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Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof

A vaccine composition and chicken infectivity technology, applied in the field of veterinary medicine, can solve the problems of negative reaction, high production cost, high endotoxin content, etc., and achieve the effect of reducing negative reaction, production cost and side reaction

Active Publication Date: 2014-06-11
PU LIKE BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 1. The VP2 protein obtained by the prior art mostly exists in the form of inclusion bodies, and the production cost is high
[0008] 2. The protein purified by the prior art has a high content of endotoxin, which will cause serious negative reactions

Method used

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  • Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof
  • Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof
  • Infectious bursal disease virus subunit antigen-containing vaccine composition, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Construction of pColdⅢ_IBDV_VP2 expression system

[0049] 1. Experimental materials

[0050] Plasmid extraction kit was purchased from Tiangen Biology; T4 DNA Ligase was purchased from BioLab; EcoR1, Sal1 restriction endonuclease, pColdⅢ_DH5α strain were purchased from TaKaRa; agarose gel recovery kit was purchased from Tianze Biology, and other reagents were Analytical pure.

[0051] 2. Experimental steps

[0052] 2.1 Preparation of VP2 cDNA

[0053] 2.1.1 Extraction of total RNA

[0054] The brief process of the total RNA is as follows: grind the bursa of SPF chickens infected with the supervirulent LQ9 strain of chicken infectious bursal disease virus with a grinder. Take 200 μL of disease material and add TE (10mM Tris, 1mM EDTA, pH8.0) to 500 μL, add 5 μL of proteinase K and 50 μL of 10% (W / V) sodium dodecylsulfonate (SDS), and bathe in water at 56°C for 3 hours . Add an equal volume of phenol / chloroform (1:1, V / V) to extract three times, and extr...

Embodiment 2

[0079] Embodiment 2: IBDV VP2 protein preparation

[0080] 1. Experimental materials: pColdⅢ-VP2 / E.Coli BL21 (DE3) strain, IPTG, PBS.

[0081] 2. Experimental method

[0082] 2.1 Prepare LB medium containing 50-100 μg / ml ampicillin.

[0083] 2.2. Inoculate the culture medium containing the pColdⅢ-VP2 / E.Coli BL21 (DE3) strain prepared in Example 1, the inoculum amount is 1% (V / V), and culture with shaking at 37°C.

[0084] 2.3. When OD600=0.4-0.6, place it at 15°C for 30 minutes.

[0085] 2.4. Add isopropyl-β-D-thiogalactopyranoside (IPTG) so that the final concentration is 0.1-1.0mM, and culture with shaking at 15°C for 24 hours.

[0086] 2.5. After the cultivation, collect the bacteria, and use PBS (sodium chloride, 8g, potassium chloride, 0.2g, disodium hydrogen phosphate, 1.44g, potassium dihydrogen phosphate, 0.24g, adjust pH 7.4, constant volume 1L) The bacteria were resuspended, ultrasonically disrupted, and centrifuged to take out the previous agarose diffusion test...

Embodiment 3

[0089] Example 3: Escherichia coli expresses VP2 protein endotoxin clearance

[0090] 1. Experimental materials:

[0091] The antigen prepared in Example 2; endotoxin detection Limulus test kit and endotoxin standard were purchased from Xiamen Limulus Reagent Company, and other reagents were of analytical grade.

[0092] 2. Experimental method

[0093] 2.1 Triton X-114 solution for removing endotoxin from crude VP2 protein

[0094] Add 0.5ml of the solution to be treated and Triton X-114 (5μl) at a final concentration of 1% (v / v) to a 1.5ml centrifuge tube, and vortex. Samples were placed on ice for 5 minutes. After vortexing the cooled samples, the centrifuge tubes were immediately placed in a 37°C water bath for 5 min to allow a new two-phase formation. Then, the samples were centrifuged at 37°C for 60 s. After centrifugation, the target protein will remain in the upper layer, while the detergent containing endotoxin will remain at the bottom of the centrifuge tube in t...

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Abstract

The present invention provides an infectious bursal disease virus (IBDV) subunit antigen-containing vaccine composition, which contains an immune amount of recombinant VP2 protein and an adjuvant. According to the present invention, with the subunit gene engineering vaccine composition, the infectious bursal disease can be effectively prevented and controlled; and with the purification technology adopted by the recombinant antigen protein, the endotoxin content in the vaccine composition is significantly reduced, the side reaction of the chicken individuals is significantly reduced, and the safety of the vaccine is substantially increased.

Description

technical field [0001] The invention relates to a vaccine composition containing chicken infectious bursal disease virus subunit antigen, which belongs to the field of veterinary medicine. Background technique [0002] Infectious bursal disease is a highly contagious disease caused by infectious bursal disease virus (IBDV). It is mainly characterized by enlarged cyst of Fabricius and liver damage, which can cause immunosuppression in chicks. The disease is distributed worldwide and occurs occasionally in our country, causing serious economic losses. [0003] Chicken infectious bursal disease live vaccines are mainly divided into inactivated vaccines of infectious bursal disease capsule tissue and inactivated vaccines of infectious bursal virus cell culture; there are more than 20 types of inactivated vaccines currently on the market. Including 2512HEP, LZD228, Lukert, 228E, D78 and B2 etc. [0004] VP2 of IBDV contains at least three groups of epitopes, namely neutralizin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/70C07K14/08C07K1/14A61K39/12A61P31/14
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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