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IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine

A subunit vaccine, bursal disease technology, applied in the direction of viral peptides, antiviral agents, viral antigen components, etc., can solve the complex procedures of recombinant silkworm baculovirus, expensive insect cell culture medium, and unfavorable farm acceptance, etc. problems, to achieve the effect of intuitive and convenient screening, easy operation, and simplification of tedious steps

Inactive Publication Date: 2013-06-26
PU LIKE BIO ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Researchers at home and abroad have used various expression systems to express IBDV VP2 protein to prepare IBDV VP2 protein subunit vaccines. For example, Rong Jun et al. used E. coli expression system, Macreadie et al. used yeast expression system to express, and Bayliss et al. System, Darteil et al. used recombinant turkey herpes virus expression system, and Sheppard et al. used recombinant poultry adenovirus expression system to express IBDV VP2 protein. However, most of these methods require a large number of cultured cells, which are expensive and are not conducive to farm acceptance.
Although Dybbing et al. successfully used recombinant Autographa californica nuclear polyhedrosis virus AcNPV to express IBDV VP2 protein in insect cells, the insect cell culture medium is still expensive
[0004] Although Lu Mijia et al. used the recombinant Bombyx mori baculovirus / bombyx mori larvae expression system to express IBDV VP2 protein, the procedures for constructing recombinant Bombyx mori baculovirus are complex, the virus recombination is complex and the success rate is low, and it needs to be produced in difficult-to-raise silkworm cells. Reproduction of recombinant viruses in medium-sized cultures also limits the application of recombinant viruses in the production of IBDV VP2 protein and IBD subunit vaccines

Method used

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  • IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine
  • IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine
  • IBDV (Infectious Bursal Disease Virus) VP2 protein and IBD subunit vaccine

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Embodiment 1

[0030] This embodiment describes the method for obtaining the infectious bursal disease virus (IBDV) VP2 gene provided by the present invention. The cloning method is nested RT-PCR, comprising the following steps:

[0031] (1) Extraction of Genomic RNA of Infectious Bursal Disease Virus (IBDV) LYZS Strain:

[0032] The IBDV LYZS strain virus was extracted from the bursal disease material clinically diagnosed as chicken infectious bursal disease (IBD) from Luoyang area. After homogenization, it was extracted according to the TIANamp virus RNA of Tiangen Biochemical Technology (Beijing) Co., Ltd. Kit instructions for the extraction of IBDV genomic RNA.

[0033] (2) Cloning of VP2 gene of IBDV LYZS strain:

[0034]1. Design a pair of primers located in the peripheral region of the VP2 gene sequence according to the sequence of the IBDV strain registered in GenBank to select the conserved region for amplifying a large fragment containing the VP2 gene, with the purpose of determin...

Embodiment 2

[0039] The present embodiment has described the method for producing Infectious Bursal Disease Virus (IBDV) VP2 albumen, and it expresses above-mentioned gene protein with silkworm or chrysalis, and comprises the steps:

[0040] (1) Transform the recombinant vector pFastBac-VP2 obtained above into the Escherichia coli competent cell DH10Bac (Invitrogen, Catalog: 10361012) containing the full-length baculovirus genome shuttle vector, recombine in the Escherichia coli cell, and pass the cyanobacteria Bacmid was screened for the recombinant shuttle vector, and the recombinant shuttle vector was named Bacmid-VP2.

[0041] (2) Transfect insect sf9 cells (Invtrogen, Catalog: 11496015) with the recombinant shuttle vector Bacmid-VP2 described in step (1), and produce recombinant baculovirus in the cells, named vBac-VP2: prepare 5 × 10 5 cells / ml of sf9 cell suspension, insert 2ml / well of sf9 cells into a 6-well cell culture plate, and let stand at 27°C for more than 1 hour to allow th...

Embodiment 3

[0045] This example describes the IBDV genetically engineered subunit vaccine provided by the present invention. It is an injection vaccine: including the above-mentioned dilution of hemolymph of silkworm larvae infected with the recombinant virus, immune adjuvants, preservatives and stabilizers, etc., the above-mentioned silkworm hemolymph is diluted with PBS (0.01M, pH=7.4) When the concentration of agar expansion antigen of VP2 protein reaches 1:16, the obtained dilution is mixed with immune adjuvant at 1:1~1:3 (V:V), and the immune adjuvant includes mineral oil, Siben-80, spit Wen-80, aluminum stearate and ISCOM etc. The ratio of hemolymph diluent and immune adjuvant can be 1:2. The production method is as follows: collect the above silkworm hemolymph, process it with a high-pressure homogenizer, treat it twice at 4°C and 800 bar pressure, centrifuge at 12,000 rpm for 30 minutes, collect the supernatant, and dilute the above-mentioned blood by 200 times with PBS (0.01M, p...

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Abstract

The invention relates to an IBDV (Infectious Bursal Disease Virus) VP2 protein. The mass production of the expressive IBDV VP2 protein in a silkworm larva or a pupae is succeeded for the first time by performing the subcutaneous injection of a restructured autographa californica nuclear polyhedrosis virus into the silkworm larva or the pupae. However, in the prior art, the expression of the restructured autographa californica nuclear polyhedrosis virus containing the IBDV VP2 in the silkworm larva or the pupae always cannot be performed successfully. The invention further provides a method for producing the IBDV VP2 protein and an IBD subunit vaccine by utilizing a silkworm bioreactor. The IBDV VP2 protein can be applied to the preparation of the vaccine for treating and preventing the IBD as well as the preparation of an IBD diagnostic reagent.

Description

technical field [0001] The present invention relates to an infectious bursal disease virus VP2 protein and a preparation method thereof, in particular to a method for preparing infectious bursal disease subunit vaccine by using the infectious bursal disease virus VP2 protein, Belongs to the field of biotechnology. Background technique [0002] Infectious bursal disease (IBD) is one of the three major infectious diseases that endanger the poultry industry in the world. my country is a big country in the production of poultry eggs and poultry meat in the world, and its production scale ranks first in the world. The epidemic situation of the disease in our country is complex, and it is listed as a second-class severe infectious disease for key defense by the Ministry of Agriculture. Infectious bursal disease virus (IBDV) mainly attacks chicks aged 3 to 10 weeks, especially chickens within 4 weeks of age. The mortality rate of sick flocks reaches 5%~30%. The organ damaged by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/08C12N15/40C12N15/866A61K39/12A61P31/14G01N33/68G01N33/569C12R1/93
Inventor 张许科孙进忠白朝勇
Owner PU LIKE BIO ENG
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