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CN118388607A - Recombinant A-type avibacterium paragallinarum HMTP protein, vaccine composition and application thereof - Google Patents

Recombinant A-type avibacterium paragallinarum HMTP protein, vaccine composition and application thereof Download PDF

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CN118388607A
CN118388607A CN202410769555.6A CN202410769555A CN118388607A CN 118388607 A CN118388607 A CN 118388607A CN 202410769555 A CN202410769555 A CN 202410769555A CN 118388607 A CN118388607 A CN 118388607A
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hmtp
avibacterium paragallinarum
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李国攀
荣俊
尹梓瑞
荣雪宁
匡红艳
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Jingzhou Changxin Biotechnology Co ltd
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Jingzhou Changxin Biotechnology Co ltd
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Abstract

The invention discloses a recombinant A-type avibacterium paragallinarum HMTP protein, a vaccine composition and application thereof, and belongs to the technical field of biological products for animals. The invention cuts 147 amino acid residues at the N end of A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO.3 to obtain a core sequence of 148-666 amino acid sequences, and then fuses antigen epitope sequences at the N end of the core sequence to obtain recombinant A-type avibacterium paragallinarum HMTP protein. Furthermore, after the recombinant A-type avibacterium paragallinarum HMTP protein and the recombinant A-type avibacterium paragallinarum aroA protein are mixed according to a specific proportion, the prepared genetic engineering subunit vaccine composition has better protection effect on infectious rhinitis (A type) of chickens.

Description

Recombinant A-type avibacterium paragallinarum HMTP protein, vaccine composition and application thereof
Technical Field
The invention belongs to the technical field of biological products for livestock, and particularly relates to a recombinant A-type avibacterium paragallinarum HMTP protein, a vaccine composition and application thereof.
Background
Infectious rhinitis of chickens is a disease of the respiratory tract of birds caused by infection with avian bacilli of paragallinarum, most commonly in bred chickens and laying hens of 1 to 4 months of age. The egg yolk peritonitis of the bred chickens is generated and largely eliminated, so that the follicles of the laying hens are softened, hematoma and egg yield are rapidly reduced. Chicken infected with parachicken bacillus will not die generally, but the mortality rate of the sick chicken increases significantly once mixed infection with other pathogenic microorganisms occurs.
Avian paragallinarum can be classified into A, B and C serotypes according to the plate agglutination assay. All three serotypes are detected in China, and the infection rate is increased, wherein the A type is the serotype with the longest existing time and the most serious hazard in China. At present, the prevention and control of infectious rhinitis of chickens mainly adopts parachicken avian bacillus inactivated vaccine. However, there are three major problems with the production, preparation and use of avibacterium paragallinarum inactivated vaccine: (1) NAD and serum are required to be added in the in-vitro culture of the avian bacillus paragallinarum, so that the requirements on nutrition are high, the culture is difficult, and the cost is high; (2) The serotype of the strain of the inactivated vaccine for infectious coryza of chicken is possibly inconsistent with that of the epidemic strain, and the cross protection capability cannot be provided; (3) The inactivated vaccine for infectious rhinitis of chicken contains a large amount of endotoxin, bacterial virulence proteins, other additives and the like, and serious side reactions are easy to cause after inoculation. These factors lead to the current unsatisfactory prevention and control of the disease in China.
HMTP210 is the major hemagglutinin factor of avibacterium paragallinarum and is also an important virulence factor. The complete HMTP210 antigen is capable of folding to form a trimeric structure and induces the body to produce protective antibodies. However, HMTP210 has a large molecular weight, contains a large number of hydrophobic amino acids at the N-terminus, is difficult to express by a prokaryotic expression system, and is low in expression level and easy to form inclusion bodies.
In order to solve the above problems, it is now common practice to delete the N-terminal part of the amino acid in HMTP210, and this treatment results in an increase in the expression level and solubility, and also in maintenance of the normal trimeric structure. However, after deleting the amino acid at the N-terminal part of the HMTP210 protein, some important epitopes are lost, so that the genetic engineering subunit vaccine prepared from the antigen has the problem of reducing the protective effect on infectious rhinitis (type A) of chickens to different degrees.
Disclosure of Invention
The invention aims to provide a recombinant A-type avibacterium paragallinarum HMTP protein, a vaccine composition and application thereof. The invention cuts 147 amino acid residues at the N end of the A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO.3 to obtain a core sequence of 148-666 th amino acid sequences, and then fuses antigen epitope sequences at the N end of the core sequence to obtain recombinant A-type avibacterium paragallinarum HMTP protein.
In a first aspect, the invention provides a recombinant A-type avibacterium paragallinarum HMTP protein, which is obtained by cutting 147 amino acid residues from the N end of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 to obtain a core sequence of 148-666 amino acid sequences, and then fusing an epitope sequence at the N end of the core sequence; wherein the antigen epitope sequence is at least one of 6 th to 12 th amino acid sequences, 22 th to 36 th amino acid sequences, 60 th to 67 th amino acid sequences, 75 th to 86 th amino acid sequences, 93 th to 100 th amino acid sequences and 103 th to 112 th amino acid sequences of the A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO. 3.
In the invention, the inventor researches and discovers that the 1 st-147 th amino acid sequence of the N-terminal of the A-type avibacterium paragallinarum HMTP210 protein is removed, and the recombinant A-type avibacterium paragallinarum HMTP protein obtained after the antigen epitope sequence is fused has obviously increased soluble expression quantity, and the immunogenicity of the recombinant A-type avibacterium paragallinarum HMTP protein is further improved after the antigen epitope sequence is fused.
Specifically, in the invention, the antigen epitope sequence can be 1 or 2 or 3 or 4 or 5 or 6 of amino acid sequences of 6 th to 12 th amino acid sequences, 22 th to 36 th amino acid sequences, 60 th to 67 th amino acid sequences, 75 th to 86 th amino acid sequences, 93 th to 100 th amino acid sequences and 103 th to 112 th amino acid sequences of the A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO.3, for example. When the various epitope sequences of the A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO.3 are selected, the connection sequences of the various epitope sequences can be combined arbitrarily.
