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Preparation method of fusion mutant of Ebola virus glycoprotein and matrix protein

A technology for Ebola virus and matrix protein, applied in the field of preparing fusion mutants of Ebola virus glycoprotein and matrix protein

Active Publication Date: 2017-08-11
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on Ebola virus protein vaccines mainly focuses on the subunit vaccine research based on Ebola virus glycoprotein GP and the research on virus-like particle vaccines based on GP, ​​VP40 / GP, NP, VP40. There is no report on the fusion expression of the relevant protective antigen segment in the protein and the Ebola virus matrix protein VP40

Method used

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  • Preparation method of fusion mutant of Ebola virus glycoprotein and matrix protein
  • Preparation method of fusion mutant of Ebola virus glycoprotein and matrix protein
  • Preparation method of fusion mutant of Ebola virus glycoprotein and matrix protein

Examples

Experimental program
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Embodiment 1

[0088] Example 1, Construction of Ebola virus glycoprotein and matrix protein fusion mutant expression vector

[0089] 1. Acquisition of Ebola virus glycoprotein gene and matrix protein gene

[0090] By means of artificial synthesis, the full-length gene of EBOV-GP protein and the full-length gene of EBOV-VP40 protein (>KM034549|Zaire_ebolavirus_isolate_Hsapiens-wt / SLE / 2014 / ManoRiver-EM095B_|Homo_sapiens|01-Jun-2014) were synthesized, and according to Pichia pastoris preferred codons for codon optimization. At the same time, in order to synthesize a gene capable of expressing the full-length GP protein when synthesizing the EBOV-GP gene, an "A" was artificially added to the position where RNA editing occurred. The synthesis work Entrusted to Nanjing Jinruisi Biotechnology Co., Ltd. for synthesis. Wherein, the full-length gene of EBOV-GP protein is shown in sequence 1 in the sequence listing; the full-length gene of EBOV-VP40 protein is shown in sequence 5 in the sequence list...

Embodiment 2

[0101] Example 2, Expression, Purification and Identification of Ebola Virus Glycoprotein and Matrix Protein Fusion Mutant

[0102] 1. Construction and screening of recombinant yeast

[0103] About 10 μg of the expression plasmid pPICZ-GP1 Core fusion VP40 constructed in Example 1 was single-point linearized with the restriction endonuclease BglII, and the enzyme digestion system (50 μL) was as follows: expression plasmid PICZ-GP1 Core fusion VP40 43 μL; BglII 2 μL ; 10×NEB3.1 buffer 5μL.

[0104] Samples were taken after enzyme digestion at 37°C for 1 hour, and separated by 1% agarose gel electrophoresis to analyze whether the plasmid was linearized completely. The separation results showed that the completely linearized enzyme digested products were recovered with a spin-column type DNA fragment recovery kit, and the linearized plasmid was finally eluted with 30 μL of pure water.

[0105] The method for preparing yeast electroporation competent cells in the following steps...

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Abstract

The invention discloses a preparation method of a fusion mutant of Ebola virus glycoprotein and matrix protein by saccharomycetes. The preparation method includes steps of 1), introducing gene for encoding the fusion mutant of Ebola virus glycoprotein and matrix protein to a receptor yeast cell to obtain the recombined yeast cell for expressing the gene; 2), orderly cultivating and breaking the recombined yeast cell to obtain the fusion mutant of Ebola virus glycoprotein and matrix protein from the broken product. The fusion mutant of Ebola virus glycoprotein and matrix protein is a recombined protein obtained by fusing an Ebola virus glycoprotein core zone reserved with an Ebola virus glycoprotein receptor bond zone and a glycan cap zone to an N terminal of the Ebola virus matrix protein. The fusion mutant of Ebola virus glycoprotein and matrix protein has a polymer structure and has galactosylated modification; therefore, the fusion mutant is potential to be a vaccine for preventing Ebola hemorrhagic fever.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for preparing fusion mutants of Ebola virus glycoprotein and matrix protein, in particular to a method for preparing Ebola virus glycoprotein with polymer structure and glycosylation modification by using yeast Method for fusion mutants with matrix proteins. Background technique [0002] Ebola virus is a single-stranded negative-strand RNA virus with a genome length of about 19K. The whole genome can encode seven proteins: NP, VP35, VP40, GP, VP30, VP24 and L protein. Among them, the VP40 matrix protein is the most abundant protein in the Ebola virus matrix. Its main function is to participate in the formation of the virus skeleton. The VP40 matrix protein plays an important role in the assembly and release of the virus in the host cell. Its main characteristics That is, auto-oligomerization can occur, and the dimer is the basic unit of its oligomerization. The envelope glyc...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/81C07K19/00C07K1/22C07K1/18C07K1/16A61K39/295A61K39/12A61K31/14
CPCA61K39/00C07K14/005C07K2319/00C12N2760/14122C12N2760/14134
Inventor 吴军吴慕胜刘波
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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