CN110170049B - Cytokine gene adjuvant for chicken coccidiosis live vaccine - Google Patents

Cytokine gene adjuvant for chicken coccidiosis live vaccine Download PDF

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CN110170049B
CN110170049B CN201910473538.7A CN201910473538A CN110170049B CN 110170049 B CN110170049 B CN 110170049B CN 201910473538 A CN201910473538 A CN 201910473538A CN 110170049 B CN110170049 B CN 110170049B
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pci
chicken
plasmid
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CN110170049A (en
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郑明学
贡鑫
郝飞飞
白瑞
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Shanxi Agricultural University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0003—Invertebrate antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a cytokine gene adjuvant for chicken coccidiosis live vaccine, which is a fusion gene recombinant plasmid pCI-chIL-4-chIL-2 constructed by taking chicken interleukin 4 (chIL-4) and chicken interleukin 2 (chIL-2) as objects. The recombinant plasmid constructed by the invention can transfect the cecum epithelial cells, the chick embryo fibroblasts and the myoblasts, and express and secrete the IL-2-IL-4 fusion protein. The chicken coccidiosis live vaccine is subjected to drip immunization and intramuscular injection of the cytokine gene adjuvant, so that the chicken spleen lymphocyte proliferation can be promoted, the chicken coccidiosis live vaccine has obvious synergism, and the adverse effect of vaccine immunization on chicken weight gain can be reduced.

Description

Cytokine gene adjuvant for chicken coccidiosis live vaccine
Technical Field
The invention relates to an adjuvant for chicken coccidiosis live vaccines, in particular to an adjuvant matched with chicken coccidiosis live vaccines, and the adjuvant is a fusion gene recombinant plasmid constructed by fusion of chIL-2 and chIL-4 genes and has a synergistic effect on chicken coccidiosis live vaccines.
Background
Chicken coccidiosis is a very serious global parasitic disease caused by eimeria parasitism in chicken intestinal tracts. The chicken coccidiosis live vaccine is used for immunoprophylaxis and control of chicken coccidiosis, and has become the most promising safe, stable and efficient method for preventing and controlling the chicken coccidiosis.
However, the current live vaccine for chicken coccidiosis has slow immunoprotection, long immunoprotection idle period and easy infection and morbidity of chicken farm with severe coccidiosis pollution before immunity is generated. Meanwhile, adverse reactions of damaging intestinal mucosa can be generated during live vaccine immunization, so that the conversion rate of chicken feed is reduced, and the weight gain of the fast large broiler chickens is affected. This greatly limits its wide application in broilers, not to mention it is unfortunate.
The immune effect of the chicken coccidiosis live vaccine is positively correlated with the reduction of the chicken feed conversion rate, so that the immune effect of the chicken coccidiosis live vaccine can be improved while the chicken coccidiosis live vaccine inoculation dosage is reduced, and the chicken coccidiosis live vaccine is a key for solving the problem.
Cytokines (cytokines) are a class of proteins or small molecule polypeptides secreted by immune cells and other cells, with immunomodulatory and immunoadjuvant effects. Cytokines can activate and regulate immune cells in vivo, and play an adjuvant role by regulating or enhancing the immune effect of the vaccine through different action links. Cytokines have many advantages as immunoadjuvants: 1) The operation is simple, the preparation is easy, and the eukaryotic expression plasmid can be inserted for mass preparation; 2) The sustainability, the plasmid constructed by the cell factor can enable the factor to express for a long time in the animal body, thus achieving the effect of continuously enhancing the immune effect; 3) Rapidity, which can directly act on antigen presenting cells to rapidly perform immune reaction; 4) Is safe and nontoxic, and has no side effect to organism as immune effector in organism.
Interleukin 2 (IL-2) is one of the most widely used immunological adjuvants at present, and can promote the activation and proliferation of macrophages, natural killer cells, T lymphocytes and B lymphocytes. Under the action of IL-2, monocytes are converted into dendritic cells and combined with other cytokines, so that the movement of the dendritic cells and the aggregation of the dendritic cells in the lung and the skin are promoted, the antigen presenting capacity of the dendritic cells is enhanced, and the immune response is regulated. IL-2 also enhances CTL cell activity, promotes cell contact, induces cells to secrete cytokines, and also synergistically acts with other cytokines to induce proliferation of B lymphocytes to produce antibodies and differentiate memory cells. IL-2 as a new adjuvant can not only avoid adverse reactions of conventional adjuvants, but also remarkably improve the immune efficacy of viral, bacterial and parasitic vaccines, improve antibody levels, prolong the duration of antibodies, and reduce side effects of partial vaccines. Many domestic and foreign researches report that the IL-2 recombinant protein has an immune enhancement effect in chicken embryos and chicken bodies, can enhance the protective capability after vaccination and improve the resistance of organisms to coccidium. The recombinant chicken IL-2 protein can reduce coccidium infection pathological changes and oocyst yield and improve relative weight gain. IL-2 recombinant proteins act rapidly but not permanently, whereas IL-2 eukaryotic expression plasmids can maintain higher levels of immunity and a long-lasting immunization time.
Interleukin 4 (IL-4) was found by Howard in 1982 and successfully cloned in 1986. It is an immunocompetent regulator with multiple biological functions, and plays an important role in the immune process. IL-4 can promote B cell mediated IgE immune response, induce T cell proliferation to differentiate Th2 cells, enhance humoral immunity, inhibit secretion of macrophage IFN-gamma, stimulate proliferation of mast cells, activate macrophages and enhance antigen presenting ability of B cells. IL-4 has the effect of activating dendritic cells to present antigens.
IL-2 has been reported to have a broad effect of up-regulating immune response in immune networks, and can shorten the time for immune production and enhance immune function. IL-4 can enhance antigen presenting ability of macrophages, guide monocytes to differentiate toward DC, enhance antigen presenting ability, and shorten immune generation period.
The recombinant fusion protein is a new product with multifunctional fusion protein by combining cell factor and the coding gene of special functional protein by using genetic engineering and protein engineering technology. The fusion protein can play a synergistic effect by fusing the cytokine with the functional active domain of other molecules, so that the biological activity of the protein is greatly enhanced.
