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Recombination attenuated salmonella typhimurium carrier vaccine of expression IBDV (Infectious Bursal Disease Virus) immunogenic gene and preparation method thereof

A technology of Salmonella typhi and vector vaccine, applied in the field of bioengineering, can solve the problems of no effective method, attenuated vaccine and inactivated vaccine immunization failure, etc., to avoid cumbersome processes and defects, good immunogenicity and immune protection effect , the effect of stimulating mucosal immune response

Inactive Publication Date: 2010-12-22
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no practical and effective method for the control of IBD disease. The vaccines to prevent and control IBD mainly include inactivated vaccines and attenuated vaccines. In recent years, IBDV mutant strains and super-virulent strains have become popular, and traditional attenuated vaccines and inactivated vaccines have failed. happens often

Method used

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  • Recombination attenuated salmonella typhimurium carrier vaccine of expression IBDV (Infectious Bursal Disease Virus) immunogenic gene and preparation method thereof
  • Recombination attenuated salmonella typhimurium carrier vaccine of expression IBDV (Infectious Bursal Disease Virus) immunogenic gene and preparation method thereof
  • Recombination attenuated salmonella typhimurium carrier vaccine of expression IBDV (Infectious Bursal Disease Virus) immunogenic gene and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0045] 1.1 Design of primers

[0046] Referring to the genome sequence of IBDV OKYM strain (GenBank: D49706.1), a pair of specific primers (P1 / P2) was designed. Upstream primer P1: 5′ CCG GAATTCGC ATGACAAACCTGCAAGATC-3'5' end plus EcoR I restriction site. Downstream primer P2: 5′-ACG GTC GAC GCCTTAGGGCCCGGATTATG-3', 5' end plus a Sal I restriction site. The expected amplified fragment is 1356bp, containing the complete VP2 gene.

[0047] 1.2 Amplification of viral genome

[0048] The wild virus of the IBDV Shaanxi isolate stored at -80°C was taken out, and the viral RNA was extracted according to the Trizol extraction kit. Take 3 μL of RNA as a template, add 1 μL of VP2 upstream and downstream primers P1 / P2, 4 μL of dNTP, 0.5 μL of RNase-Inhibitor, 0.5 μL of AMV, 4 μL of 5×AMV Buffer, add DEPC H2O to 20 μL, and incubate at 42°C for 1 hour to obtain a cDNA template;

[0049] The PCR reaction system is as follows: 10×PCR Buffer 5 μL, dNTPs 4 μL, P1 / P2 1 μL, rTaq 1 μL, cD...

Embodiment 2

[0102] Recombinant bacteria X4550 (pYA3341-VP2) and empty plasmid bacteria X4550 (pYA3341) were respectively inoculated in LB liquid medium containing 20 μg / ml NA, shaken at 37°C for 18 hours, centrifuged to discard the supernatant, collected the bacteria, and added 0.01M ( After resuspending the cells in PBS solution (pH 7.2), add 5× protein loading buffer, boil for 15 minutes, collect the supernatant by centrifugation, and perform SDS-PAGE electrophoresis on a 12% gel. Prepare SDS-PAGE polyacrylamide gel. After the gel is completely polymerized, pull out the sample comb, wash the sample hole with negative buffer and dry the water in the hole with filter paper. The sample to be tested and the protein molecular weight standard are loaded at the same time, and the electrophoresis is stopped when the Coomassie brilliant blue reaches the bottom of the gel. The gel was fixed in the fixative solution for 30-60 minutes, heated to 60°C for Coomassie brilliant blue staining for 20 min...

Embodiment 3

[0105] SDS-PAGE electrophoresis was performed according to the above method, and after the protein was separated by SDS-PAGE, it was electrotransferred to PVDF membrane. 5% skimmed milk powder or 3% bovine serum albumin buffer (10mmol / L Tris Cl, pH7.5, 150mmol / L NaCl, 0.05% Tween20) for blocking at 37°C for 2h, washing buffer (10mmol / L Tris Cl pH7. 5. Wash 3 times with 150mmol / L NaCl, 0.05% Tween20), 10-15min each time, dilute chicken anti-IBDV positive serum with blocking buffer (1:300) to cover the membrane, feel at room temperature for 2h, wash 3-5 Once, horseradish peroxidase-labeled rabbit anti-chicken IgG (1:2000) was sensed at room temperature for 2 hours, washed 3 to 5 times, each time for 10 to 15 minutes, and finally washed once with distilled water, clipped out the PVDF membrane and dried slightly , prepare ECL (enhanced chemiluminescence) working solution, incubate the membrane at room temperature under visible light for several minutes, wrap the imprinted membrane...

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Abstract

The invention discloses a recombination attenuated salmonella typhimurium carrier vaccine of expression IBDV (Infectious Bursal Disease Virus) immunogenic gene, a preparation method and application thereof, in particular relates to oral attenuated salmonella typhimurium. The recombination attenuated salmonella typhimurium comprises a sequence disclosed by SEQ ID NO.1, and the recombination virus can express IBDV capsid protein VP2. The preparation method comprises the following steps of: inoculating the attenuated salmonella typhimurium in an LB substrate for culturing 18 hours; regulating bacterial concentration as 1010CFU / ml; and preparing the safe and effective oral attenuated salmonella typhimurium carrier vaccine for preventing IBD (Infectious Bursal Diseases).

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a recombinant attenuated Salmonella typhimurium vector vaccine expressing IBDV immunogen gene and a preparation method thereof. Background technique [0002] IBD (Infectious Bursal Disease) is an acute, highly contagious infectious disease that is harmful to chicks caused by IBDV (Infectious Bursal Disease Virus), and is one of the main infectious diseases that endanger the poultry industry in the world. It mainly injures the central immune organ of chicks - the bursa of Fabricius, leading to immunosuppression, thereby enhancing the body's susceptibility to other pathogens and reducing its responsiveness to other vaccines. [0003] There are currently two serotypes of IBD occurring in the world: serotype I and serotype II. Serum type II strains have no pathogenicity to chickens, and serum type I can be divided into classic strains (also known as standard serum type I), va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K48/00C12N15/40C12N15/74C12N1/21A61P31/14
Inventor 童德文许信刚张琪
Owner NORTHWEST A & F UNIV
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