Type 1 bovine viral diarrhea virus-like particle as well as preparation and application thereof

A virus-like, particle-based technology, applied in applications, viruses, antiviral agents, etc., can solve the problems of short immune duration and low immune activity, and achieve good immune effect, good compatibility, and improved efficiency

Active Publication Date: 2018-08-28
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Inactivated vaccines are relatively safe, but have the pro

Method used

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  • Type 1 bovine viral diarrhea virus-like particle as well as preparation and application thereof
  • Type 1 bovine viral diarrhea virus-like particle as well as preparation and application thereof
  • Type 1 bovine viral diarrhea virus-like particle as well as preparation and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: Construction of a kind of BVDV-1 structural protein gene recombinant baculovirus

[0034] 1. Construction of pFBD-BVDV-1 recombinant baculovirus transfer vector

[0035] According to the sequence of the structural protein coding gene of type 1 BVDV strain GS4 (partially submitted to GenBank, accession number: KC700344), an upstream primer (SEQ ID No.3) 5'-GCC with a restriction site was designed GGATCC BamHI ATG TCCGACACAAATACAGAAGG-3' and downstream primer (SEQ ID No.4) 5'-CTC AAGCTT HindIII CTAs ACCCGAGGTCATTTGTTCTG-3', with start codon and stop codon (italics) added to the upstream primer and downstream primer, respectively, for type 1 BVDV structural protein C-E rns - Amplification of genes encoding E1-E2. By reverse transcription polymerase chain reaction (RT-PCR), using type 1 BVDV viral RNA as a template, using upstream primer (SEQ ID No.3) and downstream primer (SEQ ID No.4) to amplify C-E rns -E1-E2 coding genes, recovery of target frag...

Embodiment 2

[0041] Example 2: Expression and preparation of type 1 BVDV VLPs in insect cells

[0042] Seed sf-9 cells in 5 cm 2 Cell culture flasks were inoculated with 1 MOI of F3stock seed virus when the cells grew into a 90% monolayer, and the supernatant was collected and the cells were ultrasonically lysed at 72 hours, and the cell debris was removed by centrifugation at 4000g for 30 minutes at 4°C, and the Amicon Ultra- Concentrate the supernatant to 2 mL in 15 ultrafiltration centrifuge tubes, then add 10-60% sucrose density gradient and centrifuge at 4°C and 35,000 rpm for 2 hours in a Beckman ultracentrifuge (SW41 rotor), take 1 mL and label the order to prepare E2 protein Western detection was performed. For the detection of samples containing E2 protein, take 10 μL and drop it on a 200-mesh copper grid covered with a support film and a carbon film, absorb at room temperature for 3 minutes, stain with 3% phosphotungstic acid after drying, and use FEI Tecnai G 2 F20 transmissi...

Embodiment 3

[0043] Example 3: Preparation of type 1 bovine viral diarrhea virus-like particle vaccine and assay of immune efficacy

[0044] The F3stock seed virus in Example 1 was used to inoculate sf9 cells at an MOI of 1, and the diseased cells were collected 72 hours after inoculation, ultrasonically disrupted and centrifuged at 4°C, 6000g for 10 min to remove cell debris, and the BCA protein concentration assay kit (Beiyuntian Biology) was used to inoculate sf9 cells. technology company) to measure the protein concentration of the crude antigen, then inactivate it with 10mM binary ethyleneimine (BEI) for 48h, add a final concentration of 15mM sodium thiosulfate for neutralization, and fully emulsify with Merckinade SDA-201 biphasic oil adjuvant for at least Drop in cold water in the form of oil droplets without spreading. The prepared type 1 bovine viral diarrhea virus-like particle vaccine is aseptically dispensed quantitatively and stored at 4°C.

[0045] Two BVDV antibody-negative...

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Abstract

The invention discloses a type 1 bovine viral diarrhea virus (BVDV-1) virus-like particle as well as a preparation method and application thereof. A pFBD-BVDV-1 recombinant baculovirus transfer vectoris built by cloning structural protein C, Erns, E1 and E2 coding genes of BVDV-1 to pFastBacDual; a recombinant baculovirus vector Bac-BVDV-1 is obtained through the transposition in colibacillus DH10Bac; insect cells are transfected; recombinant baculovirus Baculo-BVDV-1 is obtained; the structural protein C, Erns, E1 and E2 of the insect cells for expressing BVDV-1 are infected and are automatically assembled into BVDV-1 virus-like particles (VLP) in the cells. The obtained virus-like particle is more similar to the morphostructure of the natural BVDV virus particles; after the inoculationinto animals, the bodies can be stimulated to generate good immune reaction; a better immune effect can be reached. The obtained virus-like particle can be directly purified by using the sucrose density gradient centrifugation. Compared with other expression systems, the virus-like particle has the advantages that the complicated process of extracellular assembly after the respectively purification of the structural protein is avoided; the efficiency is favorably improved; the cost is reduced; in addition, in the production process, the infective BVDV living viruses are not needed; the safetyis improved.

Description

technical field [0001] The invention relates to a recombinant baculovirus, a virus-like particle of a virus and its preparation and application. Specifically, the invention relates to a type 1 bovine viral diarrhea virus genetically recombined baculovirus, a type 1 bovine virus Virus-like particle of acute diarrhea, preparation method and application of the virus-like particle. Background technique [0002] Bovine Viral Diarrhea - Mucosal Disease (BVD-MD) is an infectious disease caused by Bovine Viral Diarrhea Virus (BVDV), which seriously endangers the cattle industry. The disease was first discovered by Olafson in 1946 in cattle in Ithaca, the capital of Tompkins County, New York State, USA. In 1957 and 1960, researchers isolated the non-cytopathic strain NY-1 and the cytopathic strain Oregon C24V, and used the virus neutralization test to confirm that bovine viral diarrhea and mucosal disease are caused by the same pathogen With different clinical symptoms, the disease...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N15/866A61K39/12A61P31/14
CPCA61K39/12A61P31/14C12N7/00C12N15/86C12N2710/14043C12N2770/24323C12N2770/24334C12N2800/105
Inventor 高闪电独军政田占成殷宏常惠芸
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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