Recombinant fusion proteins can be classified into cytokine-antibody fusion proteins, cytokine-toxin fusion proteins, dual cytokine fusion proteins, cytokine and receptor fusion proteins, cytokine-other molecule fusion proteins, and the like, according to biological functions. The research of a plurality of domestic scholars discovers that the fusion protein co-expressed by two different cytokine genes can exert respective biological activities, has better immunoadjuvant effect, has very broad scientific significance and application prospect, opens up more new ways and brings new hopes for preventing and controlling animal diseases.
Disclosure of Invention
The invention aims to provide a cytokine gene adjuvant for a chicken coccidiosis live vaccine, which is used together with the chicken coccidiosis live vaccine, so that the immune effect of the chicken coccidiosis live vaccine can be improved, the side effect of the chicken coccidiosis live vaccine can be reduced, and particularly the weight gain of chickens can be influenced.
The cytokine gene adjuvant for chicken coccidiosis live vaccine is recombinant plasmid containing chicken IL-4-chIL-2 fusion gene and constructed by taking chicken IL-4 (chIL-4) gene and chicken IL-2 (chIL-2) gene as objects and connecting the genes on eukaryotic expression plasmid.
More specifically, the cytokine gene adjuvant for chicken coccidiosis live vaccine is fusion gene recombinant plasmid pCI-chIL-4-chIL-2 constructed by eukaryotic expression plasmid pCI, chIL-4 gene and chIL-2 gene.
The invention also provides a construction method of the cytokine gene adjuvant for chicken coccidiosis live vaccine, which is to design chIL-4 gene with 453bp gene fragment and chIL-2 gene with 429bp gene fragment respectively, clone the genes on plasmid pUC57, transform Top 10 engineering bacteria, extract pUC57-chIL-4 plasmid and pUC57-chIL-2 plasmid after enrichment; then respectively connecting pUC57-chIL-4 plasmid and pUC57-chIL-2 plasmid with eukaryotic expression plasmid pCI by double enzyme digestion, transforming DH5 alpha competent cells by the connection products, and extracting bacterial liquid DNA after enrichment to obtain eukaryotic expression plasmids pCI-chIL-4 and pCI-chIL-2 respectively; then, the eukaryotic expression plasmid pCI-chIL-4 and pUC57-chIL-2 are subjected to double enzyme digestion, an IL-2 DNA fragment is connected to the pCI-chIL-4, DH5 alpha competent cells are transformed by the connection product, bacterial liquid DNA is extracted after enrichment, and the fusion gene recombinant plasmid pCI-chIL-4-chIL-2 is constructed.
The invention adds in the upstream of the gene according to the chicken IL-4 gene sequence (sequence number: AJ 621249.1) published by Gen BankEcoRI enzyme cutting site, removing stop codon TGA of chicken IL-4 gene downstream of gene, adding linker sequence, addingKpnI enzyme cutting site, designing and synthesizing chIL-4 gene with target gene fragment about 453bp.
Linker sequence:
GGAGGCGGGGGTAGCGGAGGCGGGGGTAGCGGAGGCGGGGGTAGC[(G 4 S) 3 ]。
furthermore, the present invention was based on the chicken IL-2 gene sequence (SEQ ID NO: AF 000631.1) published by Gen Bank, and was added upstream of the geneKpnI enzyme cutting site, removing termination codon TAA of chicken IL-2 gene downstream of gene, addingSalI enzyme cutting site, designing and synthesizing chIL-2 gene of target gene fragment about 429bp.
According to the invention, the synthesized chIL-4 gene and the synthesized chIL-2 gene are cloned to a pUC57 vector to prepare a pUC57-chIL-4 plasmid and a pUC57-chIL-2 plasmid respectively.
Further, the invention is to double enzyme cut pUC57-chIL-4 plasmid and eukaryotic expression plasmid pCI, the enzyme cut product is identified by agarose gel electrophoresis and the target bands of about 453bp (chIL-4) and 4006bp (pCI) are recovered, eukaryotic expression plasmid pCI is connected with IL-4 DNA fragment to prepare eukaryotic expression plasmid pCI-chIL-4.
Similarly, the invention is to double enzyme cut pUC57-chIL-2 plasmid and eukaryotic expression plasmid pCI, the enzyme cut product is identified by agarose gel electrophoresis and the target bands of about 429bp (chIL-2) and 4006bp (pCI) are recovered, eukaryotic expression plasmid pCI is connected with IL-2 DNA fragment to prepare eukaryotic expression plasmid pCI-chIL-2 plasmid.
Finally, pUC57-chIL-2 plasmid and eukaryotic expression plasmid pCI-chIL-4 are digested, the digested products are identified and recovered by agarose gel electrophoresis to obtain target bands of about 429bp (chIL-2) and 4459bp (pCI-chIL-4), and eukaryotic expression plasmid pCI-chIL-4 is connected with IL-2 DNA fragment to prepare fusion gene recombinant plasmid pCI-chIL-4-chIL-2.
Furthermore, in order to characterize the influence of the cytokine gene adjuvant for the live vaccine of chicken coccidiosis on the immunity effect of the live vaccine of chicken coccidiosis and reduce side effects, the invention also constructs a fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP by connecting an EGFP gene on the constructed fusion gene recombinant plasmid pCI-chIL-4-chIL-2.
And carrying out gene sequencing identification on the constructed fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGPF, wherein the sequencing result shows that the construction of the pCI-chIL-4-chIL-2-EGFP is successful, and the gene sequence is as follows.
pCIGAATTCATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAACGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGCAAAAAGGAAACCTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAACAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAGGAGGCGGGGGTAGC GGAGGCGGGGGTAGCGGAGGCGGGGGTAGC GGTACCATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAACGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGCAAAAAGGAAACCTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAACAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAGTCGAC EGFP
The recombinant plasmid of the pCI-chIL-4-chIL-2-EGFP fusion gene constructed in the above way transfects the cecum epithelial cells, the chick embryo fibroblasts and the myoblasts, and the relative expression quantity and the protein quantity of mRNA are measured by Q-PCR and a fluorescence spectrophotometer, so that the result shows that the recombinant plasmid of the pCI-chIL-4-chIL-2-EGFP fusion gene can promote proliferation of the chick spleen lymphocytes, and express and secrete IL-2 and IL-4 fusion proteins.
