Preparation of CHO cell expressed infectious bovine rhinotracheitis virus protein gD and subunit vaccine thereof and application

A subunit vaccine, rhinotracheitis virus technology, applied in vaccines, viruses, viral peptides, etc., can solve problems such as economic losses, and achieve the effects of high safety, good immunogenicity, and sufficient supply

Pending 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

Epidemiological surveys show that the average infection rate of IBRV in the 14 major cattle raising provinces in my country is 33.3%, which has caused huge economic losses to our cattle raising industry

Method used

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  • Preparation of CHO cell expressed infectious bovine rhinotracheitis virus protein gD and subunit vaccine thereof and application
  • Preparation of CHO cell expressed infectious bovine rhinotracheitis virus protein gD and subunit vaccine thereof and application
  • Preparation of CHO cell expressed infectious bovine rhinotracheitis virus protein gD and subunit vaccine thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Codon optimization of gD protein of bovine infectious rhinotracheitis virus and construction of pEE12.4-OPTI-gD recombinant plasmid

[0033] By codon-optimizing the nucleotide sequence of the gD protein of bovine infectious rhinotracheitis virus, the OPTI-gD sequence was obtained, as shown in SEQ ID NO.2, and this work was commissioned to Nanjing GenScript Biotechnology Co., Ltd.

Embodiment 2

[0034] Example 2: Construction of pEE12.4-OPTI-gD recombinant plasmid

[0035] 2.1 PCR amplification of the target fragment OPTI-gD

[0036] 2.1.1 PCR reaction

[0037] (1) Primer design and synthesis

[0038] Upstream primer: 5'-CGGAAGCTTGCCGCCACCATGCTGCCTACCCCCAGCTCCAAG-3'

[0039] Downstream primer: 5'-GGCGAATTC TTAGTGATGGTGATGGTGATGT-3'

[0040] (2) Add 50 μL of the sample system, as shown in the table below:

[0041]

[0042] PCR amplification program:

[0043]

[0044] 2.1.2 Gel recovery of PCR products

[0045] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0046] (2) Take the weight of the marked empty EP tube, and record the value;

[0047] (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;

[0048] (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 gently turn...

Embodiment 3

[0112] Example 3: Establishment of transfection of pEE12.4-OPTI-gD recombinant plasmid into CHO-K1 cells and monoclonal screening

[0113] 3.1 CHO-K1 cell transfection

[0114] (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.

[0115] (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.

[0116] (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.

[0117] (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.

[0118] (5) Transfer the digested cells to a...

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Abstract

The invention discloses preparation of CHO cell expressed recombinant infectious bovine rhinotracheitis virus protein gD and a subunit vaccine thereof and an application and belongs to the technical fields of animal vaccines and veterinary biologicals. The condition that the vaccine can generate relatively high humoral immunity in bovine bodies is proven. The object of the invention is to providea preparation method capable of industrially producing the infectious bovine rhinotracheitis virus recombinant subunit vaccine on a large scale. The reparation method for the recombinant subunit vaccine comprises the following steps: 1) cloning an eukaryotic expression vector containing a protein gD coding gene; 2) transfecting CHO cells, and obtaining suspending CHO cell strains, which stably andefficiently express the protein gD, in a selecting, screening and acclimatizing manner; 3) subjecting the cell strains obtained in the step 2) to fermented culture, and carrying out purification, soas to obtain recombinant protein gD; and 4) uniformly mixing the recombinant protein gD and ISA 201 VG 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 up to 2g / L to 3g / 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 and efficiently secreting and expressing bovine infectious rhinotracheitis virus gD protein and a preparation method and application of a bovine infectious rhinotracheitis virus gD protein subunit vaccine, belonging to animal vaccine and veterinary biological product technology field. Background technique [0002] Infectious Bovine Rhinotracheitis (IBR) is an acute, febrile, contact infectious disease caused by Infectious Bovine Rhinotracheitis virus (IBRV). , rhinitis, sinusitis, and upper respiratory tract inflammation. The virus can also cause damage to the reproductive system of cows, leading to miscarriage, stillbirth, etc., and sometimes enteritis, calf encephalitis, conjunctivitis and keratitis. The disease has a great impact on milk production, fattening rate and fecundity of dairy cows, and acute IBR respiratory tract infection can be secondary to fatal bacterial pneumonia in cattle. The disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/06C12N15/85C12N5/10A61K39/265A61P31/22G01N33/569
CPCA61K39/12A61K2039/552C07K14/005C12N15/85C12N2710/16034C12N2710/16051C12N2800/107G01N33/56994G01N2333/06
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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