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Recombinant lactobacillus strain for fusion expression of infectious bursal disease virus VP2 protein and salmonella outer membrane protein and application of recombinant lactobacillus strain

A technology of recombinant lactic acid bacteria and chicken infectivity, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problem of unsatisfactory effect

Active Publication Date: 2017-08-29
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on the construction of recombinant lactic acid bacteria IBDV-VP2 live vector vaccine using lactic acid bacteria is expected to have made preliminary progress, but the effect is not very satisfactory, and further exploration and research are needed

Method used

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  • Recombinant lactobacillus strain for fusion expression of infectious bursal disease virus VP2 protein and salmonella outer membrane protein and application of recombinant lactobacillus strain
  • Recombinant lactobacillus strain for fusion expression of infectious bursal disease virus VP2 protein and salmonella outer membrane protein and application of recombinant lactobacillus strain
  • Recombinant lactobacillus strain for fusion expression of infectious bursal disease virus VP2 protein and salmonella outer membrane protein and application of recombinant lactobacillus strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Construction and identification of embodiment 1 recombinant lactic acid bacteria

[0037] 1. Construction of recombinant lactic acid bacteria vector

[0038] The genes encoding chicken infectious bursal virus virus VP2 protein and Salmonella outer membrane protein (OMP) were optimized according to the codon preference table of lactic acid bacteria, and the optimized genes were named OptiVP2 and OptiOMP respectively. The optimized chicken Infectious bursal virus virus VP2 gene sequence OptiVP2 is shown in SEQ ID NO: 3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4, and the optimized outer membrane protein (OMP) of Salmonella gallinarum The gene sequence of OptiOMP is shown in SEQ ID NO:5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:6. The optimized OptiVP2 and OptiOMP gene sequences are connected through the link sequence (shown in SEQ ID NO.7) to obtain the nucleotide sequence encoding the VP2-OMP fusion protei...

Embodiment 2

[0049] Example 2 Detection of the adhesion characteristics of the fusion protein of VP2 protein expressed by recombinant lactic acid bacteria r-L.lactis-OMPH-VP2 of the present invention and OMP

[0050] Inoculate the recombinant lactic acid bacteria strain r-L.lactis-OMPH-VP2 in Elliker-medium medium, add 0.5% lactose, culture statically at 30°C overnight, subculture according to 1:50, and culture the bacteria density to OD 600 When it is 0.3-0.5, add nisin to induce culture, and the concentration of Nisin is 10ng / mL for culture. After static induction culture for 5 to 6 hours, the cell lysate supernatant is obtained after concentration and lysis, which contains the fusion protein of expressed OMP and VP2 protein .

[0051] At the same time, the recombinant lactic acid bacteria r-L.lactis-VP2 (the preparation method is the same as that of r-L.lactis-OMPH-VP2, which only expresses the VP2 protein of chicken infectious bursal virus virus) and the recombinant lactic acid bacteri...

Embodiment 3

[0053] The determination of the immunogenicity of embodiment 3 recombinant lactic acid bacteria of the present invention

[0054] Take out the recombinant Lactococcus lactis strain r-L.lactis-OMPH-VP2 prepared in Example 1 and the recombinant lactic acid bacteria r-L.lactis-VP2 that only express chicken infectious bursal virus virus VP2 protein (preparation method and The preparation of r-L.lactis-OMPH-VP2 is the same, the only difference is that it does not contain the OMP expression sequence), respectively streaked on the solid L-Elliker medium, cultured at 30°C, and after 10h-12h a single colony grows, use incinerated Bacteria were picked with a spear tip, inoculated in liquid L-Elliker medium, and cultivated to logarithmic phase; transferred to liquid L-Elliker medium at a ratio of 1:100 by volume and cultured at 30°C for 12h-14h , and then transferred to liquid L-Elliker medium at a ratio of 1:50 by volume, and cultured at 30°C for about 2h-3h. When OD600=0.4-0.5, add Nis...

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Abstract

The invention discloses a recombinant lactobacillus strain for fusion expression of infectious bursal disease virus VP2 protein and salmonella outer membrane protein and an application of the recombinant lactobacillus strain. The recombinant lactobacillus strain contains an optimized gene sequence for encoding the infectious bursal disease virus VP2 protein and a gene sequence for encoding the salmonella outer membrane protein (OMP). An experiment proves that the recombinant lactobacillus strain is capable of carrying out efficient fusion expression on the VP2 protein and the OMP, and exists in cytoplasm in a soluble form. The recombinant lactobacillus strain is directly applied to immunization of chickens as a vaccine, and the experiment result shows that the recombinant lactobacillus strain is capable of effectively inducing an organism to generate a specific immune response, and a specific neutralizing antibody is mainly generated, so that the immunized chicken obtain 100% protection of resisting a highly pathogenic vvIBDV lethal attack, and the residual virus in the body can be eliminated to achieve the target of clearance immunity. Therefore, a new technical method is provided for prevention and treatment of infectious bursal disease.

Description

technical field [0001] The present invention relates to a kind of recombinant lactic acid bacteria strain and its fused expression protein and application, particularly to a kind of recombinant lactic acid bacteria strain which fuses and expresses chicken infectious bursal virus virus VP2 protein and Salmonella outer membrane protein OMP, and also relates to the Use of the recombinant lactic acid bacteria strain and the fusion protein of chicken infectious bursal virus VP2 protein and Salmonella outer membrane protein OMP obtained by fusion expression in preventing chicken infectious bursal disease and salmonella infection. The invention belongs to the technical field of medicine or veterinary medicine. Background technique [0002] Chicken infectious bursal disease (Infectious bursal disease, IBD) is an important viral disease caused by infectious bursal disease virus (Infectious bursal disease virus, IBDV) that can cause chicken body immunosuppression, seriously endangerin...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/62C07K19/00A61K39/295A61K39/112A61K39/12A61P31/04A61P31/14C12R1/01
CPCA61K39/0275A61K39/12A61K2039/523A61K2039/542A61K2039/70C07K14/005C07K14/255C07K2319/00C12N15/62C12N15/746C12N2720/10022C12N2720/10034C12N2800/22
Inventor 崔红玉王笑梅
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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