzed by gray scale analysis, and the protein content was not less than 1 g / L, the endotoxin content was less than 20,000 EU / ml, and no bacteria grew in the sterility test of the three proteins.

[0056] 2. Preparation of Aqueous Phase Mix the recombinant antigen proteins HMTpA, HMTpB, and HMTpC in a ratio of 1:1:1 so that the antigen content in the vaccine is 50 μg per dose. Take 96 doses of the mixed antigen solution, add 4 doses of sterilized Tween-80, and mix well until Tween-80 is completely dissolved.

[0057] 3. Preparation of Oil Phase Take 94 parts of white oil for injection, add 2 parts of aluminum stearate, and stir while adding until it is completely transparent, then add 6 parts of Span-80, mix well, and sterilize for standby.

[0058] 4. The emulsified aqueous phase and oil phase are emulsified at a volume ratio of 1:2. The emulsification steps are as follows: First, the oil phase is input into the emulsification tank and stirred, then the aqueous phase is slowly added, and stirring is continued to fully mix the oil phase and the aqueous phase. Then, in-line emulsification is carried out in the emulsification tank. After emulsification, the dosage form and stability are detected.

[0059] 5. Final inspection of the emulsified vaccine The emulsified vaccine is a white emulsion, and the dosage form is water-in-oil type. 10 ml of the vaccine is drawn and added to a centrifuge tube, and centrifuged at 3000 r / min for 15 minutes. It is observed that no aqueous phase precipitates at the bottom of the tube. After inspection, no bacteria grow.

[0060] Example 5 Immunogenicity analysis of recombinant outer membrane protein of Avibacterium paragallinarum

[0061] Forty-two-day-old chickens were immunized with the trivalent inactivated vaccine (type A + type B + type C) against infectious coryza prepared in the laboratory at a dose of 0.5 ml per chicken. Thirty days after immunization, chickens in the immunized group and the control group were challenged with type A DCA03 strain, DCA04 strain, type B DCB02 strain, DCB03 strain, type C DCC04 strain, and DCC05 strain isolated clinically. The results showed that the protection ratios of the trivalent vaccine against different isolates of type A, type B, and type C Avibacterium paragallinarum were all 10 / 10, indicating that the vaccine product had good cross-immunoprotective effects against isolates of different serotypes of Avibacterium paragallinarum.

[0062] Table 4 Results of challenge protection tests with different isolates

[0063]

[0064]

[0065] Example 6 Comparative study with similar products

[0066] 1. Safety comparison test between the "trivalent vaccine" and the "commercial vaccine" Twenty 21-day-old SPF chickens were randomly divided into 2 groups, with 10 chickens in each group. The "trivalent vaccine" and the "commercial vaccine" were each subcutaneously injected into the necks of 1 group of 21-day-old SPF chickens at a dose of 1.0 ml per chicken, and another 10 chickens were not immunized as a control. After observing for 14 days, the mental state, food intake, water intake, respiration, feathers, feces, movement, etc. of the test chickens and the control chickens of the "trivalent vaccine" were all normal; no local reactions such as swelling, ulceration, nodules, and granulomas caused by the vaccine appeared at the injection site. Granulomas and a large amount of vaccine residues were found during the autopsy of the immunized group of the "commercial vaccine". The results showed that the "trivalent vaccine" prepared from recombinant protein of Avibacterium paragallinarum had high safety, good vaccine absorption effect, and better safety than the whole-bacterium commercial vaccine. The results are shown in Table 5.

[0067] Table 5 Safety comparison test between the "trivalent vaccine" and the "commercial vaccine"

[0068]

[0069] Note: " / " indicates not performed; *: "Normal at the injection site" means that no local reactions caused by the vaccine such as swelling, ulcer, nodule, granuloma, etc. occurred at the injection site. "√" indicates normal.

[0070] 2. Comparative test on the efficacy of "trivalent vaccine" and "commercial vaccine": 60 42-day-old SPF chickens were randomly divided into 2 groups, with 30 chickens in each group. Each group was subcutaneously inoculated with "trivalent vaccine" and "commercial vaccine", 0.5 ml per chicken, and another 30 chickens were not inoculated as a control. 30 days after inoculation, 10 immunized chickens and 10 control chickens were taken, and 0.2 ml of DCA03 strain bacterial solution was injected into the infraorbital sinus of each chicken. Another 10 immunized chickens and 10 control chickens were taken, and 0.2 ml of DCB02 strain bacterial solution was injected into the infraorbital sinus of each chicken. Another 10 immunized chickens and 10 control chickens were taken, and 0.2 ml of DCC05 strain bacterial solution was injected into the infraorbital sinus of each chicken. The morbidity and protection were counted 7 days after challenge, and the challenge protection ratio was analyzed. The results showed that after challenge of the immunized groups of "trivalent vaccine" and "commercial vaccine", the challenge protection ratios of the A-type strain were 10 / 10 and 9 / 10 respectively, the challenge protection ratios of the B-type strain were 10 / 10 and 7 / 10 respectively, and the challenge protection ratios of the C-type strain were 10 / 10 and 5 / 10 respectively. 9 / 10 - 10 / 10 of the control chickens got sick after challenge. The results indicated that the "trivalent vaccine" prepared from recombinant protein of Avibacterium paragallinarum had better immune effect than the whole-bacterium commercial vaccine. The results are shown in Table 6.