In some embodiments, the epitope sequences include amino acid sequences 75-86 and amino acid sequences 103-112 of the A-type avibacterium paragallinarum HMTP210 protein as shown in SEQ ID NO. 3; the fusion operation includes: the linker was used for ligation, and the amino acid sequence of the linker was shown in SEQ ID No. 4.
In the invention, the inventor further researches that the amino acid sequence 75-86 and the amino acid sequence 103-112 of the A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO.3 are relatively close in position and larger in epitope, so that the two epitope sequences are preferably fused, and the obtained recombinant A-type avibacterium paragallinarum HMTP protein has higher soluble expression quantity and better immunogenicity. Linker is used for connecting 75-86 amino acid sequence and antigen epitope sequence of A type avian pullorum disease HMTP210 protein shown in SEQ ID NO.3, and connecting a plurality of antigen epitope sequences.
In some embodiments, the amino acid sequence of the recombinant A-type avibacterium paragallinarum HMTP protein is shown in SEQ ID NO. 1.
The recombinant A-type avian paragallinarum HMTP protein provided by the invention can be a natural, recombinant or synthetic active polypeptide, and the active polypeptide can be a natural purified product, a chemically synthesized product or a product generated from a prokaryotic host (such as escherichia coli) or a eukaryotic host (such as yeast and higher plants) by using a recombinant technology.
In some embodiments, the recombinant A-type avibacterium paragallinarum HMTP protein is obtained by introducing a recombinant vector containing a coding gene thereof into an expression host (such as escherichia coli BL21 (DE 3)) to obtain a recombinant genetic engineering strain, culturing the recombinant genetic engineering strain, and carrying out induced expression.
In a second aspect, the invention provides a nucleic acid molecule encoding the recombinant A-type avibacterium paragallinarum HMTP protein, wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.5 or SEQ ID NO. 6; wherein the nucleotide sequence shown as SEQ ID NO.6 is obtained by codon optimization of the nucleotide sequence shown as SEQ ID NO. 5.
In some preferred embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 6.
In the invention, in order to increase the expression quantity and the solubility level of prokaryotic expression, the nucleotide sequence shown as SEQ ID NO.5 is subjected to codon optimization; specifically, codon optimization comprises optimizing one or more of the amino acid residues encoding positions 59, 61, 89, 90, 102, 179, 237, 339, 346, 366, 367, 381, 407, 460 and 509; preferably, the codon optimisation comprises: the codon ATA at position 59 is replaced by the codon ATT which can be normally recognized by the tRNA of the escherichia coli, the codon CTA at position 179 is replaced by the codon CTG which can be normally recognized by the tRNA of the escherichia coli, the codon AGA at position 237 is replaced by the codon AGC which can be normally recognized by the tRNA of the escherichia coli, the codon CTA at position 381 is replaced by the codon CTG which can be normally recognized by the tRNA of the escherichia coli, and the codon CTA at position 407 is replaced by the codon CTG which can be normally recognized by the tRNA of the escherichia coli.
After the above codon optimization, the nucleotide sequence shown as SEQ ID NO.6 is obtained.
The above nucleic acid molecules provided by the present invention can be obtained by PCR amplification or artificial synthesis.
In a third aspect, the present invention provides a biomaterial selected from any one of the following: a1 An expression cassette comprising the above nucleic acid molecule; a2 A recombinant vector comprising the above nucleic acid molecule; a3 A recombinant cell comprising the above nucleic acid molecule or the above recombinant vector.
In the invention, the expression cassette can also comprise a regulatory element and an enhancing element; the recombinant vector comprises a cloning vector and an expression vector, wherein the cloning vector is used for copying related sequences, the expression vector is used for expressing related genes, and the vector used for constructing the expression vector can be PET28a; the recombinant cell may be E.coli BL21 (DE 3).
In a fourth aspect, the invention provides a genetically engineered subunit vaccine for infectious rhinitis (type a) in chickens, comprising any of the recombinant avian paragallinarum HMTP proteins described above.
In some embodiments, the genetically engineered subunit vaccine further comprises a pharmaceutically acceptable carrier, including an adjuvant.
In some preferred embodiments, the adjuvant is ISA71VG.
In some preferred embodiments, the content of recombinant A-type avibacterium paragallinarum HMTP protein is greater than or equal to 25 μg/mL, for example, 25 μg/mL, 50 μg/mL, 100 μg/mL, and the preferred volume ratio of recombinant A-type avibacterium paragallinarum HMTP protein to adjuvant is 1:3.
In a fifth aspect, the invention provides a genetically engineered subunit vaccine composition for infectious rhinitis (type a) in chickens, comprising any of the recombinant avibacterium paragallinarum HMTP protein, recombinant avibacterium paragallinarum aroA protein, and a pharmaceutically acceptable carrier; wherein the amino acid sequence of the recombinant A-type avibacterium paragallinarum aroA protein is shown as SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule for encoding the recombinant A-type avibacterium paragallinarum aroA protein is shown as SEQ ID NO. 7.
The recombinant A-type avibacterium paragallinarum aroA protein provided by the invention can be a natural, recombinant or synthetic active polypeptide, wherein the active polypeptide can be a natural purified product, a chemically synthesized product or a product generated from a prokaryotic host (such as escherichia coli) or a eukaryotic host (such as yeast and higher plants) by using a recombinant technology.
In some embodiments, the recombinant A-type avibacterium paragallinarum aroA protein is obtained by introducing a recombinant vector containing a coding gene thereof into an expression host (such as escherichia coli BL21 (DE 3)) to obtain a recombinant genetic engineering strain, culturing the recombinant genetic engineering strain, and carrying out induced expression.
The nucleic acid molecule for encoding the recombinant A-type avibacterium paragallinarum aroA protein provided by the invention can be obtained by a PCR amplification or artificial synthesis method.
In some preferred embodiments, the content of recombinant A-type avibacterium paragallinarum HMTP protein in the genetic engineering subunit vaccine composition is more than or equal to 25 mug/mL, and the content of recombinant A-type avibacterium paragallinarum aroA protein is more than or equal to 25 mug/mL.
In some more preferred embodiments, the recombinant A-type avibacterium paragallinarum HMTP protein is greater than or equal to 100 μg/mL and the recombinant A-type avibacterium paragallinarum aroA protein is greater than or equal to 50 μg/mL.
