CN117838890A

CN117838890A - C-type avibacterium paragallinarum subunit vaccine and application thereof

Info

Publication number: CN117838890A
Application number: CN202311816011.2A
Authority: CN
Inventors: 欧阳征亮; 高任; 李飞跃; 黄文彬; 顾嘉运; 邓智昕; 陈瑞爱; 吴达兴; 何恒昌; 高翔; 孙银焕; 梁铭治
Original assignee: Zhaoqing Dahuanong Biological Pharmaceutical Co Ltd
Current assignee: Zhaoqing Dahuanong Biological Pharmaceutical Co Ltd
Priority date: 2023-12-26
Filing date: 2023-12-26
Publication date: 2024-04-09

Abstract

The invention belongs to the technical field of biology, discloses a C-type avibacterium paragallinarum subunit vaccine and application thereof, and particularly discloses application of an HMTp210 gene in preparation of products for preventing and/or treating related diseases infected by avibacterium paragallinarum. The invention discovers that the serous type C parachicken bacillus is mainly divided into 2 groups in the aspect of molecular biology for the first time, wherein 138bp HMTp210 gene deletion mainly exists. The constructed recombinant engineering bacteria with or without 138bp HMTp210 gene are used for preparing corresponding subunit vaccines and carrying out a virus attack experiment, and the result shows that the engineering bacteria with 138bp HMTp210 gene mainly deleted are prepared for obtaining the vaccine with low antigen content and high antigen content and have good protection effect on cross immunity of different strains in serum C type.

Description

C-type avibacterium paragallinarum subunit vaccine and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a C-type avibacterium paragallinarum subunit vaccine and application thereof.

Background

Infectious rhinitis (Infectious coryza, IC) is a bacterial disease of the respiratory tract of chickens caused by infection of avibacterium paragallinarum (Apg), which is a worldwide epidemic and can occur all the year round, especially in spring and autumn, and is characterized by swelling of the face, inflammation of the nasal cavity, unsmooth breathing, and the like, which are accompanied with the symptoms of egg drop. In 1920, beach reported the disease first, and in 1932 De Blieck isolated Haemophilus paragallinarum for the first time. The Page method and the Kume method are the most commonly used serotyping methods for avian paragallinarum, wherein the Page method classifies Apg into A, B and C three serotypes by plate agglutination assay, wherein the Kume rule is to divide avian paragallinarum into serogroups A, B and C by hemagglutination inhibition assay (HI), serogroup A comprising serosubtypes A-1, A-2, A-3, A-4 (corresponding to Page serotype A); serogroup B includes serosubtype B-1 (corresponding Page serogroup B); serogroup C includes serosubtypes C-1, C-2, C-3, and C-4 (corresponding to Page serogroup C), and studies indicate that there is no cross-protection between the three serotypes, and that cross-protection within the different serotypes is also different, with good cross-protection between serotype A isolates, partial cross-protection between serotype B isolates, and low cross-protection between serotype C isolates.

Hemagglutinin (HA) is a virulence factor of most pathogenic microorganisms, and is capable of specifically binding to receptors on mammalian or avian erythrocytes, helping the pathogenic microorganisms to adhere to host cells and causing hemagglutination to occur. Three hemagglutinin proteins are found on the surface of the strain of avian paragallinarum, but only HA-L HAs immunogenicity and serotype specificity, and is the most suitable antigen component for developing subunit vaccines. Noro (2008) researches HA epitopes of type A and type C avibacterium paragallinarum, and discovers that a gene of a serum type A avibacterium paragallinarum coding HA epitope is positioned on Hpa5.1 gene segment, and the expressed protein is about 180ku; the gene encoding HA epitope of serotype C is located mainly on the Hpc5.5 gene fragment and expresses a protein of approximately 196ku. After the recombinant expression protein is immunized on chicken flock, the recombinant protein can generate better protection effect on bacterial strains with the same serotype.

With the continuous and intensive research on the avian secondary bacillus hemagglutinin protein, a high variable region exists in the hemagglutinin protein Hmtp210 gene sequence of the A-type and C-type avian secondary bacillus, the high variable region shows serum specificity, the antigenicity of the expressed protein of the variable region is best, the infection capacity of the avian secondary bacillus with the serum type can be obviously reduced after the recombinant expressed protein of the high variable region is used for immunizing chickens, and the protection rate is as high as 83% -100%. Research shows that the constructed avian bacterial strain mutant for knocking out Hmtp gene has no hemagglutination activity and can not induce organism to generate specific antibody, westernblot detection shows that the immunogenicity of the mutant is superior to other areas of hemagglutinin protein, and specific serotypes exist, and the homology of the mutant is up to 99.8% through comparing the gene sequences of high variable regions of avian bacterial strain Hmtp with the serotypes.

