CN117512010A - Construction and application of IBV S1 and Cps MOMP gene bivalent epitope vaccine - Google Patents
Construction and application of IBV S1 and Cps MOMP gene bivalent epitope vaccine Download PDFInfo
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- CN117512010A CN117512010A CN202311577083.6A CN202311577083A CN117512010A CN 117512010 A CN117512010 A CN 117512010A CN 202311577083 A CN202311577083 A CN 202311577083A CN 117512010 A CN117512010 A CN 117512010A
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Abstract
The invention discloses construction and application of an IBV S1 and Cps MOMP gene bivalent epitope vaccine. The method mainly comprises the steps of screening the IBV S1 gene and the Cps MOMP gene through online biological software, and connecting the screened epitopes by using connecting peptide to construct a new peptide segment M with high immunogenicity and small side effect. The M, S and MOMP tandem genes were respectively connected to eukaryotic expression vector pEGFP-N1 to construct 3 groups of recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP. After entering the organism, the immune system can firstly start the self cell and humoral immunity function, and then start the immune reaction through the specific antigens of IBV S1 and Cps MOMP, thereby realizing the polygenic synergistic immune effect and providing test basis and theoretical support for the research of gene vaccines of IBV and Cps.
Description
Technical Field
The invention belongs to the field of genetic bioengineering, and relates to a method for predicting and designing dominant B, T cell epitopes of chicken Infectious Bronchitis Virus (IBV) S1 protein and avian Chlamydia psittaci (Cps) MOMP protein, synthesizing and constructing recombinant eukaryotic plasmid pEGFP-N1, in particular to a method for obtaining IBV S1 gene and Cps MOMP gene epitope prediction sequence (M) and constructing recombinant pEGFP-N1 vector by using bioinformatics and an application thereof.
Background
Infectious Bronchitis (IB) and avian chlamydia (Ac) are the most common respiratory diseases in poultry and often present with mixed infections, causing serious economic losses to the poultry industry. Currently, the prevention of IBV and Cps is mainly achieved by using inactivated and attenuated vaccines. Because the traditional vaccine has the defects of immunosuppression, non-neutralization epitope, potential disease treatment area, easy toxin return and the like, the traditional vaccine is unfavorable for disease control, and therefore, the research of the novel vaccine is particularly critical. Epitope vaccines exhibit some advantages including high specificity, good safety, easiness in production and storage, strong stability and the like, so that the epitope vaccines are increasingly focused in the field of vaccine research. In addition, the genetic engineering bigeminal vaccine can reduce the stress and save the cost when the animal is immunized, and the aim of preventing multiple animals by one needle is fulfilled.
The aim of the research is to carry out epitope screening on IBV S1 genes and Cps MOMP genes through online biological software, the screened epitopes are connected through connecting peptide, a new peptide segment M with high immunogenicity and small side effect is constructed, the new peptide segment M can firstly start self cell and humoral immunity function after entering an organism, and then the immune reaction is started through IBV S1 and Cps MOMP specific antigens, so that the polygenic synergistic immune effect is realized, and experimental basis and theoretical support are provided for gene vaccine research of IBV and Cps.
Disclosure of Invention
The invention aims to solve the technical problems that the infectious bronchitis and the chlamydia disease of chickens are the two most common infectious diseases in the poultry industry, have similar clinical symptoms, are not easy to distinguish and are mixed infection. Avian chlamydia can infect humans through avian infections, not only causing serious economic losses to the poultry industry, but also causing a threat to human health. Therefore, there is a real need to develop a vaccine that can prevent both diseases together. Relevant reports on the prediction and design of IBV S1 epitope and Cps MOMP epitope exist at present, but research reports on multi-epitope DNA vaccine based on IBVS1 and Cps MOMP are not found yet. The test aims at establishing a multi-epitope DNA vaccine which can simultaneously prevent infectious bronchitis and avian chlamydia.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention firstly discloses three groups of recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP, which are characterized in that the nucleotide sequence of the inserted genes of the plasmids is shown as SEQ ID NO. 1-3.
The invention further discloses a construction method of the recombinant plasmid, which is characterized by comprising the following steps:
1) Firstly, predicting dominant antigen epitopes of an IBV S1 gene and a Cps MOMP gene, connecting the screened epitopes, analyzing physicochemical properties, hydrophilicity, protein secondary and tertiary structures, and constructing a peptide segment which has good antigenicity and no side effect and is named as M;
2) Constructing 3 groups of recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP by taking pEGFP-N1 as a vector; and the recombinant eukaryotic expression plasmid is introduced into competent cells by a cold-hot alternation methodDH5α；
3) The recombinant plasmid is transfected into DF-1 cells to successfully express fluorescence, and Western Blotting and IFA tests prove that the target protein is successfully expressed;
4) The recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP are used for immunizing 7-day-old chicks, and the specific antibody, the cytokine and the T lymphocyte molecular level are detected;
5) Pathological tissue sections and organ index measurements were performed.
The invention also discloses a recombinant eukaryotic expression plasmid of the IBV S1 and Cps MOMP genes bigeminal epitope genes. And constructing a recombinant eukaryotic expression vector pEGFP-N1 and evaluating the immunity level of the chicks.
The invention also discloses application of the construction method of the recombinant eukaryotic expression vector pEGFP-N1 of the IBV S1 and Cps MOMP and the epitope protein M in preventing infectious bronchitis and avian chlamydia. The experimental results show that: the constructed epitope protein has stable structure, good immunogenicity and no side effect; the recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP are verified to be consistent with the expected size, and can be normally expressed when transfected into DF-1 cells. The levels of anti-IBV IgG and anti-Cps IgG antibodies were significantly increased in the chicken serum of the 3 plasmid-immunized group after four-immunization (P < 0.01) compared to the control group (pEGFP-N1 and PBS groups); the levels of cytokines IFN-gamma and IL-4, and the levels of T lymphocyte antigens CD3+, CD4+ and CD8+ in chicken serum were significantly or very significantly higher than in the control group (pEGFP-N1 group and PBS group) (P <0.05 or P < 0.01). The result shows that the constructed multi-epitope DNA vaccine pEGFP-M can excite humoral immunity and cellular immune response of chickens, and lays a foundation for developing the multi-epitope vaccine for simultaneously preventing infectious bronchitis and avian chlamydia.