In the invention, the inventor further researches and discovers that the prepared genetic engineering subunit vaccine composition has better protection effect on infectious rhinitis (type A) of chickens after the recombinant type A avibacterium paragallinarum HMTP protein and the recombinant type A avibacterium paragallinarum aroA protein are mixed.
In a sixth aspect, the invention provides a method for preparing the chicken infectious rhinitis (A-type) genetic engineering subunit vaccine composition, comprising the following steps: s1, respectively constructing a recombinant vector containing a nucleic acid molecule for encoding recombinant A-type avibacterium paragallinarum HMTP protein and a nucleic acid molecule for encoding recombinant A-type avibacterium paragallinarum aroA protein, and then transforming the recombinant vector into a host cell to obtain a recombinant cell; s2, respectively culturing the recombinant cells, carrying out induced expression to obtain a culture, and separating and purifying the culture to obtain recombinant A type avibacterium paragallinarum HMTP protein and recombinant A type avibacterium paragallinarum aroA protein respectively; s3, mixing the recombinant A-type avibacterium paragallinarum HMTP protein and/or the recombinant A-type avibacterium paragallinarum aroA protein with an adjuvant to obtain the chicken infectious rhinitis (A-type) genetic engineering subunit vaccine composition.
In the invention, the culture method and the culture conditions have no special requirements, and the normal growth of the recombinant cells is ensured. And the method for separating the recombinant A-type avibacterium paragallinarum HMTP protein and the recombinant A-type avibacterium paragallinarum aroA protein from the culture is a conventional method in the field.
In some preferred embodiments, the recombinant avibacterium paragallinarum HMTP protein a is purified by a weak cation exchange chromatography method; the recombinant A-type avibacterium paragallinarum aroA protein is purified by a weak anion exchange chromatography method.
In some embodiments, the medium used in the preparation of recombinant avibacterium paragallinarum HMTP protein, recombinant avibacterium paragallinarum aroA protein is a medium in the art that can express the protein, preferably LB medium.
In a seventh aspect, the invention provides the use of any one of the recombinant avian paragallinarum type a HMTP protein, the nucleic acid molecule, the biological material, the avian infectious rhinitis (type a) genetic engineering subunit vaccine, and the avian infectious rhinitis (type a) genetic engineering subunit vaccine composition described above in the preparation of a medicament for preventing and/or treating avian infectious rhinitis (type a).
The beneficial effects of the invention are as follows: compared with the prior art, the invention cuts 147 amino acid residues at the N end of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 to obtain a core sequence of 148-666 amino acid sequences, and fuses an epitope sequence at the N end of the core sequence to obtain the recombinant A-type avibacterium paragallinarum HMTP protein.
Drawings
FIG. 1 is a SDS-PAGE map of proteins expressed before and after codon optimization of the HMTP gene of avian Paramygdalina A in example 2 of the present invention, wherein lane M: protein markers; lane 1: a negative control; lane 2: expressing soluble protein before codon optimization (HMTP); lane 3: expressing total protein before codon optimization (HMTP); lane 4: expressing the soluble protein after codon optimization (HMTPopti); lane 5: expressing the total protein after codon optimization (HMTPopti);
FIG. 2 is a SDS-PAGE diagram of purified recombinant A-type avian pullorum HMTP protein according to example 2 of the present invention, wherein lane M: protein markers; lane 1: a negative control; lanes 2 and 3: recombinant A-type avian bacterium paragallinarum HMTP protein before purification; lane 4: purified recombinant A-type avian bacterium paragallinarum HMTP protein;
FIG. 3 is a SDS-PAGE diagram of recombinant A-type avibacterium paragallinarum aroA protein purified in example 3 of the present invention, wherein, lane M: protein markers; lane 1: a negative control; lane 2: recombinant A-type avibacterium paragallinarum aroA protein before purification; lane 3: the recombinant A-type avian parachicken bacillus aroA protein after purification.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
The experimental methods, which are not specified 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 Experimental guidelines, molecular cloning, ALaboratoryManual, robert F-Weaver, molecular biology, etc., or according to the methods suggested by the manufacturers of kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.
EXAMPLE 1A cloning of the HMTP Gene of avian Paramygdalin, sequence optimization, construction of expression Strain and purification of prokaryotic expression
1.1 Cloning and sequencing of the full-Length A-parachicken bacillus HMTP210 Gene
1.1.1 Extraction of genome of A-type avian paragallinarum HP-8 Strain
Taking 0.2ml of A-type avibacterium paragallinarum HP-8 bacterial liquid, centrifuging for 5min at 8000g, discarding the supernatant, and adding 50 mu L of ddH 2 O for resuspension; boiling at 100deg.C for 10min, centrifuging at 8000g for 5min, and collecting supernatant and storing at-20deg.C. The supernatant contains the genome of the A-type avibacterium paragallinarum HP-8 strain and is a template for PCR amplification.
1.1.2 PCR amplification of full-length A-type avian paragallinarum HMTP210 gene fragment
First, a primer for amplifying a full-length A-type avibacterium paragallinarum HMTP210 gene fragment is synthesized, and the specific steps are as follows:
HMTP F1:5'-ATTCGGATACCTCAATGAC-3'(SEQ ID NO.10)
HMTP R1:5'-CCCCTCGAGTTATTGAGTGCTAGATGCTGTAG-3'(SEQ ID NO.11)
the italic part is the introduced protecting base and the underlined part is the cleavage site of the introduced Xho I. The genome of the extracted A-type avibacterium paragallinarum HP-8 strain is used as a template, and HMTP F1/HMTP R1 is used as a primer to amplify the HMTP210 gene fragment of the full-length A-type avibacterium paragallinarum. The amplification system is as follows:
Setting temperature cycle parameters: 95 ℃ for 2min;35 cycles (95 ℃,20s;60 ℃,20s;72 ℃,60 s); 72℃for 5min.