In recent years, the occurrence rate of infectious rhinitis of laying hens and breeding hens is obviously increased, the occurrence rate of non-immunized chicken flocks and immunized chicken flocks is mainly related to the non-correspondence of the serosubtype of the vaccine at the present stage, and the development of the infectious rhinitis vaccine for chickens with wide immune protection spectrum and good safety is needed.

Disclosure of Invention

The object of the first aspect of the present invention is to provide the use of the HMTp210 gene for the preparation of a product for the prevention and/or treatment of diseases associated with infection by avibacterium paragallinarum.

The object of the second aspect of the present invention is to provide an HMTp210 protein.

It is an object of a third aspect of the present invention to provide a nucleic acid molecule encoding the HMTp210 protein of the second aspect of the invention.

The object of the fourth aspect of the present invention is to provide a biological material which is identical to the nucleic acid molecule of the third aspect of the present invention.

The object of the fifth aspect of the present invention is to provide the use of the HMTp210 protein of the second aspect of the present invention, the nucleic acid molecule of the third aspect of the present invention and/or the biological material of the fourth aspect of the present invention in the preparation of a vaccine for infectious rhinitis in chickens.

The sixth aspect of the invention aims to provide a vaccine for infectious rhinitis of chicken.

The seventh aspect of the invention aims to provide a method for improving the cross immune response of a vaccine to avian bacillus paragallinarum type C.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides application of an HMTp210 gene in preparing a product for preventing and/or treating diseases related to infection by avian paragallinarum, wherein the HMTp210 gene is an HMTp210 gene with a part of gene fragments deleted, and the nucleotide sequence of the part of gene fragments is shown as SEQ ID NO. 3.

In some embodiments of the invention, the nucleotide sequence of the HMTp210 gene is shown in SEQ ID NO. 9, or a sequence which has more than 95% identity and the same or similar biological activity as the HMTp210 gene.

In some embodiments of the invention, the products include vaccines and pharmaceuticals.

In a second aspect of the invention, an HMTp210 protein is provided, and the amino acid sequence of the protein is shown as SEQ ID NO. 13.

In a third aspect of the invention there is provided a nucleic acid molecule encoding the HMTp210 protein of the second aspect of the invention.

In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 9.

In a fourth aspect of the present invention, there is provided a biological material related to the nucleic acid molecule of the third aspect of the present invention, the biological material being any one of (1) to (3);

(1) An expression cassette comprising a nucleic acid molecule of the third aspect of the invention;

(2) A recombinant vector comprising the nucleic acid molecule of the third aspect of the invention or the expression cassette of (1);

(3) A recombinant cell comprising a nucleic acid molecule of the third aspect of the invention, the expression cassette of (1) or the recombinant vector of (2).

In some embodiments of the invention, the cell is E.coli.

In some embodiments of the invention, the recombinant vector is a plasmid vector, a viral vector, or a cellular vector.

In some embodiments of the invention, the plasmid vector may be an optional plasmid, the viral vector may be an optional virus, and the cellular vector does not include propagation material.

In a fifth aspect, the invention provides the use of the HMTp210 protein of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention and/or the biological material of the fourth aspect of the invention in the preparation of a vaccine for infectious rhinitis in chickens.

In some embodiments of the invention, the avian infectious rhinitis vaccine is for preventing avian infectious rhinitis caused by avian bacillus paragallinarum.

In some embodiments of the invention, the chicken infectious rhinitis vaccine is a subunit vaccine or an inactivated vaccine.

In a sixth aspect of the invention there is provided a vaccine for infectious rhinitis in chickens comprising the HMTp210 protein of the second aspect of the invention, the nucleic acid molecule of the third aspect of the invention and/or the biomaterial of the fourth aspect of the invention.

In some embodiments of the invention, the chicken infectious rhinitis vaccine further comprises an adjuvant.

In some embodiments of the present invention, when the infectious rhinitis vaccine is a subunit vaccine, the vaccine is prepared by cloning the HMTp210 gene of the first aspect of the present invention into a vector, transforming a host bacterium, fermenting, inducing, purifying, emulsifying, and the like.

In some embodiments of the invention, the vector is a PET28b plasmid.

In some embodiments of the invention, the host bacterium is E.coli.