The invention cultures DH5 alpha competent cell bacterial liquid containing fusion gene recombinant plasmid pCI-chIL-4-chIL-2, dissolves the fusion gene recombinant plasmid pCI-chIL-4-chIL-2 after extraction and purification in ddH 2 And (3) preparing the cytokine gene adjuvant.
Preferably, in the cytokine gene adjuvant prepared by the invention, the concentration of the fusion gene recombinant plasmid pCI-chIL-4-chIL-2 is 2-4 mg/ml.
Further, when in use, the prepared cytokine gene adjuvant is treated with ddH 2 The solution was diluted to a concentration of pCI-chIL-4-chIL-2 of 1. Mu.g/. Mu.l.
The cytokine gene adjuvant prepared by the invention is used for being combined with chicken coccidiosis live vaccine and is used for immunization of chicken coccidiosis.
The specific application method is that the chicken coccidiosis live vaccine drip immunization is carried out, and simultaneously, the cytokine gene adjuvant is injected into the muscle, and the injection amount is 50-200 mug/chicken coccidiosis live vaccine drip immunization calculated by fusion gene recombinant plasmid.
Preferably, the injection amount is 100. Mu.g/g of the recombinant plasmid fusion gene.
The cytokine gene adjuvant prepared by the invention can promote proliferation of chicken spleen lymphocytes, has obvious synergism on chicken coccidiosis trivalent four-strain live vaccines, and can reduce adverse effects of immunization of the vaccine on chicken weight gain.
Drawings
FIG. 1 shows the results of the double cleavage assay of plasmids pUC57-chIL-4 and pUC57-chIL-2. In the figure: m is DL 5000 DNA maker.
FIG. 2 shows the results of double digestion of plasmids pCI-chIL-4, pCI-chIL-2 and pCI-chIL-4-chIL-2. In the figure: 1 is pCI-chIL-4;2 is pCI-chIL-2;3 is pCI-chIL-4-chIL-2; m is DL 5000 DNA maker.
FIG. 3 shows the results of double restriction enzyme identification of eukaryotic expression plasmids pCI-chIL-4-EGFP, pCI-chIL-2-EGFP and pCI-chIL-4-chIL-2-EGFP. In the figure: 1.2 is pCI-chIL-4-EGFP; 3. 4 is pCI-chIL-4-chIL-2-EGFP; 5. 6 is pCI-chIL-2-EGFP; m is DL 5000 DNA maker.
FIG. 4 shows the transfection efficiency of the cecal epithelial cells of the chick embryos transfected with each eukaryotic expression plasmid.
FIG. 5 is a graph showing the transfection effect of each eukaryotic expression plasmid on the transfection of chicken embryo cecum epithelial cells.
FIG. 6 is a graph showing the result of electrophoresis of PCR products of IL-2 and IL-4. In the figure: 1 and 5 are IL-2;2 and 6 are IL-4;3 is chicken beta-actin; 4 is murine beta-actin; m is DL 1000 DNA maker.
FIG. 7 shows the relative expression levels of mRNA from the cecal epithelial cells of transfected chick embryos.
FIG. 8 shows the protein expression levels of the transfected chick embryo cecal epithelial cells.
FIG. 9 is the secretion of expressed protein from the cecum epithelial cells of transfected chick embryos.
FIG. 10 shows the transfection efficiency of chicken embryo fibroblasts transfected with each eukaryotic expression plasmid.
FIG. 11 is a graph showing the transfection effect of each eukaryotic expression plasmid on chicken embryo fibroblasts.
FIG. 12 shows the relative expression levels of mRNA from transfected chick embryo fibroblasts.
FIG. 13 shows the protein expression levels of transfected chick embryo fibroblasts.
FIG. 14 shows the secretion of the protein expressed by the fibroblasts of transfected chick embryos.
FIG. 15 shows the transfection efficiency of mouse myoblasts transfected with each eukaryotic expression plasmid.
FIG. 16 is a graph showing the effect of transfection of mouse myoblasts with each eukaryotic expression plasmid.
FIG. 17 shows the relative expression levels of mRNA from myoblasts of transfected mice.
FIG. 18 shows the protein expression levels of the myoblasts of the transfected mice.
FIG. 19 is a graph showing the secretion of proteins expressed by myoblasts from transfected chicken mice.
FIG. 20 shows the lymphoproliferative activity of expression products of cecal epithelial cells transfected with chick embryos at different times.
FIG. 21 is a comparison of lymphoproliferative activity of expression products of cecal epithelial cells transfected with respective eukaryotic expression plasmids.
FIG. 22 shows the lymphoproliferative activity of the expression product of the fibroblasts transfected with chick embryos at different times.
FIG. 23 is a comparison of lymphoproliferative activity of the expression products of the eukaryotic expression plasmids transfected into chick embryo fibroblasts.
FIG. 24 shows lymphoproliferative activity of the expression product of myoblasts from mice transfected at different times.
FIG. 25 is a comparison of lymphoproliferative activity of the expression products of myoblasts of mice transfected with each eukaryotic expression plasmid.
Detailed Description
The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention in any way. Various modifications and variations of the present invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
The gene sequences of the genes involved in the embodiments of the present invention have been disclosed, and thus the present invention does not provide a gene sequence table of the genes involved.
Example 1: design and Synthesis of chicken IL-4 and IL-2 genes.
According to the chicken IL-4 gene sequence (sequence number AJ 621249.1) published by Gen Bank, additional nucleotide sequences are added at the upstream of the gene EcoR IEnzyme cutting site, removing stop codon TGA of chicken IL-4 gene downstream of gene, adding Linker sequence, adding Kpn ICleavage site. The size of the target gene fragment is about 453bp.
Linker sequence:
GGAGGCGGGGGTAGCGGAGGCGGGGGTAGCGGAGGCGGGGGTAGC[(G 4 S) 3 ]。
according to the chicken IL-2 gene sequence (sequence number AF 000631.1) published by Gen Bank, add on the upstream of the gene Kpn IEnzyme cutting site, removing termination codon TAA of chicken IL-2 gene downstream of gene, adding Sal ICleavage site. The size of the target gene fragment is about 429bp.