The invention is described in more detail below:
the construction and application of the IBV S1 and Cps MOMP gene bivalent epitope vaccine are characterized by comprising the following steps:
1) For the amino acid sequences of IBV S1 and Cps MOMP proteins provided on NCBI, accession numbers are respectively: AF193423.1 and L25436.1. Screening dominant epitopes of B, T cells of two genes by using online biological prediction software, connecting the dominant epitopes by using a connecting peptide and naming the dominant epitopes as M; predicting the secondary structure, physicochemical property, hydrophilicity, antigenicity and the like of M.
2) Respectively constructing 3 groups of recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP by taking pEGFP-N1 as a vector; and the recombinant eukaryotic expression plasmid is introduced into competent cells by a cold-hot alternation methodDH5α。
3) The recombinant plasmid is transfected into DF-1 cells, and the expression condition of the recombinant plasmid at the cellular level is verified by Western Blotting and IFA tests.
4) Animal test: recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP were used to immunize 7-day-old chicks at a dose of 200. Mu.g, and two weeks after each immunization, fin vein blood was collected, and total immunization was performed 4 times, and the control group was injected with PBS. Detection of IL-4, IFN-gamma and CD3 by ELISA + 、CD4 + 、CD8 + Content of the components.
IBV S1 and Cps MOMP gene bigeminal epitope gene recombination eukaryotic expression plasmid.
Construction of a eukaryotic expression vector pEGFP-N1 by using a bigeminal epitope gene recombination of IBV S1 and Cps MOMP genes and evaluation of chicken immune effect.
The recombinant plasmids are pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP. Wherein the pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP gene sequences are as follows:
pEGFP-M：
CTCGAGGCCACCATGGGAATGGCTTGGTCTAAAAGCCAGTTTTGTAGCGCTCATTGCAATTTTGGACCTGGACCTGGCACTGGATCTGAACAGGTGGAGAATCAGTTTTATGTGAAGCTGACAAATGGACCAGGACCAGGAGCTGTGGTGAATAGTACAAATTATTCTAATAATGCTGGCAGCGCTCCTGGACCAGGCCCAGGACATGGATCTAGAATTCAGACTAGAACAGAGCCACTGGTGTTGACCCAGGGACCTGGACCAGGAACAGGAGAGGCTAGACTGATTAATGAAAGGGCTGCCCATATGAACGCTGGACCTGGCCCAGGAGAAGGCGCATCAGGAGATCCTTGCGATCCTTGCAGTACATGGTGTGATGGACCTGGACCTGGACTGGGCGAAGCTACAATGCTGGATACAAGCAACAAGTTCTCCGATTTCGGCCCACTGAGCACCAACTTCACCTTCACCAACGTGGGACCCGGCCCTGGCAGCGCCTTCAGACCCCCCAACGGCTGGCACCTGGGCCCAGGCCCCGGCAGCGCTCACTGCAACTTCAGCGAGATCGGCCCCGGCCCCGGCCAGTACGCCCAGAGCAACCCCAAGATCGGCCCAGGCCCAGGCAGCGCCGCCTTCAACCTGGTGGGCCTGGGCCCCGGCCCTGGCCTGAGCTACAGGCTGAACATGCTGGGCCCCCTGAGCGCCAGCAACGGCTACTTCAAGGCCAGCAGCGCCGCCTTCAACCTGGGCCCCGGACCCGGCCTGGGCGCCGAGTTCCAGTACGCCCAGAGCAACCCCAAGATCGAGGATTACAAGGATGACGACGATAAGGTCGAC
pEGFP-S1-MOMP：
CTCGAGGCCACCATGTTGGGGAAGTCACTGTTTTTAGTGACCATTTTGTGTGCACTATGTAGTGCAAATTTGTTCGATTCTGCTAATAATTATGTGTACTACTACCAAAGTGCCTTTAGGCCTCCAAATGGATGGCATTTGCAAGGGGGTGCTTATGCAGTAGTGAATTCCACTAATTATAGTAATAATGCAGGTTCTGCACCTCAGTGCACTGTTGGTGTTATTAAGGACGTCTATAATCAAAGTGCGGCTTCTATAGCTATGACAGCACCTCTTCAGGGTATGGCTTGGTCTAAGTCACAATTTTGTAGTGCACACTGTAACTTTTCTGAAATTACAGTTTTTGTCACACATTGTTATAGTAGTGGTAGCGGGTCTTGTCCTATAACAGGCATGATTCCACGTGATCATATTCGTATTTCTGCAATGAAAAATGGTTCTTTATTTTATAATTTAACAGTTAGCGTATCTAAATACCCTAATTTTAAATCTTTTCAATGTGTTAACAACTTCACATCTGTTTATTTAAATGGTGATCTTGTTTTTACTTCCAACAAAACTACTGATGTTACGTCAGCAGGTGTGTATTTTAAAGCAGGTGGACCTGTAAATTATAATATTATGAAAGAATTTAAGGTTCTTGCTTACTTTGTTAATGGTACAGCACAAGATGTAATTTTGTGCGATAATTCCCCCAAGGGTTTGCTAGCCTGTCAATATAACACTGGCAATTTTTCAGATGGCTTTTATCCTTTTACTAATAGTACTTTAGTTAGGGAAAAGTTCATTGTCTATCGCGAAAGTAGTGTTAATACTACTCTGGCGTTAACTAATTTCACTTTTACTAATGTAAGTAATGCACAGCCTAATAGTGGTGGTGTTAATACTTTTCATTTATATCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTGTCATTTCTGAGTCAGTTTGTGTATAAGGCAAGTGATTTTATGTATGGGTCTTACCACCCTAGTTGTTCTTTTAGACCAGAAACCATTAATAGTGGTTTGTGGTTTAATTCCTTGTCAGTTTCTCTTACTTATGGACCCCTACAGGGAGGGTGTAAGCAATCTGTTTTTAGTGGTAAGGCAACGTGTTGTTATGCCTACTCTTATAATGGCCCAAGGGCATGTAAAGGTGTTTATTCAGGTGAATTAAGCATGAATTTTGAATGTGGATTGCTGGTTTATGTTACTAAGAGTCATGGCTCTCGTATACAGACTAGAACGGAGCCCTTAGTATTAACGCAACACAATTATAATAATATTACTTTAGATAAGTGTGTTGCTTATAATATATATGGCAGAGTAGGCCAAGGTTTTATTACTAATGTGACTGATTCTGCTGCTAATTTTAGTTATTTAGCAGATGGTGGGTTAGCTATTTTAGATACGTCGGGTGCCATAGATGTTTTTGTTGTAAAGGGCAGCTATGGTCTTAATTATTACAAGGTTAATCCTTGTGAAGATGTTAACCAACAGTTTGTAGTGTCTGGTGGCAATATAGTTGGCATTCTTACTTCTAGAAATGAAACAGGTTCTGAACAGGTTGAGAACCAGTTTTATGTTAAGTTAACCAATAGCTCACATCGTCGCAGGCGTTCTATTGGCCAAAACGTAACAACTTGCCCTTATGTTGATTACAAGGATGACGACGATAAGGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGAAAAAACTCTTGAAATCGGCATTATTATTTGCCGCTACGGGTTCCGCTCTCTCCTTACAAGCCTTGCCTGTAGGGAACCCAGCTGAACCAAGTTTATTAATCGATGGCACTATGTGGGAAGGTGCTTCAGGTGATCCTTGCGATCCTTGCTCTACTTGGTGTGATGCTATCAGCATCCGCGCAGGATACTACGGAGATTATGTTTTCGATCGTGTATTAAAAGTTGATGTGAATAAAACTTTCAGCGGCATTGGCAAGAAACCCACAGGATCCTCTCCAAATGACTTTAAAAATGCTGAAGATAGACCCAACGTCGCTTATGGCAGACATTTGCAAGACTCCGAATGGTTTACAAATGCAGCTTTCTTAGCGTTAAATATCTGGGATCGTTTTGATATTTTCTGCACATTAGGCGCTTCTAATGGGTACTTCAAAGCTAGTTCTGCGGCATTCAATCTCGTTGGTTTGATTGGTGTTAAAGGAAGCTCCTTAACAAATGACCAACTTCCCAACGTAGCCATCACTCAAGGCGTTGTTGAGTTTTACACAGATACAACGTTCTCTTGGAGCGTAGGTGCACGTGGAGCTCTATGGGAATGTGGTTGCGCAACTTTAGGAGCTGAATTCCAATACGCTCAATCTAATCCTAAAATTGAAATGTTGAATGTAATCTCCAGCCCAGCACAATTTGTGGTTCACAAGCCTAGAGGATACAAGGGAACGTCCGCCAACTTTCCTTTACCTGCAAATGCAGGCACAGAGGCTGCTACGGATACTAAATCTGCAACACTCAAATATCATGAATGGCAAGTTGGTCTAGCACTCTCTTACAGATTGAACATGTTAGTTCCTTACATTGGCGTAAACTGGTCACGAGCAACTTTTGATGCCGACACTATCCGCATCGCTCAACCTAAATTGGCCTCTGCTGTTATGAACTTGACCACATGGAACCCAACCCTTTTAGGGGAAGCCACAATGCTTGATACTTCCAATAAATTCAGTGACTTCTTACAAATCGCTTCGATTCAGATCAACAAAATGAAGTCTAGAAAAGCTTGCGGTTTAGCTATTGGTGCAACGTTAATCGACGCCGACAAATGGTCAATCACTGGTGAAGCACGCTTAATCAATGAAAGAGCTGCTCACATGAATGCTCAATTCAGATTCGATTACAAGGATGACGACGATAAGGTCGAC
pEGFP-M-S1-MOMP：
ATGGGAATGGCTTGGTCTAAAAGCCAGTTTTGTAGCGCTCATTGCAATTTTGGACCTGGACCTGGCACTGGATCTGAACAGGTGGAGAATCAGTTTTATGTGAAGCTGACAAATGGACCAGGACCAGGAGCTGTGGTGAATAGTACAAATTATTCTAATAATGCTGGCAGCGCTCCTGGACCAGGCCCAGGACATGGATCTAGAATTCAGACTAGAACAGAGCCACTGGTGTTGACCCAGGGACCTGGACCAGGAACAGGAGAGGCTAGACTGATTAATGAAAGGGCTGCCCATATGAACGCTGGACCTGGCCCAGGAGAAGGCGCATCAGGAGATCCTTGCGATCCTTGCAGTACATGGTGTGATGGACCTGGACCTGGACTGGGCGAAGCTACAATGCTGGATACAAGCAACAAGTTCTCCGATTTCGGCCCACTGAGCACCAACTTCACCTTCACCAACGTGGGACCCGGCCCTGGCAGCGCCTTCAGACCCCCCAACGGCTGGCACCTGGGCCCAGGCCCCGGCAGCGCTCACTGCAACTTCAGCGAGATCGGCCCCGGCCCCGGCCAGTACGCCCAGAGCAACCCCAAGATCGGCCCAGGCCCAGGCAGCGCCGCCTTCAACCTGGTGGGCCTGGGCCCCGGCCCTGGCCTGAGCTACAGGCTGAACATGCTGGGCCCCCTGAGCGCCAGCAACGGCTACTTCAAGGCCAGCAGCGCCGCCTTCAACCTGGGCCCCGGACCCGGCCTGGGCGCCGAGTTCCAGTACGCCCAGAGCAACCCCAAGATCGAGGATTACAAGGATGACGACGATAAGGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCCCTGCAGATGTTGGGGAAGTCACTGTTTTTAGTGACCATTTTGTGTGCACTATGTAGTGCAAATTTGTTCGATTCTGCTAATAATTATGTGTACTACTACCAAAGTGCCTTTAGGCCTCCAAATGGATGGCATTTGCAAGGGGGTGCTTATGCAGTAGTGAATTCCACTAATTATAGTAATAATGCAGGTTCTGCACCTCAGTGCACTGTTGGTGTTATTAAGGACGTCTATAATCAAAGTGCGGCTTCTATAGCTATGACAGCACCTCTTCAGGGTATGGCTTGGTCTAAGTCACAATTTTGTAGTGCACACTGTAACTTTTCTGAAATTACAGTTTTTGTCACACATTGTTATAGTAGTGGTAGCGGGTCTTGTCCTATAACAGGCATGATTCCACGTGATCATATTCGTATTTCTGCAATGAAAAATGGTTCTTTATTTTATAATTTAACAGTTAGCGTATCTAAATACCCTAATTTTAAATCTTTTCAATGTGTTAACAACTTCACATCTGTTTATTTAAATGGTGATCTTGTTTTTACTTCCAACAAAACTACTGATGTTACGTCAGCAGGTGTGTATTTTAAAGCAGGTGGACCTGTAAATTATAATATTATGAAAGAATTTAAGGTTCTTGCTTACTTTGTTAATGGTACAGCACAAGATGTAATTTTGTGCGATAATTCCCCCAAGGGTTTGCTAGCCTGTCAATATAACACTGGCAATTTTTCAGATGGCTTTTATCCTTTTACTAATAGTACTTTAGTTAGGGAAAAGTTCATTGTCTATCGCGAAAGTAGTGTTAATACTACTCTGGCGTTAACTAATTTCACTTTTACTAATGTAAGTAATGCACAGCCTAATAGTGGTGGTGTTAATACTTTTCATTTATATCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAATTTGTCATTTCTGAGTCAGTTTGTGTATAAGGCAAGTGATTTTATGTATGGGTCTTACCACCCTAGTTGTTCTTTTAGACCAGAAACCATTAATAGTGGTTTGTGGTTTAATTCCTTGTCAGTTTCTCTTACTTATGGACCCCTACAGGGAGGGTGTAAGCAATCTGTTTTTAGTGGTAAGGCAACGTGTTGTTATGCCTACTCTTATAATGGCCCAAGGGCATGTAAAGGTGTTTATTCAGGTGAATTAAGCATGAATTTTGAATGTGGATTGCTGGTTTATGTTACTAAGAGTCATGGCTCTCGTATACAGACTAGAACGGAGCCCTTAGTATTAACGCAACACAATTATAATAATATTACTTTAGATAAGTGTGTTGCTTATAATATATATGGCAGAGTAGGCCAAGGTTTTATTACTAATGTGACTGATTCTGCTGCTAATTTTAGTTATTTAGCAGATGGTGGGTTAGCTATTTTAGATACGTCGGGTGCCATAGATGTTTTTGTTGTAAAGGGCAGCTATGGTCTTAATTATTACAAGGTTAATCCTTGTGAAGATGTTAACCAACAGTTTGTAGTGTCTGGTGGCAATATAGTTGGCATTCTTACTTCTAGAAATGAAACAGGTTCTGAACAGGTTGAGAACCAGTTTTATGTTAAGTTAACCAATAGCTCACATCGTCGCAGGCGTTCTATTGGCCAAAACGTAACAACTTGCCCTTATGTTGATTACAAGGATGACGACGATAAGGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTATGAAAAAACTCTTGAAATCGGCATTATTATTTGCCGCTACGGGTTCCGCTCTCTCCTTACAAGCCTTGCCTGTAGGGAACCCAGCTGAACCAAGTTTATTAATCGATGGCACTATGTGGGAAGGTGCTTCAGGTGATCCTTGCGATCCTTGCTCTACTTGGTGTGATGCTATCAGCATCCGCGCAGGATACTACGGAGATTATGTTTTCGATCGTGTATTAAAAGTTGATGTGAATAAAACTTTCAGCGGCATTGGCAAGAAACCCACAGGATCCTCTCCAAATGACTTTAAAAATGCTGAAGATAGACCCAACGTCGCTTATGGCAGACATTTGCAAGACTCCGAATGGTTTACAAATGCAGCTTTCTTAGCGTTAAATATCTGGGATCGTTTTGATATTTTCTGCACATTAGGCGCTTCTAATGGGTACTTCAAAGCTAGTTCTGCGGCATTCAATCTCGTTGGTTTGATTGGTGTTAAAGGAAGCTCCTTAACAAATGACCAACTTCCCAACGTAGCCATCACTCAAGGCGTTGTTGAGTTTTACACAGATACAACGTTCTCTTGGAGCGTAGGTGCACGTGGAGCTCTATGGGAATGTGGTTGCGCAACTTTAGGAGCTGAATTCCAATACGCTCAATCTAATCCTAAAATTGAAATGTTGAATGTAATCTCCAGCCCAGCACAATTTGTGGTTCACAAGCCTAGAGGATACAAGGGAACGTCCGCCAACTTTCCTTTACCTGCAAATGCAGGCACAGAGGCTGCTACGGATACTAAATCTGCAACACTCAAATATCATGAATGGCAAGTTGGTCTAGCACTCTCTTACAGATTGAACATGTTAGTTCCTTACATTGGCGTAAACTGGTCACGAGCAACTTTTGATGCCGACACTATCCGCATCGCTCAACCTAAATTGGCCTCTGCTGTTATGAACTTGACCACATGGAACCCAACCCTTTTAGGGGAAGCCACAATGCTTGATACTTCCAATAAATTCAGTGACTTCTTACAAATCGCTTCGATTCAGATCAACAAAATGAAGTCTAGAAAAGCTTGCGGTTTAGCTATTGGTGCAACGTTAATCGACGCCGACAAATGGTCAATCACTGGTGAAGCACGCTTAATCAATGAAAGAGCTGCTCACATGAATGCTCAATTCAGATTC
infectious Bronchitis (IB) and avian chlamydia (Ac) are the most common respiratory diseases in poultry and often present with mixed infections, causing serious economic losses to the poultry industry. Currently, the prevention of IBV and Cps is mainly achieved by using inactivated and attenuated vaccines. Because the traditional vaccine has the defects of immunosuppression, non-neutralization epitope, potential disease treatment area, easy toxin return and the like, the traditional vaccine is unfavorable for disease control, and therefore, the research of the novel vaccine is particularly critical. Epitope vaccines exhibit some advantages including high specificity, good safety, easiness in production and storage, strong stability and the like, so that the epitope vaccines are increasingly focused in the field of vaccine research. In addition, the genetic engineering bigeminal vaccine can reduce the stress and save the cost when the animal is immunized, and the aim of preventing multiple animals by one needle is fulfilled.