In a seventh aspect of the invention, there is provided a method of enhancing the cross-immune response of a vaccine to avibacterium paragallinarum type C, comprising the steps of: knocking out or silencing part of gene fragments in Hmtp210 gene sequences of the C-type avian parachicken bacillus, wherein the nucleotide sequences of the part of gene fragments are shown as SEQ ID NO. 3; or (b)

Prokaryotic expression of Hmtp210 protein of the second aspect of the invention.

In some embodiments of the invention, the vaccine is an avibacterium paragallinarum vaccine.

The beneficial effects of the invention are as follows:

the invention discovers that the C-type avibacterium paragallinarum is mainly divided into 2 groups in the aspect of molecular biology for the first time, wherein 138bp HMTp210 gene deletion mainly exists. The constructed recombinant engineering bacteria with or without 138bp HMTp210 gene are used for preparing corresponding subunit vaccines and carrying out a virus attack experiment, and the result shows that the engineering bacteria with 138bp HMTp210 gene deletion are used for preparing vaccines with low antigen content and high antigen content and have good protection effect on cross immunity of different strains in serum C type. The engineering bacteria without the segment have higher protection effect on the virus attack of the strain, and have poorer cross immunity effect on other strains, which suggests that the HMTp210 gene of the avian infectious bursal disease virus serotype C is deleted by a 138bp sequence shown as SEQ ID NO. 3, and the cross immunity effect on different strains in the serotype C can be effectively improved.

Drawings

FIG. 1 shows the sequence alignment of HMTp210 gene of type C isolate.

FIG. 2 shows the expression of HMTp210 protein of engineering bacteria ICC-C-L (left) and ICC-C-S (right).

FIG. 3 shows the amino acid sequence comparison results of HMTp210 proteins of ICC-C-L and ICC-C-S of engineering bacteria.

Detailed Description

The following describes the present invention in further detail by way of specific examples.

It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The subunit vaccine of the A serotype and the B serotype prepared in advance has ideal protection effect on different strains in the same serotype, and the subunit vaccine prepared by Hmtp210 genes of different strains of the C serotype has different cross immunity effects on different strains of the C serotype. Further according to 18 serotype C clinical isolates isolated in a laboratory where the inventor is located, PCR amplification (primer ICC-general-F: 5'-CCATTAACGGCTCACAGCTTTATGC-3' (SEQ ID NO: 1)) is carried out on the HMTp210 gene hypervariable region sequences (about 1800 bp) of 16 strains, ICC-general-R: 5'-CTCAATAGAAATATTTGGCTGTAATG-3' (SEQ ID NO: 2)) and sequence comparison is carried out, and the serotype C clinical isolates are mainly divided into 2 groups at home and abroad in terms of molecular biology, wherein the genotype 1 group mainly has a deletion of 138bp continuously, and the genotype 2 group does not have a deletion of 138bp (FIG. 1). The nucleotide sequence of the 138bp gene fragment which is mainly deleted is as follows: AAGTTACCGGCGAAAAGTCCAGCAACACCACCGGCAACTCTTTTACGATTGGCCT GGATGATGCGACCCTGAACAAAATTAACAACGCCGTGAACCAGGATCTGTCTAATC TGGGCGAAGACGGAAAGAACGCCATTA (SEQ ID NO: 3). In order to verify whether the deletion of 138bp, which is mainly deleted in the HMTp210 gene, affects the cross-immunity effect of different strains in the serotype C, the inventor respectively constructs recombinant genetic engineering bacteria ICC-C-S and ICC-C-L. Wherein, the recombinant genetic engineering bacteria ICC-C-L is obtained by constructing the HMTp210 gene of the conventional serum C type avibacterium paragallinarum, and ICC-C-S is obtained by constructing the HMTp210 gene of the serum C type avibacterium paragallinarum with 138bp deletion.

The specific process is as follows:

(1) HMTp210 genome extraction

(1) 100mL of the bacteria overnight culture was centrifuged at 5000rpm for 10 minutes, the supernatant was removed, the pellet after centrifugation was transferred to a 50mL centrifuge tube of 10-fold volume of extraction buffer, and the tissue powder was mixed uniformly with gentle agitation using a glass rod and incubated for 1h in a 37℃water bath.

(2) Proteinase K was added to the centrifuge tube to a final concentration of 100. Mu.g/mL, then mixed well with gentle agitation using a glass rod, and placed in a 50℃water bath for 3h, with gentle agitation of the solution from time to time.

(3) After the solution was slowly cooled to room temperature, it was dispensed into 1.5mL centrifuge tubes, and an equal volume of Tris-Cl (pH 8.0) equilibrated phenol (0.5M) was added, and the tubes were slowly inverted for about 10min to form an emulsion, which was centrifuged at 12000rpm for 15min at room temperature.

