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Preparation methods for CHO cell expressed recombinant bovine viral diarrhea virus protein E2 and subunit vaccine and application

A technology for bovine viral diarrhea and subunit vaccines, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problem that protein expression, folding and modification are not as good as mammalian cell expression systems, and the production process is more difficult. There are no problems such as correct structure, etc., to achieve the effect of easy mass production, low production cost, and batch-to-batch stability

Active Publication Date: 2018-05-01
NOVO BIOTECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the prokaryotic expression product is an inclusion body, which has no correct structure and poor activity; although baculovirus expression can undergo post-translational processing and modification to a certain extent, it still has certain differences from the natural antigenic protein structure of the virus. The modification is not as good as the mammalian cell expression system, and when the system produces and prepares antigens, the cells will lyse and die after being infected by the virus, which increases the difficulty of downstream purification and other production processes

Method used

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  • Preparation methods for CHO cell expressed recombinant bovine viral diarrhea virus protein E2 and subunit vaccine and application
  • Preparation methods for CHO cell expressed recombinant bovine viral diarrhea virus protein E2 and subunit vaccine and application
  • Preparation methods for CHO cell expressed recombinant bovine viral diarrhea virus protein E2 and subunit vaccine and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Bovine viral diarrhea virus E2 protein codon optimization

[0036] By codon-optimizing the nucleotide sequence of the E2 protein of bovine viral diarrhea virus GS5 strain (BVDV 1a), the OPTI-E2 sequence was obtained, as shown in SEQ ID NO.2. This work was entrusted to Nanjing GenScript Co., Ltd. The company is done.

Embodiment 2

[0037] Example 2: Construction of pEE12.4-OPTI-E2 recombinant plasmid

[0038] 2.1PCR amplification of the target fragment OPTI-E2

[0039] 2.1.1 PCR reaction

[0040] (1) Primer design and synthesis

[0041] Upstream primer: 5'-GCAAGCTTGCCGCCACCATGGGCCAGATCGTGCAGGG-3' Downstream primer: 5'-GCGAATTCTTAGTGATGGTGATGGTGATGGATGGACTCAGCAAAATAATCCCTATGGTG-3'

[0042] (2) Add 50 μL of the sample system, as shown in the table below:

[0043]

[0044] PCR amplification program:

[0045]

[0046] 2.1.2 Gel recovery of PCR products

[0047] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0048] (2) Take the weight of the marked empty EP tube, and record the value;

[0049] (3) Carefully cut out a single target DNA band from the agarose gel with a scalpel on a gel cutter and put it into a clean 1.5mL centrifuge tube;

[0050] (4) Add 600 μL PC buffer to the 1.5mL centrifuge tube in step (3), place in a 50°C water bath for about 5 minutes, and ...

Embodiment 3

[0114] Example 3: Establishment of transfection of pEE12.4-OPTI-E2 recombinant plasmid into CHO-K1 cells and monoclonal screening

[0115] 3.1 CHO-K1 cell transfection

[0116] (1) Preparation: UV sterilization in a biological safety cabinet for 30 minutes; DMEM / F12 (containing 10% serum, 1% double antibody), DMEM / F12 and PBS were placed in a 37°C water bath and preheated to 37°C.

[0117] (2) Take out the cells (10 cm cell culture dish) from the incubator at 37° C., discard the supernatant medium, wash the cells once with pre-warmed 8 mL PBS, and discard the PBS.

[0118] (3) Add 1-2mL 0.25% trypsin-EDTA to each 10cm cell culture dish, digest at room temperature for about 2 minutes, observe under the microscope that the cells shrink and become round, and appear as single cells.

[0119] (4) Add 4 mL of DMEM / F12 (containing 10% serum, 1% double antibody) to terminate the digestion reaction, and blow the cells away with a pipette.

[0120] (5) Transfer the digested cells to a...

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Abstract

The invention discloses preparation methods for CHO cell expressed recombinant bovine viral diarrhea virus protein E2 and a subunit vaccine and applications and belongs to the technical fields of animal vaccines and veterinary biologicals. The object of the invention is to provide a preparation method capable of industrially producing the bovine viral diarrhea virus recombinant subunit vaccine ona large scale. The preparation method for the recombinant subunit vaccine, provided by the invention, comprises the following steps: 1) cloning an eukaryotic expression vector containing a protein E2coding gene; 2) transfecting CHO cells, and performing selection, screening and acclimatizing to obtain suspending CHO cell strains, which stably and efficiently express the protein E2; 3) subjectingthe cell strains obtained in the step 2) to fermentation culture, and carrying out purification, so as to obtain recombinant protein E2; and 4) uniformly mixing the recombinant protein E2 and ISA 201VG thoroughly, thereby obtaining the recombinant subunit vaccine. According to the method provided by the invention, target protein can be obtained from cell culture supernatant, the yield reaches upto 500mg / L, the protein purification time is shortened, the vaccine production steps are simplified, and the vaccine production cost is greatly reduced.

Description

technical field [0001] The invention relates to a CHO cell line stably expressing bovine viral diarrhea virus E2 protein and a preparation method and application of a bovine viral diarrhea virus E2 protein subunit vaccine, belonging to the technical field of animal vaccines and veterinary biological products. Background technique [0002] Bovine viral diarrhea (BVD), also known as mucosal diaease (MD), is caused by viral diarrhea virus (BVDV), and its clinical manifestations are diarrhea, high fever, reproductive impairment, immune A disease characterized by suppression, persistent infection, and severe mucosal inflammation, erosion, and necrosis. BVDV belongs to the Pestivirus genus of the Flaviviridae family. The disease was first discovered in the United States in 1946, and then became an explosive global epidemic. It is widely spread in the United States, Britain, Germany, Switzerland, Australia, India, Japan and other large cattle-raising countries. In 1983, the dise...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12N15/40C12N15/85A61K39/12A61P31/14
CPCA61K39/12A61K2039/552C07K14/005C12N15/85C12N2770/24322C12N2770/24334C12N2800/107C12N2800/22
Inventor 钱泓吴有强卞广林张强吴素芳车影宋月鸿吕洋萍陈滨查银河
Owner NOVO BIOTECH CORP
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