The aim of the research is to carry out epitope screening on IBV S1 genes and Cps MOMP genes through online biological software, the screened epitopes are connected through connecting peptide, a new peptide segment M with high immunogenicity and small side effect is constructed, the new peptide segment M can firstly start self cell and humoral immunity function after entering an organism, and then the immune reaction is started through IBV S1 and Cps MOMP specific antigens, so that the polygenic synergistic immune effect is realized, and experimental basis and theoretical support are provided for gene vaccine research of IBV and Cps.
The construction method of the IBV S1 and Cps MOMP gene bivalent epitope vaccine has the following beneficial effects: the constructed 3-group recombinant plasmid pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP bigeminal recombinant epitope prediction vaccine has higher safety, can promote animals to generate immune response, and provides a strategy for preventing IBV and Cps in the poultry industry in China.
Drawings
FIG. 1 is a predicted map of M-sequence hydrophilicity and transmembrane region; wherein A, hydrophilic prediction; B. predicting a transmembrane region;
FIG. 2 is an M-sequence secondary structure analysis;
FIG. 3 is an M tertiary structure model;
FIG. 4 is a recombinant plasmid map; wherein A, pEGFP-N1 plasmid; B. pEGFP-M plasmid; C. pEGFP-S1-MOMP plasmid; D. pEGFP-M-S1-MOMP plasmid;
FIG. 5 is the construction and identification of recombinant plasmids;
a: identification result of recombinant plasmid pEGFP-M (M1.D2000 DNA Marker;1. Plasmid; 2. Colony PCR;3. Double enzyme digestion); b: identification result of recombinant plasmid pEGFP-S1-MOMP (M1.D2000 DNA Marker;1. Plasmid; 2. Colony PCR;3. Double enzyme digestion); c: identification result of recombinant plasmid pEGFP-M-S1-MOMP (M2.DL10001DNA Marker;1. Plasmid; 2. Colony PCR;3. Double enzyme digestion);
FIG. 6 is the result of transfection of DF-1 cells with recombinant plasmid (400X);
FIG. 7 is the results of CCK-8 experiments;
FIG. 8 is a Western Blot detection result;
FIG. 9 is the localization of recombinant plasmids in cells (400X);
FIG. 10 is anti-IBV and anti-Cps IgG antibody levels in immunized chicken serum;
FIG. 11 is the cytokines IFN-gamma and IL-4 content;
FIG. 12 is a T lymphocyte surface molecule CD3 + 、CD4 + And CD8 + Detecting;
FIG. 13 is a graph showing the result of organ weight ratio;
fig. 14 is histopathological section results (h.e., 200×).
Detailed Description
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are all commercially available.
Example 1
The invention constructs recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP by using M, S and MOMP genes, comprising the following steps:
1) Designing and synthesizing M gene sequences;
2) Constructing recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP;
3) Transfecting recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP in DF-1 cells;
4) The recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP were used to immunize 7-day-old chicks, and the immunization effect was evaluated.
The method specifically comprises the following steps:
1) The epitope was predicted and screened based on the amino acid sequence of CDS region of chicken infectious bronchitis virus S1 (AF 193423.1) gene and Chlamydia psittaci MOMP (L25436.1) protein provided in NCBI GeneBank. And selecting a sequence with higher score according to a predicted result, sequentially connecting the sequence through a flexible peptide GPGPG and a rigid peptide GPLS, finally naming the sequence as an M sequence, and analyzing the secondary structure, the transmembrane domain and the hydrophilicity of the M sequence through bioinformatics software.
2) The new peptide segment M is connected with S1 and MOMP genes in sequence by using T2A connecting peptide, amino acid sequence optimization is carried out according to experimental animals, flag tags are added at the 3' end of each segment of genes and are constructed into pEGFP-N1, and the pEGFP-M-S1-MOMP is sent to Shanghai biological limited company for synthesis, thus obtaining recombinant plasmid pEGFP-M-S1-MOMP.
3) Performing double enzyme digestion verification on the recombinant plasmid pEGFP-M-S1-MOMP, and then performing glue recovery, and connecting the product of the T4 ligase overnight to a eukaryotic expression vector pEGFP-N1 to obtain a recombinant plasmid pEGFP-M, pEGFP-S1-MOMP; and the recombinant eukaryotic expression plasmid is introduced into competent cells by a cold-hot alternation methodDH5α。
4) The recombinant plasmid is transfected into DF-1 cells, and the expression condition of the recombinant plasmid at the cellular level is verified by Western Blotting and IFA tests.
5) Animal test: recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP were used to immunize 7-day-old chicks at a dose of 200. Mu.g, and two weeks after each immunization, fin vein blood was collected, and total immunization was performed 4 times, and the control group was injected with PBS. Detection of IL-4, IFN-gamma and CD3 by ELISA + 、CD4 + 、CD8 + The content is as follows.
The IBV S1 and Cps MOMP gene epitope predictive gene M and S1, MOMP bigeminal epitope gene recombination eukaryotic expression plasmid.
The construction of the IBV S1 and Cps MOMP gene epitope prediction gene M and S1 and MOMP bigeminal epitope gene recombination eukaryotic expression vector pEGFP-N1 and the evaluation of chicken immune effect are described.