(4) Cutting off the gun head to make its outlet diameter about 0.3cm, using said gun head to transfer the viscous water phase into clean centrifugal tube, then repeating 3-4 steps for 2-3 times.

(5) With the same volume of chloroform: isoamyl alcohol (volume ratio 24:1) was extracted once, then centrifuged at 12000rpm for 15min at room temperature.

(6) Transferring the upper aqueous phase to clean1.5mL centrifuge tube of (C), while adding 0.2 volumes of 10M NH ₄ Ac, after mixing them uniformly, adding 2 volumes of anhydrous ethanol to start precooling, mixing the solution sufficiently uniformly, at this time, DNA flocculent precipitate was seen, at this time, transferring the DNA flocculent precipitate in different tubes to a 1.5mL centrifuge tube containing 1mL of anhydrous ethanol by using a sheared gun head, washing the DNA precipitate at the same time, and centrifuging at 7500rpm for 2min at room temperature.

(7) After removal of the supernatant, the solution was washed again with 1mL of absolute ethanol, then twice again with 75% ethanol, and finally the precipitated DNA was dried (operating at room temperature, 75% ethanol was removed as much as possible), and the dried precipitate was dissolved in TE (pH 8.0) and was dissolved overnight in a refrigerator at 4 ℃.

(8) The extracted DNA solution (5. Mu.L) was subjected to agarose (0.8% concentration) gel electrophoresis while the concentration and purity of the obtained sample were measured by an ultraviolet spectrophotometer and stored in a-20℃refrigerator.

(2) Amplification of fragments of interest

Specific genomic primers were designed (table 1), a PCR reaction system (table 2) was formulated using the following system, and the PCR amplification procedure was: 98 ℃ for 3min;94℃for 30sec,60℃for 30sec,72℃for 2min,30cyeles; and at 72℃for 10min. The Hmtp210 gene with or without 138bp deletion is obtained by amplification respectively.

TABLE 1 primer sequences

TABLE 2PCR reaction System

(3) Recovery and purification of PCR products

The amplified single-banded PCR product was recovered and purified using an AxygenDNA fragment purification kit, and the recovered product was sent to the bioengineering (Shanghai) Co., ltd for sequencing.

The HMTp210 gene sequence (designated ICC-DHN-YNM-1_C) without deletion of the 138bp gene fragment was as follows:

GACGGGACGATCACCTTCACCAACATTGGCGGTACCGGCCAAGATACGATCCACGATGCCATTAATAACGTTCTGACCAAACTTATCAGCCTGAGCGCGACTGAGGAAGTGGTGTCCGGTGAGGCCGTGTACGAAGCGCTTAAGAGCGCTAAGCCGACGACGGTGAGCGCGGAGGCAAATAAGGGTATCAAAGTTACCGGCGAAAAGTCCAGCAACACCACCGGCAACTCTTTTACGATTGGCCTGGATGATGCGACCCTGAACAAAATTAACAACGCCGTGAACCAGGATCTGTCTAATCTGGGCGAAGACGGAAAGAACGCCATTACCGGTCTGGTAGATGTTGTCAAAAAGGCGAACAGCCCAATTACGGTGGAAAGCTCTACGGACAACAACAAGAAGAAGACTTTCACCGTCGGTCTGGAAAAAAACATCACCGAGGTTAACAGCATCACCTTCGACAAAAGTGGTCAAGATCCGAACCAGGTTACCGGTCGTATGAGCAGCGCGGGTTTGACTTTCAAAAAGGGCGACACCACGAATGGTAGCACCACGACCTTCGCCGAGGACGGTTTGACCATCGACAGCACCACCAATTCTGCACAGACCAACCTGGTTAAAGTGAGCCGCGATGGTTTTTCCGTCAAGAACGGTTCGGATGAAAGCAAGCTGGCTCCGACTAAGCTCTCGATCGGTGCAGAGAACGCCGAGCACGTTGAAGTTACCAAGAGCGGTATTGCCCTGAAGGCGAATAATACCACCGGCAAAAGCTCGATCACCTTGTCTGATAGCGCCATCACCTTGGCTGCCGCGACCGCGGGCAATGCGATTAAACTCACCGGTGTTGCGGACGGCTCTATTACCGCGGGTTCCAAGGACGCTGTGAATGGCGGCCAGCTGCGTACCTTGCTGGGTGTCGATAGCGGCGCGAAAATCGGTGGAACCGAGAAAACGACCATTTCTGAAGCGATTAGCGATGTGAAGCAGGCACTGACCGACGCGAAGTTGGCGTATAAAGCTGACAATAAGAACTCGAAAACGGTGAAACTGACCGACGGCTTAAACTTTACCTCCACCACGAACATCGACGCGAGCGTTGAGGACAGCGGCGTTGTAAAATTTACCCTGAAAGACAAGCTGATTGGTCTGAAGACCATCGCAACCGAGTCCCTGAATGCAAGCCGTAATATCATTGCTGGCGGCACTGTAACCGTGGGTGGCGAGACAGAAGGTATTGTTTTAACGAAATCAGGTTCTGGCAACGATCGTACCCTTTCCCTGAGCGGTGCGGGCAATGCGGCGACGGACGGCATCAAGGTGTCCGGCGTGAAGGCAGGCACCGCTGATACCGACGCGGTGAACAAGGGTCAATTAGATAAATTGTTTAAAGCGATTAACGATGCGCTGGGTACAACAGACCTGGCGGTGACCAAGGACCCGAATCAAACCAGCATCTTTAATCCGATTAACGGTACGGCGCCAACGACATTCAAAGACGCGGTTGATAAGTTGACCACTGCGGTCAACACCGGTTGGGGTAGCAAAGTTGGCATTCTGGCAACCGGGATCGACGGCATCGACGCGGGTAACAAGAAAATCAGCAATGTTGCTGATGGCGACATTAGCCCGACCAGCGGTGACGTGGTTACGGGTCGCCAGCTGTACGCACTGATGCAGAAAGGTATTCGTGTTTACGGCGATGAGGTGTCCCCGACCAAGACCCAAACGACCGCA(SEQ ID NO:8)。