The chIL-4 and chIL-2 genes are synthesized and cloned on a plasmid pUC57 respectively, top 10 engineering bacteria are transformed, and plasmids pUC57-chIL-4 and pUC57-chIL-2 are extracted after enrichment.
The plasmids pUC57-chIL-4 and pUC57-chIL-2 were subjected to double restriction identification.
1) Plasmid pUC57-chIL-4 was subjected to a treatmentEcoR I andKpn i double digestion identification, 453bp fragment band and about 2.7kb vector band can be excised (see FIG. 1).
2) Plasmid pUC57-chIL-2 was subjected to a treatmentKpn I andSal i double digestion identification, can cut out a fragment band of about 429bp and a vector band of about 2.7kb (see FIG. 1).
Example 2: eukaryotic expression plasmids pCI-chIL-4, pCI-chIL-2 and fusion gene recombinant plasmids pCI-chIL-4-chIL-2 are constructed.
The plasmid pUC57-chIL-4 was usedEcoRI andKpni double cleavage, pUC57-chIL-2 useKpn I andSal i double enzyme digestion, eukaryotic expression plasmid pCI is used respectivelyEcoRI andKpn I、Kpn i andSal and I, double enzyme digestion to obtain pCI, respectively connecting the pCI with an IL-4 DNA fragment and an IL-2 DNA fragment, transforming DH5 alpha competent cells by using a connecting product, and extracting bacterial liquid DNA after enrichment to obtain eukaryotic expression plasmids pCI-chIL-4 and pCI-chIL-2.
The eukaryotic expression plasmids pcI-chIL-4 and pcI-chIL-2 were subjected to double restriction identification.
1) Eukaryotic expression plasmid pCI-chIL-4 is passed throughEcoR I andKpn i double restriction enzyme identification, a fragment band of about 453bp and a plasmid band of about 4kb (see FIG. 2) can be excised, and it was initially demonstrated that the IL-4 gene fragment had been ligated to plasmid pCI.
2) Eukaryotic expression plasmid pCI-chIL-2 is passed throughKpn I andSal i double restriction identification, a fragment band of about 429bp and a plasmid band of about 4kb (see FIG. 2) can be excised, and it was initially demonstrated that the IL-2 gene fragment had been ligated to plasmid pCI.
Eukaryotic expression plasmid pCI-chIL-4 and plasmid pUC57-chIL-2 were usedKpnI andSali double enzyme cutting, connecting the obtained IL-2 DNA fragment with pCI-chIL-4, transforming DH5 alpha competent cells by the connection product, extracting bacterial liquid DNA after enrichment to obtain fusion gene recombinant plasmid pCI-chIL-4-chIL-2。
Double enzyme digestion identification is carried out on the fusion gene recombinant plasmid pCI-chIL-4-chIL-2.
Fusion gene recombinant plasmid pCI-chIL-4-chIL-2 is passed throughEcoR I andSal i double restriction enzyme identification, about 888bp fragment band and about 4kb plasmid band can be cut (see figure 2), preliminary demonstration of IL-4-IL-2 fusion gene fragment has been connected to plasmid pCI.
Example 3: eukaryotic expression plasmids pCI-chIL-4-EGFP, pCI-chIL-2-EGFP and fusion gene recombinant plasmids pCI-chIL-4-chIL-2-EGFP are constructed.
Eukaryotic expression plasmid pCI-chIL-4 was usedKpnI andNoti double enzyme cutting, eukaryotic expression plasmid pCI-chIL-2 and fusion gene recombinant plasmid pCI-chIL-4-chIL-2SalI andNoti double enzyme digestion, plasmid pEGFP-N3 respectivelyKpnI andNot I、Sali andNoti double digestion, the digested products were identified and recovered by agarose gel electrophoresis for the target bands of about 4.4kb (pCI-chIL-4), 4.4kb (pCI-chIL-2), 4.9kb (pCI-chIL-4-chIL-2) and 750bp (EGFP), and EGFP genes were ligated to eukaryotic expression plasmids pCI-chIL-4, pCI-chIL-2 and fusion gene recombinant plasmid pCI-chIL-4-chIL-2, respectively, to construct eukaryotic expression plasmids pCI-chIL-4-EGFP, pCI-chIL-2-EGFP and fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP.
Double enzyme digestion identification is carried out on eukaryotic expression plasmids pCI-chIL-4-EGFP, pCI-chIL-2-EGFP and fusion gene recombinant plasmids pCI-chIL-4-chIL-2-EGFP.
1) Subjecting the obtained eukaryotic expression plasmid pCI-chIL-4-EGFP to treatmentKpnI andNoti double restriction enzyme identification, about 750bp and about 4.4kb fragment bands can be cut out (see FIG. 3), and the EGFP gene fragment is initially proved to be connected to the eukaryotic expression plasmid pCI-chIL-4.
2) Subjecting the obtained eukaryotic expression plasmid pCI-chIL-2-EGFP to treatmentSalI andNoti double restriction enzyme identification, about 750bp and about 4.4kb fragment bands can be cut out (see FIG. 3), and the EGFP gene fragment is initially proved to be connected to the eukaryotic expression plasmid pCI-chIL-2.
3) The obtained fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP is subjected to the processSalI andNoti double restriction enzyme identification can cut out about 750bp and about 4.9kb fragment bands (see FIG. 3), and initially prove that EGFP gene fragment has been connected to fusion gene recombinant plasmid pCI-chIL-4-chIL-2.
The constructed eukaryotic expression plasmids pCI-chIL-2-EGFP, pCI-chIL-4-EGFP and fusion gene recombinant plasmids pCI-chIL-4-chIL-2-EGFP are sent to Beijing Liuhua big gene technology Co.Ltd for sequencing.
The sequencing results were aligned with published chicken IL-4 gene sequence (SEQ ID NO: AJ 621249.1), chicken IL-2 gene sequence (SEQ ID NO: AF 000631.1) and EGFP gene sequence (SEQ ID NO: U57609.1) using NCBI BLAST, which showed that the ligated chIL-4, chIL-2 and EGFP gene sequences were all perfectly correct (100% match), indicating successful construction of eukaryotic expression plasmids pCI-chIL-2-EGFP, pCI-chIL-4-EGFP and fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP.