That is to say: dominant epitope sequences of S1 and MOMP genes are designed and named as M; m, S1 and MOMP genes are connected by T2A shearing peptide after optimized by preferential codons, and synthesized into a PEGFP-N1 vector to obtain the pEGFP-M-S1-MOMP; after being recovered by double enzyme digestion, the T4 ligase is connected with the product to a eukaryotic expression vector pEGFP-N1 overnight to obtain a recombinant plasmid pEGFP-M, pEGFP-S1-MOMP; by lipofilter TM The liposome transfection method transfects DF-1 cells with 3 groups of recombinant eukaryotic plasmids; the expression of the target protein in DF-1 cells was detected by fluorescence microscopy, IFA and Western Blotting. Group 3 plasmids were immunized on 7-day-old chicks and their effects were evaluated.
The invention will be described in further detail with reference to the following specific examples:
1. materials and methods
1.1 Design and Synthesis of M sequences
The epitopes were screened for prediction based on the nucleotide sequences in the NCBI database for the IBV QX strain S1 gene sequence (GeneBank: AF 193423.1) and the Cps C strain MOMP gene sequence (GeneBank: L25436.1). Screening B cell epitopes of S1 and MOMP genes by online software ABCPred and IEBD; the online software IEBD and NetCTLpan are used for screening TH epitope and CTL epitope of S1 and MOMP genes, and candidate epitopes are sequentially connected by using a flexible peptide GPGPG and a rigid peptide GPLS and are named as M. And predicting and analyzing biological information such as physicochemical property, hydrophilicity, transmembrane region, protein secondary and tertiary structure and the like by utilizing ProtParam, exPASy, vaxiJen, TMHMM, SOPMA and other online software.
1.2 Construction and identification of recombinant plasmids
The screened antigen epitope M, S1 and MOMP genes are constructed into a eukaryotic expression vector pEGFP-N1, named pEGFP-M-S1-MOMP, and are synthesized by a division company of biological engineering (Shanghai). The plasmids were double digested with restriction enzymes Xho I, sal I and Pst I, respectively, and the digestion system is shown in Table 1. Recovering the enzyme digestion products M, S-MOMP and pEGFP-N1 by using a DNA recovery kit, and utilizing T to recover M, S-MOMP gene fragments and pEGFP-N1 large fragments 4 DNA ligase was used for ligation, and the ligation system was shown in Table 2 at 16℃overnight; and the recombinant eukaryotic expression plasmid is introduced into competent cells DH5 alpha by a cold-hot alternation method.
Table 1 double cleavage reaction System
Table 2 connection system
1.3 Identification of pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP
The recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP were verified by PCR, the primers described in Table 4 were added in the following system (Table 3), mixed well, and the target fragment was amplified in a PCR instrument. The PCR products and the cleavage products were electrophoresed on a 1% agarose gel and photographed using a gel apparatus. Simultaneously, the PCR product was sent to the company for sequencing.
TABLE 3 PCR reaction System
TABLE 4 primers for amplifying fragments of interest
1.4 Transcription and expression of recombinant plasmids
Resuscitates DF-1 cells, places them in DMEM containing 10% FBS, and takes 1.2X10 s after the cells are in good condition 6 Cells were plated in six well plates per mL. When the cell growth is 60% -70%, lipo8000 is utilized TM The plasmid was transfected with the transfection reagent, the transfection system is shown in Table 5, and the plasmid was placed at 37℃in 5% CO 2 Culturing in an incubator.
After 48 and h transfection, the original DMEM medium is discarded, and the DMEM medium is washed 1 to 2 times for 5 minutes each time with 1 XPBS. Cell lysates (PMSF: ripa=1:1000) were prepared on ice, 200 μl per well was added and lysed on ice for 30 min. Collecting the cracked proteins with a cell scraper, transferring to a clean and sterile 1.5 mL centrifuge tube, centrifuging at 12000 r/min for 5 min, collecting supernatant, adding 1×loading buffer, boiling in a metal bath at 100deg.C for 15 min, cooling, packaging, and preserving at-80deg.C.
Protein electrophoresis was performed by preparing 10% SDS-PAGE separating gel, performing transfer membrane using a semi-dry apparatus, blocking 2 h with 5% skimmed milk powder solution at room temperature by shaking table, respectively using mouse anti-Flag monoclonal antibody and mouse anti-GADPH monoclonal antibody as primary antibodies, horseradish peroxidase-labeled goat anti-mouse IgG antibody as secondary antibodies, developing color using ECL chemiluminescent solution, and photographing and preserving in a Bio-Rad gel imager.
TABLE 5 transfection System
1.5 Recombinant plasmid immunized chick
50 chickens (broilers) of 1 day old were randomly divided into pEGFP-M, pEGFP-S1-MOMP, pEGFP-M-S1-MOMP, pEGFP-N1 and PBS groups, the groupings are shown in Table 6; the first immunization was carried out by feeding to 7 days of age, and the recombinant plasmid group chicken was intramuscular injected with 200. Mu.g/150. Mu.L/chicken, and the PBS group chicken was intramuscular injected with 150. Mu.L of 1 XPBS. The boost interval was 14 d, 4 total immunizations.
Table 6 test animal groups
1.6 ELISA for detecting humoral immunity and cellular immunity level
After first-free, 28, d and 56, d randomly selecting 6 chicks/group of lower wing veins to collect blood, standing at 37 ℃ for 2 h, centrifuging at 3500 r/min for 5 min, and separating serum to be detected. According to the ELISA kit operation steps, the serum antibodies IgG to be detected of 28 d and 56 d after the first time are detected respectively, and the serum cytokine to be detected of 56 d after the first time and CD3 are detected + 、CD4 + 、CD8 + Content, absorbance at 450 nm was measured for each sample using a microplate reader.
2. Results and analysis
2.1 B, T cell epitope screening results
Dominant peptide fragments of S1 and MOMP proteins were screened out using various on-line software (tables 7, 8), and epitope protein M was constructed by sequentially performing ligation using the flexible peptide GPGPG and the rigid peptide GPLS.
