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Construction and application of Xinjiang hemorrhagic fever virus multi-epitope eukaryotic expression vector pVAX-MEPX2

A technology of eukaryotic expression vector and hemorrhagic fever virus, which is applied in the field of molecular biology and microbiology, can solve the problems of in-depth research limitations of diagnosis, prevention and treatment methods, lack of animal models, high pathogenicity, etc., and achieve improved immunity The effect of low effect, improving immune potency, and avoiding side effects

Pending Publication Date: 2019-01-25
XINJIANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the high virulence and pathogenicity of XHFV, its research needs to be carried out in high-level laboratories, resulting in the lack of suitable animal models, which limits the in-depth research on effective diagnosis, prevention and treatment methods for XHF

Method used

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  • Construction and application of Xinjiang hemorrhagic fever virus multi-epitope eukaryotic expression vector pVAX-MEPX2
  • Construction and application of Xinjiang hemorrhagic fever virus multi-epitope eukaryotic expression vector pVAX-MEPX2
  • Construction and application of Xinjiang hemorrhagic fever virus multi-epitope eukaryotic expression vector pVAX-MEPX2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of pVAX-MEPX2 vector

[0032](1) The main antigenic positive protein segment NP2 of the nucleoprotein NP identified in the early stage of the XHFV YL04057 strain is concatenated with 6 linear B cell epitopes on the glycoprotein GP (Gn and Gc) through a flexible peptide (Gly-Gly), and the design After becoming a multi-epitope chimeric gene, the MEPX2 gene was synthesized after tandem duplication into a double-copy gene fragment and optimized by Escherichia coli preferred codons;

[0033] (2) performing base synthesis on the optimized gene fragment in step (1);

[0034] (3) Carry out enzyme digestion reaction with BamHI and XhoI restriction endonucleases and eukaryotic expression vector pVAXI empty vector, and introduce the MEPX2 gene fragment obtained in step (2) to construct recombinant eukaryotic expression vector pVAX-MEPX2;

[0035] (4) Transform the obtained recombinant vector pVAX-MEPX2 into E.coli DH5α competent cells through the above step...

Embodiment 2

[0037] Example 2: Cell transfection and identification of pVAX-MEPX2 plasmid

[0038] Transfect endotoxin-free plasmids pVAX-MEPX, pVAX-MEPX2, pVAXⅠ (negative control) and pVAX-Gc-NP2 (positive control) into 293T cells grown to 90%-95% in 24-well plates, according to lipofectamine 2000 product manual to operate. After culturing for 24 hours, the gene expression was detected by indirect immunofluorescence (IFA). After the cells were fixed and permeabilized, the mixed rabbit serum of anti-Gn, Gc and NP and the goat anti-rabbit IgG antibody coupled with FITC were added in turn, and the cells were detected by fluorescence. Microscope observation. The test results are attached figure 2 As shown, the cells transfected with pVAX-MEPX, pVAX-MEPX2 and pVAX-Gc-NP2 were found to have green fluorescence under the fluorescence microscope, while the cells transfected with the empty plasmid pVAXⅠ had no fluorescent substances. figure 2 , indicating that the recombinant eukaryotic plasmi...

Embodiment 3

[0039] Example 3: Detection of Mouse Immunity and Lymphocyte Proliferation

[0040] The 6-8 week old BALB / c female mice were randomly divided into 9 groups, 8 mice in each group, and immunized once every 14 days. Groups a-d are DNA immunization groups: adjust the concentration of a large amount of extracted plasmid DNA to 1000 ng / μL with physiological saline, and inject 50 μL / mouse of the plasmid into the hind leg muscles of mice using an electric pulse gene transfer instrument. Group c is pVAX-Gc -NP2 positive control group, group d is empty pVAXⅠ as a negative control; groups e-f are protein immunization groups (rMEPX, rMEPX2 respectively): adjust the concentration of the purified recombinant protein to 1 mg / mL with PBS, and use an equal volume of Adjuvant (Freund's complete adjuvant FCA for initial immunization, and Freund's incomplete adjuvant FIA for booster immunization) was mixed by ultrasound, and 300 μL / mouse was injected subcutaneously at multiple points on the back ...

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Abstract

The invention discloses a recombinant eukaryotic expression vector for preparing Xinjiang hemorrhagic fever medicine, a eukaryotic expression vector is obtain by combining a multi-epitope double-copygene fragment MEPX2 of Xinjiang hemorrhagic fever virus and an eukaryotic expression vector pVAX I, reasonable epitope design improves the immune efficacy and has the ability to focus the immune response on conservative epitopes, and it has good immunogenicity through the immunization of animal experiments. The recombinant plasmid was used as the antibody level of DNA vaccine combined with proteinvaccine. The cellular immunity level and immunity effect were better than single antigen immunization. The multi-epitope double-copy gene fragment MEPX2 of Xinjiang hemorrhagic fever virus strain YL04057 was recombined with eukaryotic expression vector pVAX I to form eukaryotic expression vector and immunized with protein vaccine, so overcome the shortcomings of poor immunity caused by single antigen, and have good development and application prospects.

Description

technical field [0001] The present invention relates to the fields of molecular biology and microbiology. Specifically, the present invention relates to the construction and application of a multi-epitope eukaryotic expression vector of Xinjiang hemorrhagic fever virus. Background technique [0002] Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease caused by Crimean-Congo hemorrhagic fever virus (CCHFV), which belongs to the Nyaviridae Nerovirus genus. According to reports, 5-40% of patients showed severe bleeding, causing shock and even death. In China, the first case of CCHF was reported in 1965, so the disease is also known as Xinjiang hemorrhagic fever (XHF). Virus strains have been isolated from patients in the Bachu region of Xinjiang, which is considered to have the highest incidence of CCHF in my country. Due to the high virulence and pathogenicity of XHFV, its research needs to be carried out in high-level laboratories, resulting in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/85A61K31/12A61P31/14
CPCA61P31/14A61K31/12C07K14/005C12N2760/12034C12N2760/12022
Inventor 孙素荣俞欢阿来·沙力塔那李轶杰张富春
Owner XINJIANG UNIVERSITY
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