The PCR products are subjected to electrophoresis and separation by utilizing agarose gel electrophoresis technology, DNA bands with the size of 2000bp are recovered by cutting, subcloned into pTOPOT vectors and transformed into DH5 alpha competent cells, the correct strain is identified by PCR as E. coliDH alpha/pTOPOT-full LENGTH HMTP210, and the obtained product is sent to Shanghai biological engineering Co Ltd for sequence determination, BLAST comparison is carried out on the determined result, the amplified fragment is the HMTP210 gene of A type avibacterium paragallinarum, the nucleotide sequence of the HMTP210 gene is shown as SEQ ID NO.8, and the amino acid sequence of the HMTP210 protein of A type avium paragallinarum is shown as SEQ ID NO. 3.
1.2 Cutting of A-type avian bacterium paragallinarum HMTP210 gene sequence and N-terminal epitope fusion thereof
Because the full-length A-type avibacterium paragallinarum HMTP210 gene is easy to cause the phenomenon of inclusion body expression in a prokaryotic expression system, the protein can be reformed into correct folding only through complex denaturation and renaturation processes in the follow-up process, the efficiency is low and the amplification is not easy; in addition, most of the epitopes of the A-type avibacterium paragallinarum HMTP210 are mainly concentrated at 148-666 amino acid residues, and the amino acid residues play an important role in maintaining the conformation of a trimer and forming a correct antigen structure, so that the DNA sequence corresponding to 148-666 amino acids at the C end of the A-type avibacterium paragallinarum HMTP210 gene fragment is amplified by a PCR method.
First, a synthetic primer is designed, specifically as follows:
HMTP F2:5'-CCCGGATCCACTAAAGGGATCTACCTTAAAG-3'(SEQ ID NO.12)
The italic part is the introduced protecting base and the underlined part is the cleavage site for the introduced BamHI. The DNA fragment of A-type avibacterium paragallinarum HMTP (148-666) is amplified by taking pTOPOT-full LENGTH HMTP as a template and HMTP F2/HMTP R1 as a primer, and the amplified fragment size is 1557bp. The PCR products were analyzed by agarose gel electrophoresis, and the A-type avibacterium paragallinarum HMTP (148-666) fragment was recovered by gel cutting.
The recovered A-type avibacterium paragallinarum HMTP (148-666) gene fragment and pET28a plasmid were digested with BamH I and Xho I restriction enzymes, and after electrophoresis recovery of the double digested products, ligation was performed using T4 DNA ligase.
Ligation products were transformed into E.coli BL21 (DE 3) competent cells and screened using kanamycin plates. The monoclonal was picked for PCR verification and the correct strain was identified as E. coliBL21 (DE 3)/pET 28a-HMTP (148-666).
Through analysis and prediction, the nucleotide sequence of 6 epitopes at the N end of the A-type avibacterium paragallinarum HMTP210 gene is found, 2 epitope fragments (the 222 th to 258 th positions of the nucleotide sequence of the A-type avibacterium paragallinarum HMTP210 gene shown as SEQ ID NO. 8: ATTGTTAATGTTGCAGCAGGCGATGTTTCGCAAGCT, the 75 th to 68 th positions of the amino acid sequence of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO. 3: IVNVAAGDVSQA, the 307 th to 336 th positions of the nucleotide sequence of the A-type avibacterium paragallinarum HMTP210 gene shown as SEQ ID NO. 8: TTGAGCAAAGTGGCTCAATCTGTTAAGAGC, the 103 th to 112 th positions of the amino acid sequence of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO. 3: LSKVAQSVKS) are selected for fusion with the A-type avibacterium paragallinarum HMTP (148-666) fragments.
In order to fuse the antigen epitope at the N end of the A-type avibacterium paragallinarum HMTP (148-666), the antigen epitope integrity of the A-type avibacterium paragallinarum HMTP is reserved, and the purpose is achieved by introducing a DNA sequence coded by the antigen epitope at the 5' end of a forward primer of PCR. The synthetic primers were designed as follows:
HMTP(75-86)F1:5'-GATGTTTCGCAAGCTGGCGGCGGCGGCAGTACTAAAGGGATCTAC-3'(SEQ ID NO.13)
HMTP(75-86)F2:5'-CCCGGATCCATTGTTAATGTTGCAGCAGGCGATGTTTCGCAAGCT-3'(SEQ ID NO.14)
HMTP(103-112)F1:5'-CAATCTGTTAAGAGCGGTGGTGGTGGTAGCACTAAAGGGATCTAC-3'(SEQ ID NO.15)
HMTP(103-112)F2:5'-CCCGGATCCTTGAGCAAAGTGGCTCAATCTGTTAAGAGC-3'(SEQ ID NO.16)
HMTP(75-86/103-112)F1:5'-GATGTTTCGCAAGCTGGCGGCGGCGGCAGTTTGAGCAAAGTGGCT-3'(SEQ ID NO.17)
PCR amplification was performed using pET28a-HMTP (148-666) as template and HMTP (75-86) F1/HMTP R1 as primer, and the PCR product was designated "a". And then carrying out PCR amplification by taking 'a' as a template and HMTP (75-86) F2/HMTP R1 as a primer, wherein a PCR product is marked as 'b'. The DNA fragment is fused with 75-86 th epitope sequence of amino acid sequence of A type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO.3 and DNA sequence corresponding to Linker at 5' end of A type avibacterium paragallinarum HMTP (148-666), and the fragment is named A type avibacterium paragallinarum HMTP (75-86/148-666), wherein the amino acid sequence of Linker is shown in SEQ ID NO.4, and the nucleotide sequence of Linker is shown in SEQ ID NO. 9.
Amino acid sequence of Linker (GGGGS, SEQ ID NO. 4), nucleotide sequence of Linker (GGCGGCGGCGGCAGT, SEQ ID NO. 9)
PCR amplification was performed using pET28a-HMTP (148-666) as a template and HMTP (103-112) F1/HMTP R1 as primers, and the PCR product was designated "c". And then carrying out PCR amplification by taking 'c' as a template and taking HMTP (103-112) F2/HMTP R1 as a primer, wherein a PCR product is marked as'd'. And (3) electrophoresis gel cutting and purification of a target fragment, wherein the DNA fragment is fused with an antigen epitope sequence at the 103 th to 112 th positions of an amino acid sequence of an A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 and a DNA sequence corresponding to a Linker at the 5' end of the A-type avibacterium paragallinarum HMTP (148-666), and the fragment is named as the A-type avibacterium paragallinarum HMTP (103-112/148-666).
