Preparation method of O-type foot-and-mouth disease virus-like particle antigen, prepared O-type foot-and-mouth disease virus-like particle antigen and application thereof

A foot-and-mouth disease virus and particle antigen technology, which is applied in the fields of molecular biology and virology, can solve the problems of large-scale epidemics of diseases, increase the difficulty of vaccines, and the virus is not completely killed or fully attenuated, and achieves the stability of foot-and-mouth disease virus-like particles. , The effect of soluble expression with high yield and no biosafety risk

Pending Publication Date: 2022-01-21
PU LIKE BIO ENG
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are also the following disadvantages: 1. The virus must be produced in highly sealed equipment to prevent contamination of the surrounding environment; during the production process, the virus may not be completely killed or fully attenuated, which will cause the vaccine to contain highly toxic pathogens. The disease substance, which in turn causes the disease of the immunized animals and causes the disease to spread on a large scale
2. The validity period is very short, and often need to be frozen, which increases the difficulty of promoting the use of vaccines in rural areas
3. Inactivated vaccine immunity is indistinguishable from animals infected with FMDV, which restricts the export of animals
4. Production depends on FMDV, so FMDV cannot be completely eliminated
The current use of inactivated vaccines and attenuated vaccines for prevention and control can no longer adapt to the current healthy development of animal husbandry

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method of O-type foot-and-mouth disease virus-like particle antigen, prepared O-type foot-and-mouth disease virus-like particle antigen and application thereof
  • Preparation method of O-type foot-and-mouth disease virus-like particle antigen, prepared O-type foot-and-mouth disease virus-like particle antigen and application thereof
  • Preparation method of O-type foot-and-mouth disease virus-like particle antigen, prepared O-type foot-and-mouth disease virus-like particle antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Expression of embodiment 1 recombinant FMDV capsid protein SUMO-VPO, SUMO-VP3 and SUMO-VP1

[0069] 1.1 Preparation and transformation of recombinant plasmids

[0070]The O-type foot-and-mouth disease virus VP0 gene fragment shown in the sequence listing SEQ ID NO.1, the O-type foot-and-mouth disease virus VP3 gene fragment shown in the sequence listing SEQ ID NO.2, and the sequence listing SEQ ID NO were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. . The O-type foot-and-mouth disease virus VP1 gene fragment shown in 3 is connected to the pETSUMO vector respectively. Then the fragments containing RBS-SUMO-VPO, T7-RBS-SUMO-VP3 and T7-RBS-SUMO-VP1 were amplified using the successfully ligated recombinant plasmids as templates. Step by step, the fragments obtained after digestion with corresponding endonucleases were cloned into the same pET28a expression vector to obtain a recombinant plasmid containing SUMO-VPO-SUMO-VP3-SUMO-VP1.

[0071] Transform the ligat...

Embodiment 2

[0078] The influence of the Ulp1 protease of embodiment 2 different enzymatic activity units on the assembly of capsid protein

[0079] 2.1 Assembly of virus particles under different protease addition conditions

[0080] Dilute the purified protein solution containing recombinant FMDV capsid proteins SUMO-VP0, SUMO-VP3 and SUMO-VP1 to a total protein concentration of 1.0mg / mL, take 0.5ml of the above solution as a reaction system, and in five reaction systems 1U, 2U, 5U, 10U or 20U of Ulp1 protease were added respectively. Each protein sample was incubated at 4°C for 24 hours, and then nickel filler was added to adsorb SUMO protein. After treatment, the state of the protein in each reaction system was determined by high performance liquid gel filtration chromatography.

[0081] The result is as figure 2 as shown, figure 2 A-E show the results of high performance liquid gel filtration chromatography of the solution after Ulp1 protease was digested and adsorbed SUMO prote...

Embodiment 3

[0085] The impact of different salt concentrations of embodiment 3 on FMDV VLPs assembly

[0086] In this example, further investigation (NH 4 ) 2 SO 4 The effect of salt concentration on the assembly of FMDV VLPs. Specifically, unassembled recombinant FMDV capsid proteins SUMO-VPO, SUMO-VP3 and SUMO-VP1 were added to different concentrations (0.05M, 0.1M, 0.2M, 0.3M, 0.5M, 0.8M or 1.0M )(NH 4 ) 2 SO 4 Add 5U Ulp1 protease to 20mM disodium hydrogen phosphate (pH7.4) buffer solution, and incubate at 4°C for 24 hours, then add nickel filler to adsorb SUMO protein. After treatment, the high-performance liquid gel filtration chromatography was used to observe the influence of different salt ion concentrations on the assembly of FMDV VLPs.

[0087] The result is as Figure 4 As shown, the above experimental results under different salt ion concentration conditions correspond to Figure 4 A-G. Calculate the peak area, the results show that different salt ion concentrations...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
diameteraaaaaaaaaa
Login to view more

Abstract

The invention relates to a preparation method of O-type foot-and-mouth disease virus-like particles, which comprises the following steps: recombining a VP0 gene, a VP3 gene and a VP1 gene of an O-type foot-and-mouth disease virus to a pETSUMO vector, forming fusion protein genes with SUMO, amplifying the three fusion protein genes, cloning into the same pET28a expression vector to obtain a recombinant expression vector pET28a-SUMO-VP0-SUMO-VP3-SUMO-VP1, transforming the expression vector into escherichia coli to express fusion protein, and removing SUMO, VP0, VP3 and VP1 proteins from the expressed fusion protein by enzyme digestion under the condition of 10U/mL Ulp1 protease concentration to form the O-type foot-and-mouth disease virus-like particles through self-assembly. The method can be used to efficiently prepares the O-type foot-and-mouth disease virus-like particles, and is suitable for large-scale industrial production.

Description

technical field [0001] The present invention belongs to the fields of molecular biology and virology. The invention specifically relates to a method for preparing an O-type foot-and-mouth disease virus-like particle antigen, the prepared O-type foot-and-mouth disease virus-like particle antigen and applications thereof. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease that can spread quickly and over long distances. significant economic loss. Animals affected by FMD include cattle, sheep, goats and pigs. The causative agent, foot-and-mouth disease virus (Foot-and-mouth disease virus, FMDV), is a kind of aphthous virus of picornavirus family. The virus is divided into seven serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3), of which O-type FMDV is the most prevalent. Vaccine immunization is an effective measure to control the disease and protect livestock from harm. Traditional vaccines include inactivated vaccines a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/09C12N15/70A61K39/135A61P31/14
CPCC07K14/005C12N15/70A61K39/12A61P31/14C12N2770/32123C12N2770/32134C12N2770/32152A61K2039/552
Inventor 田克恭张素玲张许科
Owner PU LIKE BIO ENG
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products