the HMTp210 gene sequence (designated ICC-DHN-WZN _C) deleted of the 138bp gene fragment was as follows:

GACGGTACGATCACCTTCACCAACATTGGTGGCACCGGCCAAGACACCATTCATGATGCGATTAATAACGTTCTGACCAAACTGATTTCGTTATCGGCGACTGAGGAAGAAGTTGTGAGCGGGGAGGCCGTTTACGACGCGCTGAAGGGTGCGAAACCGACGGTTTCTGCGGAGGCAAATAAGGGTATCACCGGCTTGGTTGACGTCGTAAAAAAGGCTAATAGCCCGATTACCGTCGAGCCGAGCACCGACAACAACAAGAAGAAGACCTTTACCGTCGGTTTGATGAAGGATATTGAAGGTGTGAACAGTATCACGTTCGATAAGTCTGGTCAAGATCTGAATCAGGTTACCGGTCGCATGAGCAGCGCAGGTTTGACCTTCAAGAAGGGCGACACCACGAACGGCTCGACCACCACCTTCGCCGAAGATGGTCTGACGATTGACAGCACTACGAACAGCGCCCAAACGAACCTGGTAAAGGTGAGCCGTGATGGCTTTAGCGTTAAGAACGGCAGCGACGAAAGCAAACTTGCGTCTACCAAGCTGTCCATTGGTGCGGAGAACGCGGAACATGTTGAAGTTACCAAGTCTGGCATCGCGCTGAAAGCTGATAACACCAGCGACAAATCCAGCATCACCCTGGCGCAAGATGCGATCACGCTGGCCGGTAACGCGACTGGCACCGCGATTAAACTGACGGGTGTCGCAGATGGCAATATTACCGTCAATTCGAAAGATGCTGTGAATGGTGGTCAGCTGCGTACCTTGCTGGGTGTTGACAGCGGCGCAAAGATCGGCGGTACGGAGAAAACTACAATTTCCGAAGCCATCTCCGACGTGAAGCAGGCACTAACTGACGCCACCCTGGCGTATAAAGCTGACAACAAGAACGGCAAAACCGTGAAACTCACTGACGGCCTGAATTTTACGAGCACCACGAACATCGACGCATCTGTTGAGGATAACGGTGTCGTGAAGTTCACCCTCAAGGACAAGTTGACCGGTCTGAAAACCATCGCCACGGAGAGCCTGAACGCCAGCCAGAATATTATTGCTGGCGGTACTGTTACCGTGGGTGGTGAAACCGAGGGTATCGTGCTGACCAAAAGCGGCTCTGGTAATGATCGCACCCTCTCTTTGAGCGGCGCTGGCAACGCCGCAACCGACGGCATCAAAGTGTCCGGCGTTAAGGCAGGTACAGCGGATACCGACGCGGTGAACAAGGGCCAACTGGATAAATTATTTAAAGCGATCAACGATGCGCTTGGCACCACCGACCTGGCGGTGACTAAGAATCCGAATCAAACCAGTATCTTTAACCCGATTAACGGCACGGCTCCGACGACCTTCAAAGACGCAGTGGATAAGTTGACCACGGCTGTGAACACCGGTTGGGGTAGCAAAGTTGGTATCTTGGCGACCGGGATCGACGGCATCGATGCTGGTAACAAAAAGATCTCCAATGTTGCGGATGGTGACATCAGCCCGACCTCAGGAGATGTGGTGACTGGCCGTCAGTTGTATGCACTGATGCAGAAAGGTATTCGTGTTTACGGCGACGAGGTGTCCCCGACCAAAACTCAGACCACCGCG(SEQ ID NO:9)。

