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GB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application of gB subunit recombinant protein

A technology of porcine pseudorabies virus and pseudorabies virus, which is applied in the field of gene recombination expression in biotechnology, can solve the problems of low yield and achieve the effects of high biological safety, easy large-scale production, and high controllability

Active Publication Date: 2020-12-29
NOVO BIOTECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For application in veterinary vaccines, the yield is still relatively low

Method used

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  • GB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application of gB subunit recombinant protein
  • GB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application of gB subunit recombinant protein
  • GB subunit recombinant protein of porcine pseudorabies virus, and preparation method and application of gB subunit recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1g

[0037] Embodiment 1gB protein expression and preparation

[0038] 1.1 Selection of porcine pseudorabies virus gB protein

[0039]The surface glycoprotein gB of porcine pseudorabies virus is a polypeptide encoded by the UL27 gene. It is predicted and analyzed that 1-800 amino acids are the extracellular domain of gB protein. There is a transmembrane domain at 801-821 amino acids, and 822-916 amino acids are intracellular Area. The extracellular region of gB protein has a stable structure and good immunogenicity, and is the main antigen for producing gB subunit vaccines. At present, there is no report that the protein can be expressed and purified on a large scale in a mammalian system, which is also an important technical problem to be solved by the present invention.

[0040] 1.2 Codon optimization of porcine pseudorabies virus gB protein

[0041] The gB protein is the most conserved protein on the surface of porcine pseudorabies virus, with a homology of up to %. Our labor...

Embodiment 2

[0042] Example 2: Construction of pEE12.4-OPTI-gB recombinant plasmid

[0043] 2.1 PCR amplification of target fragment OPTI-gB

[0044] 2.1.1 PCR reaction

[0045] (1) Primer design and synthesis

[0046] Upstream primer: 5'-cgaAGCTTGCCGCCACCATGGTGGCTC-3'

[0047] Downstream primer: 5'-cgcGAATTCTCAATGGTGATGGTGATGGTGATTATGATCCACCTT-3'

[0048] (2) Add 50 μL of the sample system, as shown in the table below:

[0049]

[0050]

[0051] PCR amplification program:

[0052]

[0053] 2.1.2 Gel recovery of PCR products

[0054] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0055] (2) Take the weight of the marked empty EP tube, and record the value;

[0056] (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;

[0057] (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 gentl...

Embodiment 3

[0104] Example 3: Establishment of transfection of pEE12.4-OPTI-gB recombinant plasmid into CHO-K1 cells and monoclonal screening

[0105] 3.1 CHO-K1 cell transfection

[0106] (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.

[0107] (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.

[0108] (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.

[0109] (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.

[0110] (5) Transfer the digested cells to a...

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Abstract

The invention provides a gB subunit recombinant protein of a porcine pseudorabies virus, and a preparation method and application of the gB subunit recombinant protein. The preparation method comprises the following steps of 1) cloning an optimized gB gene sequence into an eukaryotic expression vector to obtain a recombinant plasmid containing a gB subunit protein coding gene of the pseudorabies virus; 2) transfecting the recombinant plasmid containing the gB subunit protein coding gene of the pseudorabies virus into an expression cell; 3) culturing, screening and domesticating the expressioncell in the step 2) to obtain a highly expressed cell strain; and 4) fermenting and culturing the cell strain in the step 3), and performing purification to obtain the gB subunit protein of the pseudorabies virus. The invention provides the gB subunit protein of the porcine pseudorabies virus, and the gB subunit protein can be industrially produced in a large scale; the preparation method is simple; the cost is low; and the yield is much higher than that of an existing baculovirus expression system.

Description

technical field [0001] The invention belongs to the field of biotechnology gene recombination expression. The invention relates to a gB subunit recombinant protein of porcine pseudorabies virus and its preparation method and application. Background technique [0002] Porcine pseudorabies is an acute infectious disease caused by porcine pseudorabies virus (Pseudorabies virus, PrV), which causes sow abortion, central nervous system disorder of newborn piglets, and respiratory symptoms and death of weaned piglets. Economic losses. At present, the main measure to prevent and control the disease is vaccination. The subunit vaccine does not contain nucleic acid, and the immune response produced can be distinguished from wild strains. After vaccination, it will not cause persistent infection or latent infection, and the safety is better. However. At present, the application of subunit vaccines is limited due to the high cost of protein production. [0003] PRV belongs to the h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03C12N15/85C12N5/10
CPCC07K14/005C12N15/85C12N2710/16722C12N2710/16751
Inventor 钱泓吴有强卞广林张强徐玉兰吴素芳黄丽嫒姜冰洁蔡灵芝贾宝琴
Owner NOVO BIOTECH CORP
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