PCR amplification was performed using "d" as the template and HMTP (75-86/103-112) F1/HMTP R1 as the primer, and the PCR product was designated "e". And then using "e" as a template, using HMTP (75-86) F2/HMTP R1 as a primer, and carrying out PCR amplification, wherein the PCR product is marked as "F". The electrophoresis gel cutting purification target fragment, the DNA fragment fuses the DNA sequence corresponding to the 75 th-86 th epitope sequence-Linker of the amino acid sequence of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 and the 103 th-112 th epitope sequence-Linker of the amino acid sequence of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 at the 5' end of the A-type avibacterium paragallinarum HMTP (148-666), the fragment is named as A-type avibacterium paragallinarum HMTP, the DNA sequence of the fragment is shown as SEQ NO.5, and the amino acid sequence of recombinant A-type avibacterium paragallinarum HMTP protein is shown as SEQ ID NO. 1.
And (3) digesting the A-type avibacterium paragallinarum HMTP and pET28a plasmids by using BamH I and Xho I restriction enzymes, performing electrophoretic separation and gel cutting purification on digested fragments, and connecting the digested and purified gene fragments with a digested pET28a vector. Ligation products were transformed into E.coli BL21 (DE 3) competent cells and screened using kanamycin plates. Selecting a monoclonal to carry out PCR verification, and verifying that the correct strain is named as follows: E. coliBL21 (DE 3)/pET 28a-HMTP.
1.3 Codon optimization of A-type avian paragallinarum HMTP gene
As E. coliBL21 (DE 3)/pET 28a-HMTP has lower expression level after induction, and sequence analysis shows that the A-type avibacterium paragallinarum HMTP gene contains a plurality of rare codons with lower use frequency of escherichia coli, the A-type avibacterium paragallinarum HMTP codon needs to be optimized so as to improve the expression level of recombinant protein on the premise of ensuring the soluble expression of the A-type avibacterium paragallinarum HMTP.
The codon optimization scheme is shown in the following table 1: the rare codon ATA at position 59 (the amino acid position corresponding to the A-type avibacterium HMTP after cutting) is synonymously replaced by the codon ATT which can be normally recognized by the escherichia coli tRNA, the rare codon CTA at position 179 (the amino acid position corresponding to the A-type avibacterium HMTP after cutting) is synonymously replaced by the codon CTG which can be normally recognized by the escherichia coli tRNA, the rare codon AGA at position 237 (the amino acid position corresponding to the A-type avibacterium HMTP after cutting) is synonymously replaced by the codon AGC which can be normally recognized by the escherichia coli tRNA, the rare codon CTA at position 381 (the amino acid position corresponding to the A-type avibacterium HMTP after cutting) is synonymously replaced by the codon CTG which can be normally recognized by the escherichia coli tRNA, and the rare codon CTA at position 407 (the amino acid position corresponding to the A-type avibacterium HMTP after cutting) is synonymously replaced by the codon CTG which can be normally recognized by the escherichia coli tRNA.
Table 1A codon optimization scheme of avian paragallinarum HMTP
The codon-optimized A-type avibacterium paragallinarum HMTP gene is synthesized through total genes and inserted into BamH I and Xho I sites of a pET28a vector, and the expression vector of the total gene synthesis is named pET28a-HMTPopti; the vector was transformed into BL21 (DE 3) strain and designated E. coliBL21 (DE 3)/pET 28a-HMTPopti.
EXAMPLE 2 Induction expression, purification and characterization of recombinant A-type avibacterium paragallinarum HMTP protein
E. coliBL21 (DE 3)/pET 28a-HMTP and E. coliBL21 (DE 3)/pET 28a-HMTPopti strains constructed in example 1 are activated; after inoculation according to 1% of inoculum size, culturing and shaking for 3 hours at 37 ℃; lactose was added for induction at a final lactose concentration of 30mM at 32℃for 12h. After the induction, the cells were collected by centrifugation.
The collected cells were added with 0.02mol/L PB buffer solution having a pH of 6.8, and subjected to ultrasonic disruption after resuspension. 12000g, centrifuging at 4 ℃ for 10min, and collecting supernatant, wherein the supernatant is the soluble protein. Protein content was measured by BCA method and detected by SDS-PAGE technique, and the results are shown in FIG. 1.
As can be seen from FIG. 1, the molecular weights of the E. coliBL21 (DE 3)/pET 28a-HMTP and E. coliBL21 (DE 3)/pET 28a-HMTPopti expression products are consistent with the expectations. Compared with the target protein expressed by E.coli BL21 (DE 3)/pET 28a-HMTP before codon optimization, the expression level of the E.coli BL21 (DE 3)/pET 28a-HMTPopti target protein after codon optimization is obviously increased, and the solubility level is also obviously improved.
Further, purification of recombinant a-type avibacterium paragallinarum HMTP protein was performed using weak cation exchange chromatography. The weak cation chromatography medium used for purification is Sepharose CM Fast Flow, the balance buffer is 0.02mol/L PB buffer with the pH value of 6.8, the elution buffer is 0.02mol/L PB+0.5mol/L NaCl with the pH value of 6.8, and the elution buffer is 0.02mol/L PB+1mol/L NaCl with the pH value of 6.8. The protein eluted by the eluting buffer solution is recombinant A-type avian bacterium paragallinarum HMTP protein.
The purified product was detected using SDS-PAGE technique and the results are shown in FIG. 2.
As can be seen from FIG. 2, the A-type avibacterium paragallinarum HMTP protein can be purified by a weak cation exchange chromatography method, and the operation is simple and easy to amplify.
Example 3 cloning of A A Gene of avibacterium paragallinarum, construction of expression Strain, inducible expression purification of recombinant protein
3.1 Cloning and sequencing of A-type avibacterium paragallinarum aroA gene
First, a primer for amplifying an A-type avibacterium paragallinarum aroA gene is synthesized, and the specific steps are as follows:
aroA PF1:5'-CCCGGATCCATGGAAAAATTAACGCTAC-3'(SEQ ID NO.18)
aroA PR1:5'-CCCCTCGAGTTATCGTACAATCTTCGC-3'(SEQ ID NO.19)
wherein the italic CCC base is an enzyme cutting site protecting base, and the underlined is the introduced enzyme cutting sites BamHI and Xho I.