The gene sequence of the fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP constructed by the invention is as follows:
pCIGAATTCATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAACGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGCAAAAAGGAAACCTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAACAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAGGAGGCGGGG GTAGCGGAGGCGGGGGTAGCGGAGGCGGGGGTAGC GGTACCATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAACGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGCAAAAAGGAAACCTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAACAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAGTCGAC EGFP。
example 4: transfection effect of eukaryotic expression plasmid on chick embryo cecum epithelial cells.
A1 is taken as an empty load group; a2 is pCI-chIL-2-EGFP group; a3 is pCI-chIL-4-EGFP group; a4 is pCI-chIL-4-chIL-2-EGFP group. Example 5 below is the same. The result of the comparison between the groups, ** or (b) ## Representing extremely significant differenceP＜0.01）， * Representing significant differenceP＜0.05）。
The A2 group is used for transfecting the cecal epithelial cells of the chick embryo for 24-96 hours, the cell transfection rate is firstly increased and then reduced, the difference between the time periods is not obvious, the peak value is reached in 48 hours (15.32% +/-1.62%), and the detection is not carried out in 96 hours.
The cell transfection effect of group A3 (16.21% + -1.29%) and group A4 (16.62% + -1.73%) was the same as that of group A2. No green fluorescent cells were seen for each time period in the empty group. See fig. 4 and 5 for details.
Example 5: eukaryotic expression plasmids transfect gene expression of chick embryo cecum epithelial cells.
1) mNRA expression of the transfected chick embryo cecal epithelial cells.
The results of the electrophoresis after extraction of RNA from each group of transfected cells, reverse transcription and PCR reaction are shown in FIG. 6.
In the figure, the band sizes of IL-2, IL-4, chicken beta-actin and mouse beta-actin are 155bp, 156bp, 135bp and 114bp respectively. And comparing the sequencing result with the gene sequence by Blast, wherein the homology of the genes is 99% and 99%, respectively, and the genes are proved to be IL-2 and IL-4 genes of chickens.
The dissolution curves of the target gene and the reference gene are unimodal, which indicates that the product is single and the result is reliable.
After the avian embryo cecum epithelial cells are transfected by the recombinant plasmids of A2, A3 and A4 respectively for 24-96 hours, the mRNA expression levels of chIL-2, chIL-4 and chIL-4-chIL-2 are respectively increased and then reduced, and the expression levels are extremely remarkable for 48 hoursP< 0.01) above the other time periods, see in detail fig. 7.
2) Protein expression of transfected chick embryo cecum epithelial cells.
The A2 group is used for transfecting the cecal epithelial cells of the chick embryo for 24-96 hours, the cumulative optical density value is firstly increased and then decreased, and the cumulative optical density value is extremely obvious for 72 hoursPLess than 0.01) is higher than 24 hours, and is remarkableP< 0.05) higher than 48h, up toThe peak value was undetectable for 96 hours.
The protein expression rule after the transfection of the cells of the A3 group and the A4 group is the same as that of the A2 group. No green fluorescent cells were seen for each time period in the empty group, as detailed in FIG. 8.
3) Secretion of expressed proteins by transfected chick embryo cecum epithelial cells.
The A2 group is used for transfecting the chick embryo cecum epithelial cells for 24-96 hours, the fluorescence intensity of cell supernatant is firstly increased and then decreased, and the fluorescence intensity is extremely obvious for 72 hoursP< 0.01) is higher than the remaining three time periods, reaching a peak.
The change rule of the fluorescence intensity of the supernatant after the transfected cells of the A3 group and the A4 group is the same as that of the A2 group, and the details are shown in figure 9.
Example 6: transfection effect of eukaryotic expression plasmid on chicken embryo fibroblasts.
B1 is taken as an empty load group; b2 is pCI-chIL-2-EGFP group; b3 is pCI-chIL-4-EGFP group; b4 is pCI-chIL-4-chIL-2-EGFP group. Example 7 below is the same. The result of the comparison between the groups, ** or (b) ## Representing extremely significant differenceP＜0.01）。
The B2 group is used for transfecting chicken embryo fibroblasts for 24-96 hours, the cell transfection rate is increased and then reduced, the difference between the time periods is not obvious, the peak value is reached in 48 hours (17.99% +/-1.35%), and the detection is not carried out in 96 hours.
The cell transfection effect of group B3 (16.86% + -1.55%) and group B4 (16.26% + -1.33%) was the same as that of group B2. No green fluorescent cells were seen in the empty cells for each period, as shown in FIGS. 10 and 11.
Example 7: eukaryotic expression plasmids transfect the gene expression of chicken embryo fibroblasts.
1) mNRA expression of transfected chick embryo fibroblasts.
After the chicken embryo fibroblasts are transfected by the recombinant plasmids of the groups B2, B3 and B4 for 24-96 hours, the mRNA expression levels of chIL-2, chIL-4 and chIL-4-chIL-2 are respectively increased and then reduced, and the expression levels are extremely remarkable for 48 hoursP< 0.01) above other time periods, see in detail fig. 12.
2) Protein expression of transfected chick embryo fibroblasts.
B2 group is used for transfecting chicken embryo fibroblasts for 24-96 hours, and the accumulated optical density value is firstly increased and then decreasedLow 72h cumulative optical density valueP< 0.01) is higher than 24h and 48h, reaches a peak value, and is undetectable for 96 h.
The protein expression rule after the transfection of the cells of the B3 group and the B4 group is the same as that of the cell of the B2 group. No green fluorescent cells were seen for each time period in the empty group, as detailed in FIG. 13.
3) Secretion of expressed proteins by transfected chick embryo fibroblasts.
B2 group is used for transfecting chicken embryo fibroblasts for 24-96 hours, the fluorescence intensity of cell supernatant is firstly increased and then decreased, and the fluorescence intensity of 72 hours is extremely obviousP< 0.01) is higher than the remaining three time periods, reaching a peak.
The change rule of the fluorescence intensity of the supernatant after the transfection of the cells in the B3 group and the B4 group is the same as that in the B2 group, and the details are shown in FIG. 14.
