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Recombinant lactic acid bacteria strain expressing ibdv VP2 protein and Salmonella outer membrane protein RCK and its use

A technology for recombining lactic acid bacteria and outer membrane proteins, applied in the direction of DNA/RNA fragments, fusion polypeptides, recombinant DNA technology, etc., can solve problems such as unsatisfactory effects, and achieve improved antigen presentation ability, adhesion ability, and medium cost low effect

Active Publication Date: 2021-09-17
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 lactic acid bacteria strain expressing ibdv VP2 protein and Salmonella outer membrane protein RCK and its use
  • Recombinant lactic acid bacteria strain expressing ibdv VP2 protein and Salmonella outer membrane protein RCK and its use
  • Recombinant lactic acid bacteria strain expressing ibdv VP2 protein and Salmonella outer membrane protein RCK and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Construction and identification of embodiment 1 recombinant lactic acid bacteria

[0041] 1. Construction of recombinant lactic acid bacteria vector

[0042] The genes encoding chicken infectious bursal virus virus VP2 protein and Salmonella outer membrane protein RCK were optimized according to the lactic acid bacteria codon preference table, and the optimized genes were named OptiVP2 and OptiRCK respectively. The optimized chicken infectivity The bursal virus virus VP2 gene sequence OptiVP2 is shown in SEQ ID NO: 3, the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4, and the gene sequence OptiRCK of the optimized Salmonella gallinarum outer membrane protein RCK is shown in As shown in SEQ ID NO: 5, the amino acid sequence of the encoded protein is shown in SEQ ID NO: 6. The optimized OptiVP2 and OptiRCK gene sequences were connected to obtain the nucleotide sequence OptiVP2-OptiRCK encoding the VP2-RCK fusion protein, the sequence of which is sho...

Embodiment 2

[0054] Example 2 Detection of adsorption and adhesion characteristics of recombinant lactic acid bacteria r-L.lactis-RCK-VP2 of the present invention to epithelial cells, and detection of characteristics of being internalized by epithelial cells

[0055] 1. Experimental verification of the adhesion of recombinant lactic acid bacteria r-L.lactis-RCK-VP2 to epithelial cells

[0056] (1) First, the human intestinal epithelial cell line HT29 was cultured in a 24-well plate with DMEM cell culture medium containing 10% fetal bovine serum (FBS). When 80% of the cell confluence was reached, some of the cells in the wells were digested and removed. Blow down and count the cells with a cell counter to ensure that the total number of cells in each well contains approximately 4-5×10 5 cells.

[0057] (2) Recombinant lactic acid bacteria strain r-L.lactis-RCK-VP2, recombinant lactic acid bacteria r-L.lactis-VP2 expressing chicken infectious bursal virus VP2 protein were respectively prepa...

Embodiment 3

[0067] Embodiment 3 Determination of the immunogenicity of recombinant lactic acid bacteria r-L.lactis-RCK-VP2 of the present invention

[0068] Take out the recombinant Lactococcus lactis strain r-L.lactis-RCK-VP2 prepared in Example 1 and the recombinant lactic acid bacteria r-L.lactis-VP2 that only express chicken infectious bursal virus VP2 protein (preparation method and r-L The preparation of .lactis-RCK-VP2 is the same, the only difference is that it does not contain the RCK expression sequence), respectively streaked on the solid L-Elliker medium, cultivated at 30°C, and after 10h-12h a single colony grows, use sterilized Pick the bacteria with the tip of the pipette, and transfer the liquid L-Elliker medium to the logarithmic phase; transfer to the liquid L-Elliker culture at a ratio of 1:100 after 12h-14h at 30°C Base culture at 30°C for about 2h-3h, when OD600=0.4-0.5, add Nisin with a final concentration of 5ng / mL, induce culture at 30°C for 4h-5h, centrifuge at 80...

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Abstract

The present invention discloses a recombinant lactic acid bacteria strain that fuses and expresses IBDV VP2 protein and Salmonella outer membrane protein RCK and its application. The recombinant lactic acid bacterial strain described in the present invention contains optimized coded chicken infectious bursal virus ( Gene sequence of IBDV) VP2 protein and Salmonella outer membrane protein RCK fusion protein, which can efficiently fuse and express VP2 protein and Salmonella outer membrane protein RCK, and the expressed fusion protein VP2‑RCK exists in the cytoplasm of lactic acid bacteria in a soluble form middle. The recombinant lactic acid bacteria strain of the present invention is directly used as a vaccine to immunize chickens, and the experimental results show that: the recombinant strain vaccine can effectively induce the body to produce a specific immune response, so that the immune chickens can obtain 100% protection against the lethal attack of highly pathogenic vvIBDV , and can remove the residual virus in the body to achieve the purpose of eliminating immunity. The proposal of the present invention provides a new technical means for the prevention and treatment of chicken infectious bursal disease.

Description

technical field [0001] The present invention relates to a recombinant lactic acid bacteria strain and its construction method and application, in particular to a recombinant lactic acid bacteria strain fused to express chicken infectious bursal virus VP2 protein and Salmonella outer membrane protein RCK and its construction method and application. The invention belongs to the field of medical or veterinary technology. 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 endangering the world's Chicken industry. Traditional vaccines have played a very important role in the history of the prevention and treatment of the disease. With the new changes of the disease in recent years-the emergence of serotype variants and super-virulent strains, the prevention and treatm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/62C07K19/00A61K39/295A61K39/112A61K39/12A61P31/04A61P31/14C12R1/01
CPCA61K39/0275A61K39/12A61K2039/523A61K2039/542A61K2039/70C07K14/005C07K14/255C07K2319/00C12N15/746C12N2720/10022C12N2720/10034C12N2800/22
Inventor 崔红玉李凯祁小乐高立高玉龙王笑梅
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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