(4) Double enzyme assay

The PCR is respectively carried out to obtain ICC-DHN-YNM-1_C and ICC-DHN-WZN _C complete DNA fragments and PET28b plasmid with double enzyme digestion sites, ncoI endonuclease and XhoI endonuclease are respectively carried out for 3-4 hours, and enzyme digestion products are respectively recovered and purified. The purified products ICC-DHN-YNM-1_C and PET28b after cleavage, ICC-DHN-WZN _C and PET28b were ligated overnight at 16℃to give ligation products.

(5) Coli transformation experiments

After the competent cells BHK21 are melted on ice, 10 mu L of a connection product is added into each competent cell, a centrifuge tube is flicked to be uniformly mixed, the mixture is placed on ice for more than 30min, then heat shock is carried out for 90s in a water bath at 42 ℃, the ice bath is carried out for 2-5 min, fresh LB liquid medium without antibiotics is added into each tube until the total volume is 1mL, the centrifuge tube is placed on a shaking table to be resuscitated for 1h at 37 ℃ under 200r/min, then centrifugation is carried out for 3min at room temperature and 3 000rpm, 900 mu L of supernatant medium is sucked and removed, and the rest 100 mu L of supernatant medium is gently blown and uniformly mixed and is coated on an LB (Kam+) plate. Placing the cells in a 37 ℃ incubator for inversion culture for about 12-16 hours, then picking clones, and storing the plates at 4 ℃.

(6) Bacterial liquid PCR screening positive clone and sequencing

The monoclonal bacterial plaque picked up on the plate is inoculated into a 1.5mL centrifuge tube containing 500 mu LLB liquid culture medium, shake-cultured overnight at 37 ℃ and 200rpm, 1 mu L of bacterial liquid is taken as a template, PCR amplification is carried out by using a detection primer sequence (ICC-detection-F: 5'-TAATACGACTCACTATAGG-3' (SEQ ID NO: 10), ICC-detection-R: 5'-GATATAGTTCCTCCTTTCA-3' (SEQ ID NO: 11)) according to the PCR reaction system and the reaction conditions of Table 2, and 5 mu L of PCR products are taken for agarose gel electrophoresis detection. According to the electrophoresis detection result, the positive clone obtained by screening is sent to a biological engineering (Shanghai) stock limited company for sequencing, the recombinant engineering strain with the DNA fragment of ICC-DHN-YNM-1_C with correct sequencing is named as ICC-C-L, and the recombinant engineering strain with the DNA fragment of ICC-DHN-WZN _C with correct sequencing is named as ICC-C-S.

(7) Inducible expression and purification of recombinant genetically engineered bacteria

(1) Seed preparation: and (3) streaking and resuscitating the seeds (recombinant engineering strains) constructed in the step (6), culturing on a kanamycin-resistant LB plate, adding 50mL of LB liquid culture medium into a single colony, and shake culturing at 37 ℃ and 180rpm for about 5 hours.

(2) Performing expansion culture and induction expression: transferring the liquid seed liquid according to 1% of inoculation amountInoculating into new culture medium, shake culturing at 37deg.C and 180rpm for about 5 hr to OD ₆₀₀ Induction was started when=0.6 to 0.8. IPTG with a final concentration of 0.1mM was added during induction, and the culture was continued for 5 hours to obtain a bacterial solution.

(3) And (3) harvesting and high-pressure homogenizing and cracking of thalli: after the induction, the cells were collected by centrifugation at 8000rpm for 10min in a high-speed centrifuge. The cells were resuspended in physiological saline at a ratio of 10:1 (original culture volume: current volume). Homogenizing for 3 times by using a high-pressure homogenizer at 800Bar, and collecting supernatant.