Example 8: transfection effect of eukaryotic expression plasmids in mouse myoblasts.
Taking T1 as an empty load group; t2 is pCI-chIL-2-EGFP group; t3 is pCI-chIL-4-EGFP group; t4 is pCI-chIL-4-chIL-2-EGFP group. Example 9 below is the same. The result of the comparison between the groups, ** or (b) ## Representing extremely significant differenceP＜0.01）， * Representing significant differenceP＜0.05）。
The T2 group is used for transfecting the myoblasts of the mice for 24-96 hours, the cell transfection rate is increased and then reduced, the difference between the time periods is not obvious, the peak value is reached in 48 hours (24.31% +/-1.60%), and the detection is not carried out in 96 hours.
The cell transfection effect of T3 group (25.41% + -1.65%) and T4 group (24.27% + -1.78%) was the same as that of T2 group. No green fluorescent cells were seen in the empty cells for each period, as shown in FIGS. 15 and 16.
Example 9: eukaryotic expression plasmids transfect gene expression in mouse myoblasts.
1) mNRA expression of transfected mouse myoblasts.
After the mouse myoblasts are transfected by the recombinant plasmids of the T2 group, the T3 group and the T4 group for 24-96 hours, the mRNA expression amounts of chIL-2, chIL-4 and chIL-4-chIL-2 are respectively increased and then reduced, and the expression amounts of 48 hours are extremely obviousP< 0.01) above the other time periods, see in detail fig. 17.
2) Protein expression in transfected mouse myoblasts.
The T2 group is used for transfecting the myoblasts of the mice for 24-96 hours, the cumulative optical density value is firstly increased and then decreased, and the cumulative optical density value for 72 hours is extremely obviousPLess than 0.01) is higher than 24 hours and 96 hours, and is remarkable in%P< 0.05) is higher than 48h, reaching a peak.
The protein expression rule of the T4 group cell transfected is the same as that of the T2 group cell transfected, and the 72h cumulative optical density value of the T3 group cell transfected is extremely obvious [ ]P< 0.01) is higher than the other time periods. No green fluorescent cells were seen for each time period in the empty group, as detailed in fig. 18.
3) Secretion of proteins expressed by transfected mouse myoblasts.
The T2 group is used for transfecting the myoblasts of the mice for 24-96 hours, the fluorescence intensity of cell supernatant is firstly increased and then decreased, and the fluorescence intensity of 72 hours is extremely obviousP< 0.01) is higher than the remaining three time periods, reaching a peak.
The change rule of the fluorescence intensity of the supernatant after the T3 group and the T4 group are transfected with the cells is the same as that of the T2 group, and the details are shown in FIG. 19.
The results of examples 4-9 above show that eukaryotic expression plasmids pCI-chIL-2-EGFP, pCI-chIL-4-EGFP and fusion gene recombinant plasmids pCI-chIL-4-chIL-2-EGFP can be transfected into cecum epithelial cells, fibroblasts and myoblasts, and target proteins chIL-2, chIL-4 and chIL-4-chIL-2 are expressed and secreted in the cells, and the expression and secretion amounts reach the peak for 72 hours.
Example 10: protein secretion activity of fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP.
1) Activity of expression product of transfected chick embryo cecum epithelial cells.
After the group A2, A3 and A4 plasmids are used for transfecting the chick embryo cecum epithelial cells for 24-96 hours, the proliferation activity (MTT stimulation index) of the expression products on the chick spleen lymphocytes is firstly increased and then decreased, and the proliferation activity is extremely remarkable for 72 hoursP< 0.01) is higher than the remaining three time periods, peak, see in detail fig. 20.
Furthermore, the proliferation activity (MTT stimulation index) of the expression products of the A2, A3 and A4 groups on chicken spleen lymphocytes is sequentially increased, wherein the A4 group is extremely remarkable [ (A4)P< 0.01) is higher than the A2 group and the A3 group, but the difference between the A2 group and the A3 group is not obviousP> 0.05), see the figure in detail21. In the figure: & the representative is significantly different from the A2 and A3 groups, && the representative differences were very significant compared to the A2, A3 groups.
2) Activity of expression product of supernatant of transfected chick embryo fibroblast.
After B2, B3 and B4 group plasmids transfect chicken embryo fibroblasts for 24-96 hours, the proliferation activity (MTT stimulation index) of the expression products on chicken spleen lymphocytes is firstly increased and then decreased, and the proliferation activity is extremely remarkable for 72 hoursP< 0.01) is higher than the remaining three time periods, peak, see in detail fig. 22.
Furthermore, the proliferation activity (MTT stimulation index) of the expression products of the B2, B3 and B4 groups on chicken spleen lymphocytes is sequentially increased, wherein the B4 group is extremely remarkable [ (B4)PLess than 0.01) is higher than the B2 group and the B3 group, but the difference between the B2 group and the B3 group is not obviousP> 0.05), see FIG. 23. In the figure: && the representative differences were very significant compared to the B2, B3 groups.
3) Activity of the expression product of the transfected mouse myoblasts.
After T2, T3 and T4 group plasmids are transfected into mouse myoblasts for 24-96 hours, the proliferation activity (MTT stimulation index) of the expression products on chicken spleen lymphocytes is firstly increased and then decreased, and the proliferation activity is extremely remarkable for 72 hoursP< 0.01) is higher than the remaining three time periods, peak, see in detail fig. 24.
Furthermore, the proliferation activity (MTT stimulation index) of the T2, T3 and T4 group expression products on mouse myoblasts is sequentially increased, wherein the T4 group is extremely remarkable [ ]P< 0.01) is higher than the T2 group and the T3 group, but the difference between the T2 group and the T3 group is not obviousP> 0.05), see figure 25 for details. In the figure: && the representative differences were very significant compared to the T2, T3 groups.
The result shows that the eukaryotic expression plasmids pCI-chIL-2, pCI-chIL-4 and fusion gene recombinant plasmids pCI-chIL-4-chIL-2 express secreted chIL-2, chIL-4 and chIL-4-chIL-2 can all improve the proliferation activity of chicken spleen lymphocytes in vitro, and the proliferation activity of fusion gene recombinant plasmids pCI-chIL-4-chIL-2 expressed secreted chIL-4-chIL-2 fusion protein on lymphocytes is obviously superior to that of single expressed chIL-2 or chIL-4.
