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Foot-and-mouth disease virus capsid protein tandem coexpressions and virus-like particle preparation method

A technology of foot-and-mouth disease virus and capsid protein, applied in the field of molecular biology, can solve the problems of loss of large-scale commercial application, insolubility, low expression of foot-and-mouth disease virus capsid protein, etc., achieve good protective effect, mild conditions, and process flow simple effect

Active Publication Date: 2015-03-11
SA BIOTECH (SUZHOU) PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of studies have shown that the expression of FMD virus coat protein in Escherichia coli alone or in genetically engineered bacteria obtained by co-transfecting Escherichia coli with different plasmids is very low and often insoluble, which has lost large-scale commercial use. possibility of application

Method used

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  • Foot-and-mouth disease virus capsid protein tandem coexpressions and virus-like particle preparation method
  • Foot-and-mouth disease virus capsid protein tandem coexpressions and virus-like particle preparation method
  • Foot-and-mouth disease virus capsid protein tandem coexpressions and virus-like particle preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: have the foot-and-mouth disease virus capsid protein VP3 of sequence 1 (1a, 1b, 1c), VP1, VPO (gene fusion of VP4 and VP2) tandem soluble co-expression

[0057] 1.1 is used as the preparation of the foot-and-mouth disease virus capsid protein VP3 of template, VP1, VP0 gene fragment

[0058] The full-length FMDV capsid protein gene (VP3, VP1, VP0) optimized by the codon of Escherichia coli was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The full lengths of the synthesized gene fragments were 909 bp (VPO), 657 bp (VP3), and 627 bp (VP1). The template of the foot-and-mouth disease virus capsid protein of the present invention is prepared on the basis of the artificially synthesized gene fragment of the capsid protein of the foot-and-mouth disease virus.

[0059] 1.2 Construction of tandem co-expression vector of foot-and-mouth disease virus capsid protein gene (VP3, VP1, VP0) with SUMO tag

[0060] The full-length (VP3, VP1, VPO) gene fragments ...

Embodiment 2

[0081] Embodiment 2: the affinity chromatography purification preparation of the foot-and-mouth disease virus capsid protein (VP3, VP1, VPO) with SUMO label

[0082] Resuspend the bacteria at a ratio of 1 g of bacteria to 10 mL of lysate (20 mM Tris buffer pH 7.2, 300 mM NaCl), and crush the bacteria four times with a homogenizer at a pressure of 700 bar. JA-14 turns head 13500rpm (28000g), centrifuges 35min, leaves supernatant, detects by 15% SDS-polyacrylamide gel electrophoresis, the foot-and-mouth disease virus capsid protein (VP3, VP1, VP1, VP3, VP1, The purity of VPO) is about 20%. The supernatant was filtered with a 0.45 μm pore size filter, and then chromatographically purified with a HisTrap FF column (GE Healthcare Life Sciences).

[0083] Instrument system: AKTA purification and preparative liquid chromatography system produced by GE Healthcare former Amershan Pharmacia company.

[0084] Chromatographic medium: Ni Sepharose.

[0085] Column volume: 5ml.

[0086]...

Embodiment 3

[0093] Embodiment 3: remove the molecular sieve chromatographic purification of the foot-and-mouth disease virus capsid protein (VP3, VP1, VPO) of SUMO label

[0094] The samples of the capsid protein (VP3, VP1, VPO) of foot-and-mouth disease virus (VP3, VP1, VPO) with SUMO tags obtained in Example 2 were collected and digested at 4° for 12 hours for the next step of molecular sieve chromatography purification.

[0095] Instrument system: AKTA purification and preparative liquid chromatography system produced by GE Healthcare former Amershan Pharmacia company.

[0096] Chromatography medium: HiLoad 26 / 60 Superdex 200.

[0097] Column volume: 2.6×60 cm, 318 ml.

[0098] Eluent: 20mM phosphate buffer pH 8.0, 0.2M NaCl.

[0099] Flow rate: 5ml / min.

[0100] Detector wavelength: 280nm.

[0101] The sample is 900 mL of the FMD virus capsid protein (VP3, VP1, VPO) protein solution from which the SUMO tag has been removed.

[0102] The elution procedure is as follows: after brea...

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Abstract

The invention relates to escherichia coli-derived single-plasmid-tandem soluble coexpression foot-and-mouth disease virus capsid proteins VP0 (which is a VP4 and VP2 fusion gene), VP1 and VP3, and a foot-and-mouth disease virus capsid protein virus-like particle preparation method. Foot-and-mouth disease virus capsid protein virus-like particles can be used for preparation of a foot-and-mouth disease vaccine. According to the method, a plurality of aspects of escherichia coli-derived soluble coexpression foot-and-mouth disease virus capsid protein are studied, by comprehensive use of tandem coexpression and SUMO(suggested upper merged ontology) technology with a tag for soluble coexpression of the foot-and-mouth disease virus capsid proteins VP0 (which is the VP4 and VP2 fusion gene), VP1 and VP3, the ultimate objective protein accounts for about 20% of total bacterial protein, and the foot-and-mouth disease virus capsid proteins obtained by purification can be successfully assembled into the virus like particles.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a foot-and-mouth disease virus capsid protein gene, a carrier, a bacterial strain and an expression method thereof, a vaccine containing the virus-like particle and its use in preventing foot-and-mouth disease infection. Background technique [0002] Foot-and-mouth disease is a zoonotic disease caused by foot-and-mouth disease virus infection, and individual variants of foot-and-mouth disease virus can be transmitted to humans. The symptoms of foot-and-mouth disease are blisters and rotten spots on the hoof crown, between the toes, and the skin of the heel. Some animals also have the same lesions on the oral mucosa and nasal disc. Foot-and-mouth disease is highly contagious, spreads rapidly, has a high infection and morbidity rate, and can cause a large number of deaths and serious economic losses. It is classified as a severe infectious disease internationally. In the...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N7/04A61K39/135A61P31/14C12N1/21
Inventor 袁于人
Owner SA BIOTECH (SUZHOU) PTE LTD
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