PCR amplification was performed using the genome of the A-type avibacterium paragallinarum HP-8 strain extracted in example 1 as a template and aroA PF1/aroA PR1 as a primer. The amplified products were analyzed by electrophoresis, and a band of about 1300bp was recovered by gel cutting. The DNA fragment was subcloned into the pTOPO T vector and transformed into DH5a competence, and sequenced after PCR. The sequence is confirmed to be a fowl bacillus paragallinarum A type fowl bacillus paragallinarum aroA gene by BLAST analysis, the nucleotide sequence of the fowl bacillus paragallinarum A type fowl bacillus paragallinarum aroA gene is shown as SEQ ID NO.7, and the amino acid sequence of the recombinant fowl bacillus paragallinarum aroA protein is shown as SEQ ID NO. 2.
Double-enzyme cutting of A-type avian parachicken bacillus aroA gene fragment, and connection of the fragment with pET28a vector which is treated in the same way by using T4 DNA ligase, transformation into BL21 (DE 3) competence, and identification of correct monoclonal named E. coliBL21 (DE 3)/pET 28a-aroA.
3.2 Induction expression and purification of recombinant A-type avibacterium paragallinarum aroA
(1) Activating strains: frozen E. coliBL21 (DE 3)/pET 28a-aroA strain is taken out from a refrigerator at-20 ℃ for thawing, transferred into 5mL of LB liquid medium according to the proportion of two thousandths, kanamycin sulfate with the final concentration of 50 mug/mL is added, a shaking table is set to 37 ℃ and 220rpm, shaking culture is carried out for 8 hours, bacterial liquid is streaked and inoculated into LB solid medium containing kanamycin sulfate resistance by an inoculating loop after the bacterial liquid grows, the bacterial liquid is cultured for 16 hours at 37 ℃, the grown bacterial colony is picked into 5mL of LB liquid medium containing kanamycin sulfate resistance, the shaking table is set to 37 ℃ and 220rpm, shaking culture is carried out for 8 hours, the bacterial liquid is added into 200mL of LB liquid medium containing kanamycin according to the proportion of 1 percent, the shaking table is set to 37 ℃ and 220rpm, and shaking culture is carried out for 8 hours.
(2) And (3) performing expansion culture: the successfully activated strain is added into 300mL of LB liquid medium according to the proportion of 2%, the final concentration of kanamycin sulfate is 50 mug/mL, the parameters of a shaking table are set to be 37 ℃ and 220rpm, and the shaking table is used for culturing for 3 hours, so that the OD 600 value of the strain reaches 0.8.
(3) Induction of expression: the final concentration of alpha-lactose was set to 30mmol/L, the shaking table parameters were set to 32℃and 220rpm, the induction culture was continued for 14 hours, and the cells were collected by centrifugation at 12000g for 10min using a high-speed centrifuge and weighed and recorded.
1G of E. coliBL21 (DE 3)/pET 28a-aroA cells are weighed, 40mL of 0.02mol/L PB buffer solution with a pH value of 8.0 is added according to a ratio of 1:40, and then a plastic beaker is placed into a beaker mixed with ice water, and is subjected to ultrasonic crushing for 3s at intervals of 6s for 30min.12000g, centrifugation at 4℃for 10min, collecting the supernatant, and filtering the protein solution with a 0.22 μm filter.
Purification of recombinant A-type avibacterium paragallinarum aroA protein was performed using weak anion exchange chromatography. The weak anion chromatography medium used for purification is Sepharose DEAE Fast Flow, the balance buffer is 0.02mol/L PB buffer with the pH value of 8.0, the impurity washing buffer is 0.02mol/L PB+0.5mol/L NaCl with the pH value of 8.0, the elution buffer is 0.02mol/L PB+1mol/L NaCl with the pH value of 8.0, and the elution buffer is 0.02mol/L PB+2mol/L NaCl with the pH value of 8.0. The protein eluted by the eluting buffer solution is recombinant A-type avibacterium paragallinarum aroA protein.
Protein content was determined using the BCA method and identified using SDS-PAGE method, the results of which are shown in fig. 3.
From the results in FIG. 3, it can be seen that the molecular weight of the A-type avibacterium paragallinarum aroA protein is consistent with that expected, and the A-type avibacterium paragallinarum aroA protein can be effectively purified by a weak anion exchange chromatography method, and has high purification efficiency and easy amplification.
EXAMPLE 4 safety and efficacy testing of infectious rhinitis (type A) HMTP genetically engineered subunit vaccine
4.1 Preparation of chicken infectious rhinitis (A type) HMTP gene engineering subunit vaccine
Adding a proper amount of PBS into the purified HMTP protein of the A-type avibacterium paragallinarum by weak cation exchange chromatography for dilution, adding formaldehyde with the final concentration of 0.037%, and carrying out antigen inactivation at the temperature of 4 ℃ in a refrigerator for 72 hours. After the inactivation is completed, a sterile examination is performed.
After passing the sterility test, the target protein was mixed with ISA71 VG mineral oil adjuvant at a 1:3 volume ratio at room temperature to give 200. Mu.g/mL. And (5) fully shearing by using a shearing machine at 8000rpm in an ice water bath environment for 4min, and stopping for 1min. After shearing is finished, checking the emulsification condition, sucking 1mL of vertical drop on clear water by using a 1mL liquid-transfering gun, and starting from the second drop, and not diffusing; taking 1mL of the sheared mixture, and centrifuging 3000g for 15min, wherein no demulsification condition exists. And after the emulsification inspection is qualified, the chicken infectious rhinitis (A type) HMTP genetic engineering subunit vaccine is obtained and is stored in a refrigerator at 4 ℃.
4.2 Safety test of chicken infectious rhinitis (type A) HMTP genetic engineering subunit vaccine
The safety test is carried out by using 5 SPF chickens with the age of 30 days, each chicken is immunized with 2ml of HMTP genetic engineering subunit vaccine subcutaneously in a large dose, the immunization is continuously observed within 14 days, no obvious side effect is found in the chickens, the mental state is good, and adverse effects such as induration, abscess and ulcer are not found in the injection part section. The chicken infectious rhinitis (A type) HMTP gene engineering subunit vaccine is proved to accord with the safety standard.
