Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Recombinant virus-like particle expressed based on inclusion body form, preparation method and application thereof

A technology of recombinant virus and inclusion body, applied in the field of biomedicine, can solve the problems of no biological activity, wrong structure, difficult to remove, etc., and achieve the effect of simple and easy to operate preparation method

Active Publication Date: 2019-08-06
INST OF PROCESS ENG CHINESE ACAD OF SCI
View PDF7 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem is that the surfactant is easy to bind to the protein and is not easy to remove, which seriously affects the further assembly of the assembly unit to form virus-like particles.
[0007] The preparation methods of recombinant virus-like particles disclosed in the above-mentioned prior art are all based on soluble assemblies, and the recombinant virus-like particles are directly purified by gradient density centrifugation and other methods, and the scope of application is narrow
However, in the process of vaccine preparation using virus-like particles as carriers, when antigens and viral capsid proteins are fused and expressed through exogenous expression systems, especially E. coli expression systems, it is very easy to produce inclusion bodies with structural errors and no biological activity. , recombinant virus-like particles cannot be obtained by direct purification, so it is very meaningful to develop a method for preparing recombinant virus-like particles expressed in the form of inclusion bodies that is simple to operate and widely applicable

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
  • Recombinant virus-like particle expressed based on inclusion body form, preparation method and application thereof
  • Recombinant virus-like particle expressed based on inclusion body form, preparation method and application thereof
  • Recombinant virus-like particle expressed based on inclusion body form, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Expression and Identification of Recombinant Hepatitis B Virus Core Antigen Fused with Other Antigens

[0093] The specific operation method is:

[0094] (1) Insert a major histocompatibility complex MHC class II antigen (the amino acid sequence is SEQ ID NO.3: AELVHFLLLKYRAR) in the human melanoma-related gene MAGE A3 gene into the hepatitis B virus core antigen HBc MIR by means of genetic engineering Region (inserted between HBc 78aa-79aa), ligated into plasmid pET28a, named pET28a-HBc-MAGE3II. The recombinant plasmid was subcloned into competent Escherichia coli cells BL21(DE3), and the amino acid sequence of HBc-MAGE3II is shown in SEQ ID NO.4. Inoculate to 20L fermenter for fermentation, wait for OD 600 At 5-8 hours, add 0.1mM isopropylthiogalactopyranoside (IPTG) to induce 4h to end the fermentation, and draw the thalline growth curve, such as figure 1 shown.

[0095] SEQ ID NO.4:

[0096] Mdidpykefgasvellsflpsdffpsirdlldtasalyrealespehcsphhtalrqailcwgelmnlat...

Embodiment 2

[0103] dissolution of inclusion bodies

[0104] The specific operation method is:

[0105] (1) Weigh 0.1 g of the inclusion bodies obtained in Example 1, add 1 mL of denaturant containing 8M urea, 6M guanidine hydrochloride or 1% SDS respectively, shake overnight at room temperature, collect the supernatant by centrifugation to measure the protein concentration and calculate the dissolution rate. The result is as image 3 shown.

[0106] (2) Add the inclusion bodies after washing in Example 1 to the dissolving buffer containing 20mM Tris-HCl, 1% SDS, and 0.5% β-mercaptoethanol at pH 8.0 at a ratio of 1g:1mL. The inclusion bodies are excessive and the solubility reaches saturation , using the Bradford method to measure the protein concentration, about 20mg / mL.

[0107] This example also explores the effects of different types of dissolving solvents and subsequent treatments on the secondary structure of proteins. The protein secondary structure of the processed samples was ...

Embodiment 3

[0112] extracellular assembly of inclusion bodies

[0113] The specific operation method is:

[0114] (1) Dilute the inclusion bodies dissolved in SDS obtained in Example 2 with pH 8.0 containing 15-25mM Tris-HCl and 0.4%-6% β-mercaptoethanol at 4°C. After diluting 100 times overnight, add 2-Methyl-2,4-pentanediol with a final concentration of 1M was reacted at 20°C for 2 days, and then placed at 4°C for 3 days after desalting. The desalting column used for desalting was G25 desalting column, and the buffer used for desalting was pH 7.4 20 mM Tris-HCl solution.

[0115] This example also explored the effect of different dilution factors on the protein. The specific method is: prepare the recombinant hepatitis B virus core antigen in the form of inclusion bodies fused with other antigens according to the method of Example 1-3, and wash the inclusion bodies. The inclusion bodies were dissolved with 1% SDS solution, diluted 10 times, 20 times, 50 times and 100 times respectivel...

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

No PUM Login to View More

Abstract

The invention relates to a recombinant virus-like particle expressed based on an inclusion body form, a preparation method and an application thereof. The preparation method comprises: expressing a recombinant plasmid containing a recombinant viral capsid protein gene or the recombinant viral capsid protein gene fused with another antigen genes in an exogenous expression system, and obtaining therecombinant viral capsid protein in the form of inclusion bodies or the recombinant viral capsid protein fused with the other antigens; dissolving the inclusion body with a surfactant, diluting the material, adding an amphiphilic cosolvent, and performing desalination to realize assembly of the virus-like particles to obtain the recombinant virus-like particles. The method is applicable to all recombinant virus-like particles expressed in the form of the inclusion bodies, and is simple and easy to operate, and the prepared recombinant virus-like particles have a morphology similar to that of natural viruses, and the method provides a strategy for the development of preventive vaccines and therapeutic vaccines.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant virus-like particle expressed in the form of inclusion body and its preparation method and application. Background technique [0002] Virus-like particles (VLPs) refer to hollow particles that are composed of one or more capsid proteins of viruses and do not contain viral nucleic acids, with diameters ranging from tens to hundreds of nanometers. Since VLP has a virus-like morphology and a pathway to enter the body, it can greatly improve immunogenicity and stimulate the body's strong humoral and cellular immunity, and has broad application prospects in vaccine vectors and drug delivery. Among them, hepatitis B virus core antigen (HBc) is composed of single-chain capsid protein, which can be expressed in the form of assembly in eukaryotic cells and prokaryotic cells, and is currently the most widely used VLP. Although HBc allows the insertion and rep...

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
IPC IPC(8): C07K19/00C07K14/02C07K14/015C07K14/045C07K1/34C12N15/70C12N15/81C12N15/85A61K39/12A61P31/12
CPCA61K39/12A61K2039/5258A61P31/12C07K14/005C07K2319/00C12N15/70C12N15/815C12N15/85C12N2710/16123C12N2730/10123C12N2750/14023C12N2770/14023
Inventor 刘永东苏志国张耀
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com