Example 11: preparation of fusion gene recombinant plasmid pCI-chIL-4-chIL-2.
Thawing frozen pCI-chIL-4-chIL-2 transformed DH5 alpha competent cells at room temperature, streaking and inoculating into LB agar medium (Amp) + ) Culturing at 37 ℃ for 16h. Single colonies were picked and inoculated in 5mL LB broth (Amp + ) In the process, the culture is carried out at 37 ℃ for 4 to 9 hours in a shaking way.
According to the inoculation proportion of 1%, the bacterial liquid is sucked and inoculated into LB broth (Amp+), and the bacterial liquid is subjected to shaking culture at 37 ℃ for 12-16 h.
And (3) performing alkaline lysis on the bacterial liquid to obtain the crude plasmid. Adding CTAB solution into the supernatant of the alkaline pyrolysis liquid, standing for 10min at 37 ℃, and centrifuging for 5min with 10000 g; taking the supernatant, adding equal volume of isopropanol, and centrifuging for 5min at room temperature of 10000 g. Discarding the supernatant, drying at room temperature, adding ddH 2 O dissolves the precipitate and repeats 3 times. 3mL ddH was added after 3 rd time 2 O dissolves the precipitate. 3mL of MgCl with a final concentration of 0.6mol/L was added to the above solution 2 The solution is mixed by shaking, and centrifuged at 10000r/min for 5min at room temperature. Taking supernatant, adding equal volume of isopropanol, and centrifuging at 10000r/min for 5min. The supernatant was discarded, dried at room temperature for 10min, and 1mL ddH was added 2 O is dissolved. Endotoxin content, impurity protein content, DNA concentration and purity were determined. The endotoxin content of the purified plasmid should not exceed 0.05 EU/mug plasmid, the protein content of the purified plasmid should not exceed 10 mug/mg plasmid, and the DNA purity OD 260 /OD 280 The agarose gel electrophoresis should have no obvious RNA band in the range of 1.8 to 2.0. After meeting the requirements, ddH is used 2 O the concentration of fusion gene recombinant plasmid pCI-chIL-4-chIL-2 is regulated to 2-4 mg/ml, and stored at-20 deg.C, and ddH is used when in use 2 The solution was diluted to 1. Mu.g/. Mu.l with O to prepare pCI-chIL-4-chIL-2 gene adjuvant.
Example 12: immune synergism of pCI-chIL-4-chIL-2 gene adjuvant on chicken coccidiosis trivalent four-strain live vaccine.
60 chicks with weight approaching that of 4 days old were randomly divided into 6 groups of 10 chicks each.
Wherein, the T1-T3 group is an immunity plus adjuvant group, the C1 group is an immunity control group, the C2 group is a non-immunity toxicity attack group, and the C3 group is a blank control group.
The four trivalent live vaccine drop immunizations of chicken coccidiosis were performed at 4 and 14 days of age, respectively, and the gene adjuvants (doses were calculated as fusion gene recombinant plasmids) were intramuscular injected according to the immunization protocol of table 1.
Figure 2648DEST_PATH_IMAGE001
The remaining chicks, except for group C3, were weighed on an empty stomach at 28 days of age, each with E.tenella Shanxi isolate virulent strain (Tsx) 4.0X10 4 Strong strain (Msx) 15×10 of E.maxima mountain-western isolate 4 Strong strain (Msd) 15×10 of E.maxima Shandong isolate 4 Strong strain (Asx) 20×10 of E.acervulina mountain-western isolate 4 After mixing, 1 mL/capsule is prepared, and the stomach is irrigated to remove toxic substances.
The clinical status (mental state, appetite, faeces and death) of the chickens was observed and recorded 7 consecutive days after the challenge, and blood and faecal scores were made on day 5.
At the end of day 7, weighing on an empty stomach by empty stomach, collecting feces, calculating oocyst yield, and recording scores of duodenal, jejunum and cecum lesions by section inspection; mortality, average weight gain, relative Weight Gain (RWG), average intestinal lesions score, and oocyst reduction rate after challenge were calculated.
During the immunization period of the trivalent four live vaccine strains of the chicken coccidiosis (the period is 14d after 4-28 days of the immunization), each group of chicken has good spirit, normal appetite, no death of chicken, and the relative weight gain of each group of chicken is higher than 80 percent. Wherein the average weight gain of the T2 group (100 mug gene adjuvant) chicken is obviously higher than that of the C1 groupP< 0.05), the fusion gene recombinant plasmid pCI-chIL-4-chIL-2 (100 mug/chicken) has no influence on chicken weight gain, and can reduce adverse effect of chicken coccidiosis trivalent four-strain live vaccine immunity on chicken weight gain, and specific results are shown in Table 2.
Figure 378266DEST_PATH_IMAGE002
ImmunizationAfter detoxification, the average weight gain of the T2 group (100 mug gene adjuvant) chickens is obviously higher than that of the C1 group #PLess than 0.05), the pathological change score is obviously lower than that of C1 group #P< 0.05), 94.4% lower than the C1 group, and the relative weight gain rate is higher than 90%. The anticoccidial index (ACI) of the T2 group was 189, a high anticoccidial effect was achieved, whereas the anticoccidial index (ACI) 170.23 of the C1 group was only moderate.
The specific results are shown in Table 3, and the fusion gene recombinant plasmid pCI-chIL-4-chIL-2 can obviously improve the immune effect of the chicken coccidiosis live vaccine.
Figure 798883DEST_PATH_IMAGE003
Therefore, 100 mug/along with intramuscular injection of vaccine can reduce adverse effect of chicken coccidiosis trivalent four live vaccine immunity on chicken weight gain by taking fusion gene recombinant plasmid pCI-chIL-4-chIL-2 as gene adjuvant, and obviously improve immunity effect of chicken coccidiosis live vaccine.
Example 13: effect of pCI-chul-4-chul-2 gene adjuvant on chicken coccidiosis trivalent four live vaccine immunity generation period.
The 4-day-old chicks were weighed on an empty stomach, 150 close-weight chicks were selected, and randomly divided into 15 groups of 10 chicks each.