Table 7B cell epitope peptide fragment
Table 8T cell epitope peptide fragment
2.2 Physicochemical Properties
The physicochemical properties of the M protein are predicted and analyzed by using on-line software ProtParam, and the result shows that: the M protein codes 264 amino acids in total, and the relative molecular mass of the M protein is 26.499 multiplied by 103; a theoretical isoelectric Point (PI) of 5.93; indicating that the M protein is an acidic protein; the total number of negatively charged residues (aspartic acid+glutamic acid) was 15 and the total number of positively charged residues (arginine+lysine) was 12; total number of atoms 3605, half-life 30 h, fat index 47.05, instability index 13.77; vaxiJen predicts antigenicity as 0.6758 (greater than 0.4 is antigenic). The result shows that the epitope protein M is an acidic protein, is relatively stable and has good antigenicity.
2.3 Hydrophilic, transmembrane region and phosphorylation site prediction results
ProtScale tool predictions in the on-line software ExpASY showed that the average hydrophilicity of M protein (GRAVY) was-0.463 (FIG. 1A); TMHMM prediction showed that the M protein has no transmembrane region (fig. 1B); netPhos-3.1 analysis of the amino acid position of the M protein revealed that the amino acid sequence of the M protein had 13 serine phosphorylation sites, 7 threonine phosphorylation sites, and 4 tyrosine phosphorylation sites. The results suggest that epitope protein M is a hydrophilic protein and has no transmembrane region, and a total of 24 phosphorylation sites.
2.4 Spatial structure analysis
The secondary structure of the M protein was evaluated using biological software SOPMA and showed 5.30% alpha helix, 1.89% beta sheet, 27.27% extended chain and 65.53% random coil (fig. 2). The three-level structure of M protein was predicted by using the on-line software SWISS-MODEL homology modeling method, and the highest scoring MODEL was selected from the results (FIG. 3).
2.5 Verification result of recombinant plasmid pEGFP-M, pEGFP-S1-MOMP, pEGFP-M-S1-MOMP
The PCR test shows that the band with the size corresponding to the target fragment is obtained. The recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP are successfully constructed and can be used for the next test.
2.6 Recombinant eukaryotic plasmid transfection DF-1 cell
Recombinant plasmids pEGFP-M, pEGFP-S1-MOMP, pEGFP-M-S1-MOMP and pEGFP-N1 were transfected into DF-1 cells, and the transfection was observed under a fluorescence microscope after 48. 48 h. The results showed that all 3 recombinant plasmids had green fluorescence, no-load groups had green fluorescence weaker than the target genome, and no-load groups had green fluorescence (FIG. 6), indicating successful transfection of the recombinant plasmids into DF-1 cells.
2.7 Post-transfection cell safety assessment
The safety of the recombinant plasmid was evaluated by CCK-8 method. Recombinant plasmids pEGFP-M, pEGFP-S1-MOMP, pEGFP-M-S1-MOMP and empty recombinant plasmids were transfected into DF-1 cells at concentration gradients of 50 ng, 100 ng, 200 ng and 400 ng, respectively, and OD values were measured in a microplate reader at 24 h, 48 h and 72 h after transfection, and the detection results were analyzed by SPSS (FIG. 7). The results show that the difference between groups of cell viability at each concentration is not obvious (P & gt 0.05), the cell viability is totally reduced along with the increase of the cell culture time, and compared with a control group, the total no obvious difference (P & gt 0.05) indicates that the constructed recombinant plasmid has better safety and no obvious toxic effect on cells.
2.8 Western Blot detection of protein expression
The recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP are transfected into DF-1 cells, and protein expression after 48 h is detected by Western Blot. The results showed that pEGFP-M and pEGFP-S1-MOMP exhibited specific bands at 31 kDa and 136 kDa, respectively, pEGFP-M-S1-MOMP simultaneously expressed the target bands at 31 kDa and 136 kDa, whereas the pEGFP-N1 group and the empty cell group were not seen, and the GADPH internal control was incubated with bands at 36 kDa. The constructed recombinant plasmid proteins were demonstrated to be successfully expressed in DF-1 cells, with band sizes consistent with expectations (FIG. 8).
2.9 Indirect immunofluorescence detection protein localization
The localization of the 3 recombinant plasmids in DF-1 cells was examined by indirect Immunofluorescence (IFA). Recombinant plasmids pEGFP-M, pEGFP-S1-MOMP, pEGFP-M-S1-MOMP were transfected into DF-1 cells, and empty plasmid pEGFP-N1 was used as a blank. After incubating the Flag-tagged primary antibody and the Alxa-flower 647 secondary antibody, the cells were observed for protein expression under a fluorescence microscope. The results showed that the 3 recombinant plasmid groups all showed fluorescent markers in the cytoplasm, whereas the empty groups did not see fluorescence, indicating successful expression of the recombinant plasmid protein in the cytoplasm (fig. 9).
2.10 IBV, cps specific antibody level detection results
ELISA detects IgG antibody levels against IBV and Cps in the isolated serum (see FIG. 10). As a result, it was found that the anti-IBV IgG antibodies and anti-Cps IgG antibodies were significantly higher in the sera of the three immunized groups M, C and T groups of chickens than in the control group (E and PBS groups) (P < 0.01) respectively, at 28 d and 56 d after the first immunization. However, no significant differences were found between the three recombinant plasmid groups (P > 0.05) regardless of the anti-IBV IgG antibody content or the anti-Cps IgG antibody content in the chicken serum, and more importantly, no significant differences were found between the antibody levels in the E and PBS groups (P > 0.05) at all times.
