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Subunit fusion protein tG on surface of rabies virus, and preparation method and application of subunit fusion protein tG

A fusion protein, rabies virus technology, applied in biochemical equipment and methods, viruses, viral peptides, etc., can solve the problems of inability to large-scale industrialization, inability to produce applications, and high production costs, achieving batch-to-batch stability and easy large-scale production. The effect of easy production and quality control

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

The patent "A Properly Folded Recombinant Rabies Virus G Protein Extracellular Segment and Its Potential Application" (Patent No.: CN109627294A) provides a method for producing G protein, by modifying the hydrophobic part of the protein, using insect baculovirus The system expresses and prepares G protein with correct structural folding, but the yield is very low, and the purification is complicated, so it cannot be industrialized on a large scale
Although these studies have verified that G protein is a good protective antigen, its yield is very low, and the cost of large-scale industrial production is too high to produce and apply

Method used

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  • Subunit fusion protein tG on surface of rabies virus, and preparation method and application of subunit fusion protein tG
  • Subunit fusion protein tG on surface of rabies virus, and preparation method and application of subunit fusion protein tG
  • Subunit fusion protein tG on surface of rabies virus, and preparation method and application of subunit fusion protein tG

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 G protein expression and design

[0045] 1.1 Selection of rabies virus G protein

[0046] The rabies virus surface envelope protein G protein is composed of an extracellular region whose antigenic site is located in the extracellular 1M-455N amino acids, wherein 1-19 amino acids are signal peptides, and 20-445 amino acids are mature amino acid extracellular regions. G protein is a homotrimer in structure. Although G protein, as an important protective antigen, has been deeply studied and reported as early as the 1980s, it has not been reported that it can be expressed on a large scale in a eukaryotic expression system. Purification of the protein may be caused by the unstable structure of the G protein expressed alone. In order to solve this important technical problem, the present invention introduces a zipper peptide that is easy to form a trimer at the carboxyl end of the G protein to ensure the stability of the G protein. The structure is correctly folded...

Embodiment 2

[0049] Example 2: Construction of pEE12.4-OPTI-tG recombinant plasmid

[0050] 2.1 PCR amplification of target fragment OPTI-tG

[0051] 2.1.1 PCR reaction

[0052] (1) Primer design and synthesis

[0053] Upstream primer: 5'-ACGAAGCTTGCCGCCACCATGGTGCCTCAGGTGC-3'

[0054] Downstream primer: 5'-GCGAATTCTTAATGGTGATGGTGATGGTGTGTGCGATTGC-3'

[0055] (2) Add 50 μL of the sample system, as shown in the table below:

[0056]

[0057] PCR amplification program:

[0058]

[0059] 2.1.2 Gel recovery of PCR products

[0060] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0061] (2) Take the weight of the marked empty EP tube, and record the value;

[0062] (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;

[0063] (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 the...

Embodiment 3

[0110] Example 3: Establishment of transfection of pEE12.4-OPTI-tG recombinant plasmid into CHO-K1 cells and monoclonal screening

[0111] 3.1 CHO-K1 cell transfection

[0112] (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.

[0113] (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.

[0114] (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.

[0115] (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.

[0116] (5) Transfer the digested cells to a...

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Abstract

The invention provides a subunit fusion protein tG on the surface of a rabies virus, and a preparation method and application of the subunit fusion protein tG. The preparation method comprises the following steps of 1) cloning a gene sequence shown as SEQ ID NO.4 into an eukaryotic expression vector to obtain a recombinant plasmid containing a fusion protein tG coding gene; 2) transfecting the recombinant plasmid containing the fusion protein tG coding gene into an expression cell; 3) culturing, screening and domesticating the expression cell in the step 2) to obtain a highly expressed cell strain; and 4) fermenting and culturing the cell strain in the step 3), and after purification, obtaining the subunit fusion protein tG. The tG can be expressed in a large amount in a soluble manner; the protein is stable; various problems that the G protein on the surface of the rabies virus cannot be expressed in a large scale and the like in the prior art are solved; and the preparation method issimple, and the cost is low.

Description

technical field [0001] The invention belongs to the technical field of recombinant protein expression and purification of biological products, and relates to a subunit fusion protein tG on the surface of rabies virus and its preparation method and application. Background technique [0002] Rabies is a zoonotic infectious disease caused by Rabies virus (RV), with a mortality rate as high as 100%. The clinical manifestations are hydrophobia, photophobia, mania and other symptoms. Dogs are the main host and source of infection for rabies. Rabies is distributed worldwide. The number of deaths caused by rabies is about 40,000 to 70,000 in the world every year, but most of the annual rabies occurs in developing countries, 98% of which are in Asia. my country is the country with the second highest annual rabies incidence in the world after India. Rabies has become an important epidemic that threatens the public health security of our country. [0003] Rabies virus is a member ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/205A61P31/14
CPCC07K14/005C12N15/85C12N15/62A61K39/12A61P31/14C12N2760/20122C12N2760/20134C07K2319/73C12N2800/107C12N2800/22A61K2039/552Y02A50/30
Inventor 钱泓吴有强卞广林张强徐玉兰吴素芳车影蔡灵芝贾宝琴屠莉洁
Owner NOVO BIOTECH CORP
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