A kind of recombinant gene comprising ibv multi-epitope epic and ndv F gene, recombinant expression plasmid, recombinant lactobacillus and application thereof
A recombinant gene and expression plasmid technology, applied in the fields of genetic engineering and poultry disease vaccines, can solve the problems of poor colonization ability, poor immune protection of chickens, inability to long-term colonization and colonization ability, and incomparable immune effects, and achieves good social and economic benefits. The effect of enhancing antigen immunogenicity and simple administration
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Embodiment 1
[0023] The purpose of this embodiment is to provide a recombinant gene UTEFDA, a recombinant expression plasmid and a recombinant lactobacillus.
[0024] The signal peptide sequence SpUsp45 and the cell wall anchoring domain sequence ACMA were obtained from Lactococcus lactis MG1363 (accession number: AM406671) released in Genebank, and the immune adjuvant protein sequence Tuftsin and dendritic cell-inducing peptide sequence DCpep were selected to enhance the body's response to antigens. For the immune response, the multi-epitope gene EpiC of the IBV SAIBK strain and the F gene of the avian Newcastle disease virus F48E9 strain were used as the basic materials to construct a fusion gene SpUsp45-Tuftsin-linker-EpiC-linker that can display the host surface and enhance the immunogenicity of the antigen -F-DCpep-linker-ACMA (hereinafter abbreviated as UTEFDA). The UTEFDA gene was constructed by using the homologous enzyme ligation method and overlap extension PCR technology, and it...
Embodiment 2
[0030] The purpose of this example is to identify the expression location and immune efficacy of the recombinant Lactobacillus TCMM17-UTEFDA provided in Example 1.
[0031] 1 main material
[0032] Avian infectious bronchitis virus antibody detection kit was purchased from IDEXX company; avian Newcastle disease virus antibody detection kit was purchased from Wuhan Keqian; chicken secretory immunoglobulin A (sIgA) ELISA antibody detection kit was purchased from Nanjing Jinyibai; FITC Labeled CD4+ monoclonal antibody and PE-labeled CD8+ monoclonal antibody were purchased from NOVUS Company; IBV M41 hyperimmune positive serum was purchased from China Veterinary Drug Administration.
[0033] 2 Expression and extraction of fusion protein TEFDA
[0034] 2.1 Extraction of fusion protein TEFDA
[0035] To extract TEFDA protein, the steps are as follows:
[0036] (1) Inoculate TCMM17-UTEFDA obtained in Example 1 and stored at -80°C into 5mL MRS liquid medium containing 200μg / mL Emr,...
Embodiment 3
[0049] The purpose of this example is to verify that the recombinant Lactobacillus TCMM17-UTEFDA can effectively immunize IB and ND.
[0050] 1 Chicken immunization grouping and immunization procedures
[0051] After the incubator was cleaned, it was sterilized with Biodox, and the eggs were put into the incubator at 37.6°C and the humidity was 55-60%. After 22 days of incubation, the chicks were randomly grouped and transferred to the SPF isolator for feeding and immunization. Newcastle disease-infectious bronchitis dual live vaccine (ND LaSota+IBV H120) is set as the vaccine control group, and the recombinant lactobacillus expressing IBV multi-epitope EpiC and NDV F gene provided by the invention is Lactobacillus salivarius oral vaccine TCMM17-UTEFDA as the experimental group, wild-type Lactobacillus salivarius TCMM17 as the negative control group, and PBS as the blank control group. Chickens were immunized by nasal and eye drops, and the grouping and immunization methods o...
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