4.3 Efficacy evaluation of chicken infectious rhinitis (type A) HMTP genetic engineering subunit vaccine
SPF eggs were hatched in an incubator and fed to 30 days of age. 30 SPFs of 30 days old were randomly divided into three groups, immune protection group A, challenge control group B, and blank control group C, each group of 10 SPF chickens. The neck of the immune protection group A is subcutaneously injected with 0.5ml of HMTP subunit vaccine, and the virus attack control group B and the blank control group C are injected with 0.5ml of physiological saline; after 21 days of immunization, the immunoprotection group A and the challenge control group B were injected with 0.2ml (500 CFU/0.2 ml) of the A-type avibacterium paragallinarum HP-8 strain solution in the infraorbital sinus, and the blank control group C was not challenged. Clinical symptoms were continuously observed for 7 days, and scored according to the clinical score criteria for avibacterium paragallinarum challenge of table 2 below.
TABLE 2 clinical score criteria for challenge with avian bacilli
The results of the challenge protection are shown in Table 3 below, wherein clinical symptom scores of 2 or more (2, 3,4, 5) are judged to be ill, and 1 or less (0, 1) is considered not to be ill.
TABLE 3 evaluation results of infectious coryza (A) HMTP Gene engineering subunit vaccine challenge
As can be seen from the results in Table 3, 10 chickens in the challenge control group developed no disease, none in the blank control group, and 7 chickens immunized with HMTP subunit vaccine developed no disease, 3 developed no disease, and the challenge protection rate was 70%.
EXAMPLE 5 safety and efficacy testing of an aroA genetically engineered subunit vaccine against infectious rhinitis (type A) in chickens
5.1 Preparation of chicken infectious rhinitis (A type) aroA gene engineering subunit vaccine
The recombinant aroA protein purified by weak anion exchange chromatography is diluted by adding a proper amount of PBS, formaldehyde with the final concentration of 0.037 percent is added, and antigen inactivation is carried out in a refrigerator at the temperature of 4 ℃ for 72 hours. After the inactivation is completed, a sterile examination is performed.
After passing the sterility test, 3 volumes of ISA71 VG adjuvant were added at room temperature to give a aroA antigen concentration of 200 μg/mL in the mixture. Emulsifying with high-speed shearing machine for 15min. And after the emulsification inspection is qualified, the chicken infectious rhinitis (A type) aroA genetic engineering subunit vaccine is obtained and is stored in a refrigerator at 4 ℃.
5.2 Safety test of aroA genetic engineering subunit vaccine for infectious coryza (A type) of chicken
Safety inspection is carried out by using 5 SPF chickens with the age of 30 days, each chicken is immunized with 2ml aroA genetic engineering subunit vaccine in a large dose under the skin, the immunization is continuously observed within 14 days, no obvious side effect is found in the chickens, the mental state is good, and adverse reactions such as induration, abscess and ulcer are not found in the injection part section inspection. The chicken infectious rhinitis (A type) aroA gene engineering subunit vaccine is proved to accord with the safety standard.
5.3 Evaluation of efficacy of aroA Gene engineering subunit vaccine against infectious rhinitis (type A) in chickens
SPF eggs were hatched in an incubator and fed to 30 days of age. 30 SPFs at 30 days of age were randomly divided into three groups, immune protection group D, challenge control group E, and blank control group F, each group of 10 SPF chickens. The neck of the immune protection group D is subcutaneously injected with 0.5ml aroA subunit vaccine, and the virus attack control group E and the blank control group F are injected with 0.5ml physiological saline; after 21 days of immunization, the immune protection group D and the virus-attack control group E were injected with 0.2ml (500 CFU/0.2 ml) of the A-type avibacterium paragallinarum HP-8 strain solution in the infraorbital sinus, and the blank control group F was not virus-attack. Clinical symptoms were continuously observed for 7 days and scored according to the avibacterium paragallinarum challenge clinical scoring criteria in table 2.
The results of the challenge protection are shown in Table 4 below, wherein clinical symptom scores of 2 or more (2, 3,4, 5) are judged to be ill, and 1 or less (0, 1) is considered not to be ill.
TABLE 4 evaluation results of aroA Gene engineering subunit vaccine against infectious coryza (A) in chickens
As can be seen from the results in Table 4, 10 chickens in the challenge control group developed no disease, none in the blank control group, and 4 chickens and 6 chickens immunized with aroA subunit vaccine developed no disease, with a 40% challenge protection.
EXAMPLE 6 genetically engineered subunit vaccine safety and efficacy testing of combination of infectious rhinitis (type A) HMTP and aroA in chickens
The HMTP and aroA antigens of A-type avibacterium paragallinarum were inactivated as in examples 4 and 5, the HMTP antigen protein of A-type avibacterium paragallinarum and aroA antigen protein of A-type avibacterium paragallinarum were mixed in a certain ratio, and ISA71 VG adjuvant was added in an amount of 3 times the volume of the mixed antigen, and after emulsification, genetically engineered subunit vaccines containing HMTP and aroA at different concentrations were prepared, and the content of HMTP and aroA antigen in each vaccine was as shown in Table 5 below.
Table 5 HMTP and aroA antigen content in 9 vaccines configured
SPF eggs were hatched in an incubator and fed to 30 days of age. The SPFs of 110 30 days old were randomly divided into eleven groups, combination vaccine groups 1-9, challenge control group 10, blank control group 11, 10 SPF chickens per group. Each group of immune protection groups 1-9 is subcutaneously injected into the neck of the corresponding group 1-9, and the challenge control group 10 and the blank control group 11 are injected with 0.5ml of physiological saline; after 21 days of immunization, the immune protection group 1-9 and the challenge control group 10 were injected with 0.2ml (500 CFU/0.2 ml) of the A-type avibacterium paragallinarum HP-8 strain solution in the infraorbital sinus, and the blank control group 11 was not challenged. Clinical symptoms were continuously observed for 7 days and scored according to the avibacterium paragallinarum challenge clinical scoring criteria in table 2.