Wherein, B2 group is used as blank control group; the B3 group and the B4 group are virus attack control groups; the B11-B16 groups are immune virus-counteracting groups; the TB 11-TB 16 groups are immune adjuvant toxicity counteracting groups.
According to the immunization protocol in Table 4, four trivalent strains of coccidiosis were drip immunized at 4 and 14 days of age, respectively, while 100. Mu.g/dose (based on the fusion gene recombinant plasmid) of fusion gene recombinant plasmid gene adjuvant was intramuscular injected.
Figure 209136DEST_PATH_IMAGE004
The chickens of each group, except for group B2, were treated with E.tenella Shanxi isolate virulent strain (Tsx) 4.0X10 at various times after immunization as in Table 4 4 Strong strain (Msx) 15×10 of E.maxima mountain-western isolate 4 Strong strain (Msd) 15×10 of E.maxima Shandong isolate 4 Strong strain (Asx) 20×10 of E.acervulina mountain-western isolate 4 After mixing, 1 mL/capsule is prepared, and the stomach is irrigated to remove toxic substances.
The clinical status (mental state, appetite, faeces and death) of the chickens was observed and recorded for 7 consecutive days after the challenge, the chickens were tested by last section on day 7, and the scores of the lesions of the duodenum, jejunum and cecum were observed and recorded.
Each set of results should meet the following criteria: the death rate of the virus-attack control group chicken is more than or equal to 40%, the score of at least 9 chicken duodenum lesions and cecum lesions is more than or equal to 3, and the score of 9 chicken small intestine middle lesions is more than or equal to 2. Immune challenge group and immune adjuvant challenge group efficacy test criteria: the death rate is less than or equal to 10.0 percent, and the scores of the lesions of the duodenum, the middle section of the small intestine and the cecum of at least 8 chickens are less than or equal to 1.
Figure 758804DEST_PATH_IMAGE005
The test results in Table 5 show that in the immune adjuvant virus-fighting groups, the TB 12-TB 16 groups are more than or equal to 5d after the immunity is added, and the virus-fighting all reach the efficacy test standard specified by the vaccine. In the immune toxicity attack group without adjuvant, only the groups B14-B16 are more than or equal to 7d after the administration, and the toxicity attack reaches the efficacy test standard specified by the vaccine. In contrast, in the toxicity attack control group, the mortality of the chickens in the B3 group and the B4 group is respectively 60% and 50%, the scores of the lesions of the duodenum and the cecum of 10 chickens are more than or equal to 3, and the scores of the lesions of the middle section of the small intestine of 10 chickens are more than or equal to 2. Experiments prove that the fusion gene recombinant plasmid pCI-chIL-4-chIL-2 can shorten the immune generation period of the trivalent four-strain live vaccine for chicken coccidiosis by 28.6 percent.

Claims (5)

1. A cytokine gene adjuvant for chicken coccidiosis live vaccine is characterized in that chIL-4 gene and chIL-2 gene are used as fusion genes, and fusion gene recombinant plasmid pCI-chIL-4-chIL-2 constructed by connecting eukaryotic expression plasmid pCI is connected, and the gene sequence is as follows:
pCIGAATTCATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAACGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGCAAAAAGGAAACCTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAACAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAGGAGGCGGGGGTAG CGGAGGCGGGGGTAGCGGAGGCGGGGGTAGC GGTACCATGATGTGCAAAGTACTGATCTTTGGCTGTATTTCGGTAGCAACGCTAATGACTACAGCTTATGGAGCATCTCTATCATCAGCAAAAAGGAAACCTCTTCAAACATTAATAAAGGATTTAGAAATATTGGAAAATATCAAGAACAAGATTCATCTCGAGCTCTACACACCAACTGAGACCCAGGAGTGCACCCAGCAAACTCTGCAGTGTTACCTGGGAGAAGTGGTTACTCTGAAGAAAGAAACTGAAGATGACACTGAAATTAAAGAAGAATTTGTAACTGCTATTCAAAATATCGAAAAGAACCTCAAGAGTCTTACGGGTCTAAATCACACCGGAAGTGAATGCAAGATCTGTGAAGCTAACAACAAGAAAAAATTTCCTGATTTTCTCCATGAACTGACCAACTTTGTGAGATATCTGCAAAAAGTCGAC。
2. the cytokine gene adjuvant according to claim 1, wherein the eukaryotic expression plasmid pCI is connected with the chIL-4 gene and the chIL-2 gene, and is connected with a fusion gene recombinant plasmid pCI-chIL-4-chIL-2-EGFP constructed by EGFP gene.
3. The cytokine gene adjuvant according to claim 1, wherein the purified recombinant plasmid pCI-chIL-4-chIL-2 of the fusion gene is dissolved in ddH 2 In O, preparing the cytokine gene adjuvant with the concentration of 2-4 mg/ml of fusion gene recombinant plasmid.
4. The method for constructing a cytokine gene adjuvant according to claim 1, wherein the method for constructing comprises:
1) The chIL-4 gene with 453bp synthetic gene fragment and the chIL-2 gene with 429bp synthetic gene fragment are cloned to plasmid pUC57 respectively, top 10 engineering bacteria are transformed, and pUC57-chIL-4 plasmid and pUC57-chIL-2 plasmid are extracted after enrichment;
2) The pUC57-chIL-4 plasmid and the pUC57-chIL-2 plasmid are respectively connected with eukaryotic expression plasmid pCI by double enzyme digestion, DH5 alpha competent cells are transformed by the connection products, bacterial liquid DNA is extracted after enrichment, and eukaryotic expression plasmids pCI-chIL-4 and pCI-chIL-2 are respectively obtained;
3) And (3) carrying out double enzyme digestion on eukaryotic expression plasmid pCI-chIL-4 and pUC57-chIL-2 to connect, converting DH5 alpha competent cells by the connection products, and extracting bacterial liquid DNA after enrichment to obtain the pCI-chIL-4-chIL-2 fusion gene recombinant plasmid.
5. Use of the cytokine gene adjuvant of claim 1 for the preparation of a gene adjuvant for use in combination with a live vaccine against chicken coccidiosis.
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