2.11 Serum cytokine detection results
ELISA was used to detect IFN-gamma and IL-4 levels in serum of immunized chickens (see FIG. 11). The results showed that the content of IFN- γ in the serum of recombinant plasmid group M was significantly higher than that of group C, group T and control (group E and PBS) (P < 0.01); the IFN-gamma content of groups C and T was significantly higher than that of group E (P < 0.05), and significantly higher than that of PBS (P < 0.01); while there was no significant difference between groups C and T (P > 0.05). The content of IL-4 in the serum of the recombinant plasmids of the chicken of the group M, the group C and the group T is extremely higher than that of the control group (the group E and the group PBS) (P < 0.01). Moreover, there was no significant difference in IFN-. Gamma.and IL-4 levels between the E and PBS groups (P > 0.05).
2.12 Results of the T lymphocyte molecule CD3+, CD4+ and CD8+ assays
ELISA detection of T lymphocyte surface molecule CD3 in immune chicken serum + 、CD4 + 、CD8 + Content (see fig. 12). CD3 in serum of recombinant plasmid M group immune chicken + And CD4 + The content is significantly or very significantly higher than that of the group C, the group T and the control group (group E and PBS group) (P<0.05 or P<0.01 A) is provided; the cd3+ and cd4+ content of groups C and T were significantly or very significantly higher than that of the control group (group E and PBS group) (P<0.05 or P<0.01 With no significant difference between groups C and T)Iso (P)>0.05 A) is provided; the cd8+ content in the serum of the M group immunized chickens was extremely remarkable (P<0.01 Higher than the control group (group E and PBS), the CD8+ content in the serum of the immunized chicken of the group C and the group T is significantly higher than that of the control group (group E and PBS) (P<0.05 No significant differences between the 3 recombinant plasmids (P)>0.05). Furthermore, there was no significant difference in the levels of cd3+, cd4+ and cd8+ (P > 0.05) between control group E and PBS groups.
2.13 Effect of recombinant plasmid on organs of test animals
After the four-way-free step 14 d, the chickens were sacrificed after weighing, and the heart, liver, spleen, lung and kidney were picked and weighed, and the results are shown in fig. 13. The figure shows that the organ indexes of each group have no obvious difference, and the prepared vaccine has no obvious side effect on organ organs and has better safety.
2.14 Analysis of results of tissue and pathological sections
After chick sacrifice, heart, liver, spleen, lung and kidney were picked up and rapidly placed in 4% histiocyte fixative solution, and the results after fixation, embedding, slicing and h.e. staining are shown in fig. 14. The pictures show that the cell structure of 3 groups of test groups is complete, the morphology is clear, necrosis, proliferation and obvious inflammatory changes do not occur, and the constructed recombinant plasmid is proved to generate obvious injury to animal organisms, so that the safety is higher.
3. Conclusion(s)
1) The dominant antigen epitopes of the IBV S1 gene and the Cps MOMP gene are predicted by utilizing a plurality of online software such as ABCPred, IEDB, bepipred, the screened epitopes are connected, and physicochemical properties, hydrophilicity, protein secondary structure, tertiary structure and the like are analyzed, so that a peptide segment which has good antigenicity and no side effect is successfully constructed and named as M.
2) The pEGFP-N1 is used as a vector to successfully construct 3 groups of recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP.
3) The recombinant plasmid is transfected into DF-1 cells, the foreign protein is efficiently expressed in the cells, and the cells are not obviously damaged.
4) Recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP were used to immunize 7-day-old chicks, and IL-4 and IFN- -Gamma and CD3 + 、CD4 + 、CD8 + The content shows that the content of recombinant plasmid is obviously higher than that of PBS, the constructed plasmid can induce organisms to generate cellular and humoral responses, can simultaneously generate two antibodies of IBV and Cps, and the immune effect of pEGFP-M is better than that of other two groups.
5) Pathological tissue section and organ index results show that the recombinant plasmid has no obvious inhibition effect on main organs and growth states of the chicks after immunization, and has higher safety.
The invention is characterized in that the method firstly carries out epitope screening on IBV S1 protein and Cps MOMP protein by online biological software, connects the screened epitopes by connecting peptide, constructs new protein M with high immunogenicity and low side effect, and makes the protein M efficiently excite cellular immunity and humoral immunity reaction of organisms after immunization, and research results are: lays a foundation for developing multi-epitope DNA vaccine for simultaneously preventing infectious bronchitis and avian chlamydia.
The above-described embodiments are only for illustrating the technical spirit and features of the present invention, and it is intended to enable those skilled in the art to understand the content of the present invention and to implement it accordingly, and the scope of the present invention is not limited to the embodiments, i.e. equivalent changes or modifications to the spirit of the present invention are still within the scope of the present invention.
Claims (5)
1. Three groups of recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP are characterized in that the nucleotide sequence of the plasmid insert gene is shown as SEQ ID NO. 1-3.
2. A method of constructing using the recombinant plasmid of claim 1, comprising the steps of:
1) Firstly, predicting dominant antigen epitopes of an IBV S1 gene and a Cps MOMP gene, connecting the screened epitopes, analyzing physicochemical properties, hydrophilicity, protein secondary and tertiary structures, and constructing a peptide segment which has good antigenicity and no side effect and is named as M;
2) Using pEGFP-N1 as carrier to construct 3 groupsRecombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP; and the recombinant eukaryotic expression plasmid is introduced into competent cells by a cold-hot alternation methodDH5α；
3) The recombinant plasmid is transfected into DF-1 cells to successfully express fluorescence, and Western Blotting and IFA tests prove that the target protein is successfully expressed;
4) The recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP are used for immunizing 7-day-old chicks, and the specific antibody, the cytokine and the T lymphocyte molecular level are detected;
5) Pathological tissue sections and organ index measurements were performed.
3. The IBV S1 and Cps MOMP gene bigeminal epitope gene recombinant eukaryotic expression plasmid of claim 2.
4. Construction of the IBV S1 and Cps MOMP gene bigeminal epitope gene recombinant eukaryotic expression vector pEGFP-N1 according to claim 2 and chicken immunity level evaluation.
5. Use of the recombinant plasmids pEGFP-M, pEGFP-S1-MOMP and pEGFP-M-S1-MOMP according to claim 1 for the prevention of infectious bronchitis and avian chlamydia diseases.
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