The results of the challenge protection are shown in Table 6 below, wherein a score of bed symptoms of 2 or more (2, 3, 4, 5) is judged to be ill, and 1 or less (0, 1) is considered not to be ill.
Table 6 HMTP results of toxicity challenge evaluation of genetically engineered subunit vaccine in combination with aroA
As can be seen from the results in table 6, 10 chickens in the challenge control group developed an onset, with a clinical symptom score of 3.2; none of the blank groups developed disease, and the clinical symptom score was 0.1; the vaccine combination HMTP and aroA had a challenge protection of 60%, 70%, 80%,90%, 100% respectively, and clinical symptom scores of 1.2, 1.0, 0.8, 0.6, 0.5, 0.4, 0.2, 0.1, respectively. The immune protection rate is improved and the clinical symptoms are reduced along with the increase of the HMTP and aroA antigen amount; the combination of the HMTP and aroA antigens has positive synergistic effect, and under the action of the same antigen dose, the combined vaccine of the HMTP and aroA antigens has higher toxicity attack protection than a single antigen vaccine; when the content of HMTP antigen is more than or equal to 100 mug/mL and the content of aroA antigen is more than or equal to 50 mug/mL, the vaccine has the highest virus attack protection.
In conclusion, 147 amino acid residues are sheared at the N end of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 to obtain a core sequence of 148-666 amino acid sequences, and then the N end of the core sequence is fused with an epitope sequence to obtain the recombinant A-type avibacterium paragallinarum HMTP protein. Furthermore, after the recombinant A-type avibacterium paragallinarum HMTP protein and the recombinant A-type avibacterium paragallinarum aroA protein are mixed according to a specific proportion, the prepared genetic engineering subunit vaccine composition has better protection effect on infectious rhinitis (A type) of chickens.
It should be noted that, the foregoing embodiments all belong to the same inventive concept, and the descriptions of the embodiments have emphasis, and where the descriptions of the individual embodiments are not exhaustive, reference may be made to the descriptions of the other embodiments.
The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. The recombinant A-type avibacterium paragallinarum HMTP protein is characterized in that 147 amino acid residues are sheared and removed from the N end of the A-type avibacterium paragallinarum HMTP210 protein shown as SEQ ID NO.3 to obtain a core sequence of 148 th-666 th amino acid sequence, and then an antigen epitope sequence is fused at the N end of the core sequence to obtain the recombinant A-type avibacterium paragallinarum HMTP protein;
wherein the antigen epitope sequence is at least one of 6 th to 12 th amino acid sequences, 22 th to 36 th amino acid sequences, 60 th to 67 th amino acid sequences, 75 th to 86 th amino acid sequences, 93 th to 100 th amino acid sequences and 103 th to 112 th amino acid sequences of the A-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID NO. 3.
2. The recombinant a-type avibacterium paragallinarum HMTP protein of claim 1, wherein the epitope sequence comprises amino acid sequences 75-86 and amino acid sequences 103-112 of a-type avibacterium paragallinarum HMTP210 protein shown in SEQ ID No. 3; the fusing operation includes: the ligation was performed using a linker whose amino acid sequence is shown in SEQ ID No. 4.
3. The recombinant a-type avibacterium paragallinarum HMTP protein according to claim 1 or 2, wherein the amino acid sequence of the recombinant a-type avibacterium paragallinarum HMTP protein is shown as SEQ ID No. 1.
4. A nucleic acid molecule encoding the recombinant a-type avibacterium paragallinarum HMTP protein of claim 3, wherein the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID No.5 or SEQ ID No. 6; wherein the nucleotide sequence shown as SEQ ID NO.6 is obtained by codon optimization of the nucleotide sequence shown as SEQ ID NO. 5.
5. A biomaterial, characterized in that it is selected from any one of the following:
a1 An expression cassette comprising the nucleic acid molecule of claim 4;
A2 A recombinant vector comprising the nucleic acid molecule of claim 4;
a3 A recombinant cell comprising the nucleic acid molecule of claim 4 or the recombinant vector.
6. A genetically engineered subunit vaccine for infectious coryza in chickens, comprising the recombinant a-type avibacterium paragallinarum HMTP protein of any one of claims 1-3.
7. The genetically engineered subunit vaccine of claim 6, further comprising a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier comprising an adjuvant.
8. A genetically engineered subunit vaccine composition for infectious coryza in chickens, comprising the recombinant avibacterium paragallinarum HMTP protein of any one of claims 1-3, the recombinant avibacterium paragallinarum aroA protein of type a, and a pharmaceutically acceptable carrier;
Wherein the amino acid sequence of the recombinant A-type avibacterium paragallinarum aroA protein is shown as SEQ ID NO.2, and the nucleotide sequence of the nucleic acid molecule for encoding the recombinant A-type avibacterium paragallinarum aroA protein is shown as SEQ ID NO. 7.
9. The method for preparing the chicken infectious rhinitis genetic engineering subunit vaccine composition as claimed in claim 8, which is characterized by comprising the following steps:
S1, respectively constructing a recombinant vector containing a nucleic acid molecule for encoding recombinant A-type avibacterium paragallinarum HMTP protein and a nucleic acid molecule for encoding recombinant A-type avibacterium paragallinarum aroA protein, and then transforming the recombinant vector into a host cell to obtain a recombinant cell;
s2, respectively culturing the recombinant cells, carrying out induced expression to obtain a culture, and separating and purifying the culture to obtain recombinant A type avibacterium paragallinarum HMTP protein and recombinant A type avibacterium paragallinarum aroA protein;
S3, mixing the recombinant A-type avian paragallinarum HMTP protein and/or the recombinant A-type avian paragallinarum aroA protein with an adjuvant to obtain the chicken infectious rhinitis genetic engineering subunit vaccine composition.
10. Use of the recombinant a-type avibacterium paragallinarum HMTP protein of any one of claims 1-3, the nucleic acid molecule of claim 4, the biological material of claim 5, the chicken infectious rhinitis genetic engineering subunit vaccine of claim 6, the chicken infectious rhinitis genetic engineering subunit vaccine composition of claim 8 in the preparation of a medicament for preventing and/or treating chicken infectious rhinitis.
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