[0071] Table 6 Comparative test on the efficacy of "trivalent vaccine" and "commercial vaccine"

[0072]

[0073] Compared with the vaccines on the market, the subunit vaccine prepared from Avibacterium paragallinarum has the following advantages: First, the protection rate of the protein subunit vaccine is better than that of the whole-bacterium trivalent inactivated vaccine and has a wide protection spectrum. Second, since the purified protein does not contain components such as lipopolysaccharide carried by the whole bacterium, the subunit vaccine prepared by the present invention has no side reactions, while the whole-bacterium inactivated vaccine will have side reactions such as granuloma and poor vaccine absorption. Third, in terms of preparation cost, the subunit vaccine is also lower than the conventional inactivated bacterin due to the characteristics of easy culture induction, cheap culture medium, and easy purification. Therefore, the Avibacterium paragallinarum subunit vaccine prepared in this study has good application prospects.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A trivalent inactivated vaccine for avian infectious coryza, characterized in that: The immunologically active components of the trivalent inactivated vaccine include A, B and C types of Avibacterium paragallinarum immune protective antigen proteins, wherein the amino acid sequence of the A type A Avibacterium paragallinarum immune protective antigen protein is shown in SEQ ID NO.3, and its base sequence is shown in SEQ ID NO.2, the amino acid sequence of the B type Avibacterium paragallinarum immune protective antigen protein is shown in SEQ ID NO.5, and its base sequence is shown in SEQ ID NO.4, and the amino acid sequence of the C type Avibacterium paragallinarum immune protective antigen protein is shown in SEQ ID NO.7, and its base sequence is shown in SEQID NO.

6.

2. A trivalent inactivated vaccine for avian infectious coryza according to claim 1, characterized in that: The amino acid sequences of the A, B and C types of A. paragallinarum immune protective antigen proteins are respectively formed by fusion of the immune enhancement sequence and the HMTp210 sequence of the corresponding type of A. paragallinarum.

3. A trivalent inactivated vaccine for chicken infectious coryza according to claim 2, characterized in that: The immune enhancement sequence includes a chicken granulocyte-macrophage colony stimulating factor sequence, an interleukin-2 sequence, an interleukin-1 sequence, a Thelper sequence and a chicken beta defensin 1 sequence, and the nucleotide sequence of the Thelper sequence is shown in SEQ ID NO.

1.

4. A trivalent inactivated vaccine for infectious coryza in chickens according to claim 3, characterized in that: The region 2 sequence of the HMTp210 protein that constitutes the immune protective antigen protein of Avibacterium paragallinarum type C comes from the isolated DC-C strain of Avibacterium paragallinarum type C. The DC-C strain was deposited in the China General Microbiological Culture Management Center on December 26, 2024, with the deposit number: CGMCCNO.32727; the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

5. A method for preparing an immunoprotective antigen protein of Avibacterium paragallinarum, characterized in that: The preparation method comprises the following steps of respectively expressing the HMTp210 protein region 2 sequence of type A, type B and type C Avibacterium paragallinarum separated and the nucleotide sequence formed by fusion with the enhancement sequence through secretory expression of a prokaryotic expression system, expressing soluble protein through a prokaryotic expression system, expressing soluble protein through a eukaryotic expression system or expressing soluble protein through a yeast expression system, or using artificially synthesized biologically active protein; or expressing the active protein after denaturation and renaturation of the formed inclusion body.

6. The method for preparing an Avibacterium paragallinarum immunoprotective antigen protein according to claim 5, characterized in that: The expression vector used for the HMTp210 protein region 2 sequence of Avibacterium paragallinarum and the nucleotide sequence fused with the enhancement sequence is one or more of the pET, pQE and pGEX series, and the host cells used are Escherichia coli DH5α competent cells and Escherichia coli BL21 competent cells.

7. The method for preparing an Avibacterium paragallinarum immunoprotective antigen protein according to claim 6, characterized in that: The sequence of the immune protective antigen protein of A. paragallinarum type A is fused with the Thelper sequence and the HMTp210 sequence obtained by amplifying the clinical isolate DC-A strain, the sequence of the immune protective antigen protein of A. paragallinarum type B is fused with the Thelper sequence and the HMTp210 sequence obtained by amplifying the clinical isolate DC-B strain, and the sequence of the immune protective antigen protein of A. paragallinarum type C is fused with the Thelper sequence and the HMTp210 sequence obtained by amplifying the clinical isolate DC-C strain. The DC-C strain was deposited in the China General Microbiological Culture Management Center on December 26, 2024, with the deposit number: CGMCC NO.32727; the deposit address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

8. A method for preparing the trivalent inactivated vaccine for infectious coryza in chickens as claimed in claim 4, characterized in that: The steps include: S1. obtaining A. paragallinarum type A immune protective antigen protein, A. paragallinarum type B immune protective antigen protein, and A. paragallinarum type C immune protective antigen protein by expression / synthesis respectively; S2. The three obtained recombinant proteins are mixed with an adjuvant in a ratio of 1:1:1 to obtain a genetically engineered subunit vaccine composition for infectious coryza.

9. The method for preparing the trivalent inactivated vaccine for infectious coryza in chickens according to claim 8, characterized in that: The adjuvants are oil adjuvants, aluminum gel adjuvants, water adjuvants and composite adjuvants.

10. The method for preparing the trivalent inactivated vaccine for infectious coryza in chickens according to claim 9, characterized in that: The oil adjuvant is a white oil adjuvant.

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