(4) Protein purification: the metal (Ni, ni) was first fully equilibrated with equilibration buffer (20 mM Tris-HCl,0.3M sodium chloride, 2mM imidazole, pH 8.0) ²⁺ ) After chelating the affinity chromatographic column, loading the sample according to the volume of 3 times of the column, and balancing the sample by using a balancing solution until the sample reaches 0D ₂₈₀ Invariably, 1 column volume of 0.8% Triton X-114 was added to remove endotoxin, followed by elution of the hetero protein with elution buffer I (20 mM Tris-HCl,0.3M sodium chloride, 50mM imidazole, pH 8.0) to 0D ₂₈₀ The target protein was eluted with elution buffer II (20 mM Tris-HCl,0.3M sodium chloride, 300mM imidazole, pH 8.0) constantly. The eluted antigen protein solution at the 280nM absorption peak was collected, sterilized by filtration through a 0.22 μm pore size membrane, and sampled for semi-finished product detection. The purified antigen solution is stored at 4deg.C for no more than 15 days.

The protein expression conditions of the obtained ICC-C-L and ICC-C-S engineering bacteria are shown in figure 2, and the amino acid sequence comparison results of the two are shown in figure 3.

The amino acid sequence of the protein expressed by IC-C-L is as follows:

DGTITFTNIGGTGQDTIHDAINNVLTKLISLSATEEVVSGEAVYEALKSAKPTTVS

AEANKGIKVTGEKSSNTTGNSFTIGLDDATLNKINNAVNQDLSNLGEDGKNAITGLVD

VVKKANSPITVESSTDNNKKKTFTVGLEKNITEVNSITFDKSGQDPNQVTGRMSSAGL

TFKKGDTTNGSTTTFAEDGLTIDSTTNSAQTNLVKVSRDGFSVKNGSDESKLAPTKLSI

GAENAEHVEVTKSGIALKANNTTGKSSITLSDSAITLAAATAGNAIKLTGVADGSITAG

SKDAVNGGQLRTLLGVDSGAKIGGTEKTTISEAISDVKQALTDAKLAYKADNKNSKT

VKLTDGLNFTSTTNIDASVEDSGVVKFTLKDKLIGLKTIATESLNASRNIIAGGTVTVG

GETEGIVLTKSGSGNDRTLSLSGAGNAATDGIKVSGVKAGTADTDAVNKGQLDKLFK

AINDALGTTDLAVTKDPNQTSIFNPINGTAPTTFKDAVDKLTTAVNTGWGSKVGILATG

IDGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDEVSPTKTQTTA

(SEQ ID NO:12)。

amino acid sequence of IC-C-S expressed protein:

DGTITFTNIGGTGQDTIHDAINNVLTKLISLSATEEEVVSGEAVYDALKGAKPTVSAEANKGITGLVDVVKKANSPITVEPSTDNNKKKTFTVGLMKDIEGVNSITFDKSGQDLNQVTGRMSSAGLTFKKGDTTNGSTTTFAEDGLTIDSTTNSAQTNLVKVSRDGFSVKNGSDESKLASTKLSIGAENAEHVEVTKSGIALKADNTSDKSSITLAQDAITLAGNATGTAIKLTGVADGNITVNSKDAVNGGQLRTLLGVDSGAKIGGTEKTTISEAISDVKQALTDATLAYKADNKNGKTVKLTDGLNFTSTTNIDASVEDNGVVKFTLKDKLTGLKTIATESLNASQNIIAGGTVTVGGETEGIVLTKSGSGNDRTLSLSGAGNAATDGIKVSGVKAGTADTDAVNKGQLDKLFKAINDALGTTDLAVTKNPNQTSIFNPINGTAPTTFKDAVDKLTTAVNTGWGSKVGILATGIDGIDAGNKKISNVADGDISPTSGDVVTGRQLYALMQKGIRVYGDEVSPTKTQTTA(SEQ ID NO:13)。

example 2

Protein vaccines were prepared from ICC-C-L and ICC-C-S expressed proteins constructed in example 1, and the specific preparation process was as follows:

preparing an aqueous phase: chicken infectious rhinitis expressed proteins ICC-C-L and ICC-C-S (prepared in example 1) were supplemented with PBS to 77.49ml according to low dose ICC (10 ug/feather) and high dose (30 ug/feather), respectively, and 5.83ml sterilized Tween-80 was added, and mixed well for use as a water phase. Preparing an oil phase: 158.35ml of white oil for injection is taken, 8.33ml of span is added, and the mixture is uniformly mixed and then autoclaved for standby. Emulsifying the water phase and the oil phase under the stirring of a high-speed emulsifying machine to prepare the finished vaccine. When mass production is carried out, the formulation is used for mass production.

