Fusion protein comprising Fc domain of IgG and extracellular domain of EB virus envelope glycoprotein

A technology of envelope glycoprotein and fusion protein, applied in antiviral agents, medical preparations containing active ingredients, hybrid peptides, etc., can solve the problem of lack of animal models, restrictions on the prevention and treatment of EB virus malignant diseases, and inability to infect Rodents and other problems, to achieve the effect of improving immunogenicity

Active Publication Date: 2019-05-31
SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the lack of in-depth understanding of the mechanism of Epstein-Barr virus infection, replication and lysis, and the lack of suitable animal models because it cannot infect rodents, effective specific drugs and vaccines have not yet been officially launched, which greatly restricts the current situation. The prevention and treatment effect of EB virus-related malignant diseases

Method used

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  • Fusion protein comprising Fc domain of IgG and extracellular domain of EB virus envelope glycoprotein
  • Fusion protein comprising Fc domain of IgG and extracellular domain of EB virus envelope glycoprotein
  • Fusion protein comprising Fc domain of IgG and extracellular domain of EB virus envelope glycoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of fusion protein

[0041] 1. Materials

[0042] (1) Vector and strain: shuttle vector pFastBac I, E. coli competent DH10B strain carrying bacmid;

[0043] (2) Virus packaging and protein expression cell lines: insect Sf9 cell line and H5 cell line;

[0044](3) Reagents: Cellfectin transfection reagent and reagents related to plaque analysis purchased from Invitrogen, serum-free insect cell culture medium, HisTrap and MabSelect protein purification prepacked columns purchased from GE, and other commercially available chemical reagents.

[0045] 2. Steps

[0046] (1) Link the coding sequence of the Fc domain of human IgG1 or mouse IgG2a to the ECD of the Epstein-Barr virus envelope glycoprotein gp350 by enzyme digestion 123 or ECD FL domain coding sequence (ECD 123 It is the first three amino domains at the N-terminal of the extracellular domain of gp350; ECD FL It is the 3' end of the gp350 ectodomain full length), and the secretion signal pe...

Embodiment 2

[0054] Example 2 Detection of the conformation of the neutralizing epitope in the extracellular domain of gp350 in the fusion protein

[0055] 1. Materials

[0056] (1) Reagents and consumables: commercially available chemical reagents and ELISA plates;

[0057] (2) Antibody: commercially available mouse-derived HRP-labeled secondary antibody, affinity-purified gp350 neutralizing antibody mAb72A1. Among them, mAb72A1 is secreted by the HB168 hybridoma cell line in the ATCC depository, ATCC number: 72A1 (ATCC® HB-168™).

[0058] 2. Grouping

[0059] (1) Control group: bovine serum albumin fraction IV (BSA) as the ELISA negative control group, purified non-Fc fusion gp350-ECD 123 -6His recombinant protein (6His expressed in gp350-ECD 123 fused with 6 histidine tags at the C-terminus) as a positive control group;

[0060] (2) Experimental group: four purified fusion proteins gp350-ECD 123 -Fc hIgG1 , gp350-ECD FL -Fc hIgG1 , gp350-ECD 123 -Fc mIgG2a with gp350-ECD FL ...

Embodiment 3

[0066] Example 3 Detection of specific antibody total titer and neutralizing antibody titer in immune serum of fusion protein

[0067] 1. Materials

[0068] Reagents: commercially available chemical reagents and ELISA plates, commercially available aluminum adjuvants;

[0069] Animals: Commercially purchased BALB / c mice aged 6-8 weeks.

[0070] 2. Grouping

[0071] Control group: use of purified non-Fc fusion gp350-ECD 123 -6His recombinant protein immunized mouse serum was used as a control group;

[0072] Experimental group: using purified fusion protein gp350-ECD 123 -Fc hIgG1 , gp350-ECD FL -Fc hIgG1 , gp350-ECD 123 -Fc mIgG2a with gp350-ECD FL -Fc mIgG2a The sera of immunized mice were used as the experimental group.

[0073] 3. Steps

[0074] (1) Mix the recombinant protein of the control group and the experimental group with aluminum adjuvant in equal volumes, and immunize each mouse at a dose of 20 μg. The immunization methods are divided into two types: ①...

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Abstract

The invention discloses a fusion protein comprising an Fc domain of IgG and an extracellular domain of an EB virus envelope glycoprotein. The fusion protein is represented by the following formula: P-E-F, wherein P represents a secretion signal peptide, E represents an amino acid sequence of the extracellular domain of the EB virus envelope glycoprotein gp350, and F represents the amino acid sequence of the Fc domain of the IgG. It is found for the first time that after the fusion of the Fc domain of the immunoglobulin IgG with the envelope glycoprotein gp350 from the surface of the EB virus,the immunogenicity in vivo is significantly improved. After immunization with the fusion protein, the total serum titer an immunized animal, the serum specific neutralizing antibody titer and the serum in vitro viral infection blocking efficiency are significantly higher than that of a non-fused control protein. The fusion protein using the Fc domain of the immunoglobulin IgG and the EB virus membrane glycoprotein is adopted as an evidence for the efficacy of an EB virus vaccine and has important practical and theoretical significance and application prospects for prevention and treatment of EB virus-related diseases.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and more specifically, relates to a fusion protein comprising an IgG Fc domain and an Epstein-Barr virus envelope glycoprotein extracellular domain. Background technique [0002] Epstein-Barr virus, as the first virus closely related to tumorigenesis reported by humans, is closely related to the occurrence of various lymphomas and epithelial cell carcinomas, such as mononucleosis (infectious mononucleosis, IM), Hodgkin's lymphoma (Hodgkin lymphoma, HL), post-transplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), EBV-associated gastric carcinoma (EBV-GC), etc. In addition, chronic active EBV infection (CAEBV) can also lead to extranodal natural killer / T cell lymphoma (ENK / T cell lymphoma) or hemophagocytic lymphohistiocytosis (haemophagocytic lymphohistiocytosis) ), seriously endangering personal health and life. [0003] However, due to the lack of in-depth understan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/245A61K39/395A61P31/22
CPCY02A50/30
Inventor 赵炳春曾益新张晓高灵冯琳张浩炯冯启胜
Owner SUN YAT SEN UNIV
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