Safety test of vaccine: the prepared vaccine is divided into cervical subcutaneous and chest muscle immunization methods to detect safety. The test chickens begin immunization at about 1 month of age, and the test chickens are subjected to section examination 14 days and 21 days after immunization, and the absorption condition is recorded. The test results show that: the vaccine is safe and has no side effect during low dose (0.5 mL) and overdose (1 mL) immunization, and does not cause local or systemic safety reaction to chickens. The test results are shown in Table 3.

TABLE 3 results of the total performance test of vaccines

Immunization test of vaccine: ICC-C-L protein vaccine with antigen content of 10 mug/serving and 30 mug/serving and ICC-C-S protein vaccine with antigen content of 10 mug/serving and 30 mug/serving are prepared. The four protein vaccines are respectively immunized on SPF chickens about 1 month old, and the immunization dose is 0.5mL; a blank (i.e., without any immunization) was also established. The latter month was avoided, together with SPF chickens from the control group under the same conditions, the diluted cultures of five isolated C-type strains of the clinical isolates DHN-YNM-1_C, DHN-BL-C, DHN-SCXC-C, DHN-WZN-C, DHN-SDTA-C of the company were injected with 0.2 mL/challenge only through the infraorbital sinus, wherein DHN-YNM-1_C, DHN-BL-C, DHN-SCXC-C belongs to genotype 2 group, DHN-WZN-C, DHN-SDTA-C belongs to main deletion 138 fragment genotype 1 group, and the number of incidences of each group was counted after 1 week of observation.

As shown in Table 4, the ICC-C-S protein vaccine with low antigen content and high antigen content has good protection effect on cross immunity of different strains in the avian infectious disease serogroup C, but the ICC-C-L protein vaccine has higher protection effect on virus attack of the strain, has poorer cross immunity effect on other strains, and prompts that the HMTp210 gene of the avian infectious disease group C lacks a 138bp sequence shown as SEQ ID NO:3, can effectively improve the cross immunity effect on different strains in the avian infectious disease group C, and is more suitable for serving as a target sequence of the avian infectious disease group C genetic engineering vaccine.

TABLE 4 immune Effect of ICC-C-L protein vaccine and ICC-C-S protein vaccine

While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes may be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

The application of an HMTp210 gene in preparing a product for preventing and/or treating diseases related to infection by avian paragallinarum, wherein the HMTp210 gene is an HMTp210 gene with a part of gene fragments deleted, and the nucleotide sequence of the part of gene fragments is shown as SEQ ID NO. 3.
2. The use according to claim 1, characterized in that the nucleotide sequence of the HMTp210 gene is shown in SEQ ID No. 9 or a sequence having more than 95% identity and the same or similar biological activity as the HMTp210 gene.
3. An HMTp210 protein, the amino acid sequence of which is shown in SEQ ID NO. 13.
4. A nucleic acid molecule encoding the HMTp210 protein of claim 3, preferably having the nucleotide sequence shown in SEQ ID No. 9.
5. A biological material associated with the nucleic acid molecule of claim 4, which is any one of (1) to (3);

(1) An expression cassette comprising the nucleic acid molecule of claim 4;

(2) A recombinant vector comprising the nucleic acid molecule of claim 4 or the expression cassette of (1);

(3) A recombinant cell comprising the nucleic acid molecule according to claim 4, the expression cassette according to (1) or the recombinant vector according to (2).
6. Use of the HMTp210 protein of claim 3, the nucleic acid molecule of claim 4 and/or the biological material of claim 5 for the preparation of a vaccine for infectious rhinitis in chickens.
7. A chicken infectious rhinitis vaccine comprising the HMTp210 protein of claim 3, the nucleic acid molecule of claim 4, and/or the biological material of claim 5.
8. The infectious coryza vaccine of claim 7, further comprising an adjuvant.
9. The infectious coryza vaccine of claim 7, wherein the infectious coryza vaccine of chicken is a subunit vaccine or an inactivated vaccine.
10. A method of increasing the cross-immune response of a vaccine to avibacterium paragallinarum, comprising the steps of: knocking out or silencing part of gene fragments in Hmtp210 gene sequences of the C-type avian parachicken bacillus, wherein the nucleotide sequences of the part of gene fragments are shown as SEQ ID NO. 3; or alternatively, the first and second heat exchangers may be,

prokaryotic expression of Hmtp210 protein of claim 3.