Subunit nano vaccine for preventing and treating nasopharyngeal carcinoma and preparation method of subunit nano vaccine

A nanoparticle and immune adjuvant technology, applied in the field of biomedicine, can solve the problems of ineffective immune effect and weak immunogenicity.

Active Publication Date: 2020-01-03
SUN YAT SEN UNIV
View PDF11 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] EBV will continue to express Epstein-Barr Nuclear Antigen 1 (EBNA1, Epstein-Barr Nuclear Antigen 1) after the host cell enters latent infection. The anti-tumor vaccine against EBNA1 is a hot spot in the current research field. Patents CN201310676489.X, CN201310588797.7 and CN201410817936.3 etc. have all disclosed the vaccine that comprises EB virus EBNA1 protein, but, its immunogenicity of natural EBNA1 protein is weak, often can not cause effective immune effect alone, and the glycine-alanine (GA) in EBNA1 repeats Sequence inhibits ubiquitination of antigen, thereby inhibiting antigen presentation

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
  • Subunit nano vaccine for preventing and treating nasopharyngeal carcinoma and preparation method of subunit nano vaccine
  • Subunit nano vaccine for preventing and treating nasopharyngeal carcinoma and preparation method of subunit nano vaccine
  • Subunit nano vaccine for preventing and treating nasopharyngeal carcinoma and preparation method of subunit nano vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Preparation of EBNA1△GA protein

[0082] Refer to the amino acid sequence of wild-type EBNA1 nuclear antigen protein of 4 strains of human gamma herpesvirus B95-8 (NCBI database accession number: YP_401677.1);

[0083] (1) Synthesize the target gene fragment truncated from the 92-327 sequence of the full-length EBNA1, and the C-segment is fused with a 6x histidine tag and a stop codon.

[0084](2) The synthetic gene fragment was subcloned into the prokaryotic expression vector pET30a using Ndel and Hind III as sites, and transformed into BL21 (DE3) strain.

[0085] (3) In the TB medium containing kanamycin, culture on a shaker at 37°C, when the OD600 reaches about 1.2, add IPTG, and continue to stimulate culture at 37°C for 4 hours.

[0086] (4) Collect bacteria by centrifugation, add dissolution buffer and ultrasonic-assisted bacteriostasis, centrifuge to save the supernatant, add guanidine hydrochloride to denature the target protein.

[0087] (5) The targ...

Embodiment 2

[0088] The preparation of embodiment 2 nanoparticles

[0089] 1. Preparation of nanoparticles

[0090] 1. Reagent: EBNA1△GA protein is expressed by E. coli expression system, and then obtained through dissolution and renaturation. Its amino acid sequence is shown in SEQ ID NO:1.

[0091] CpG ODN1826 is commercially available, and its nucleotide sequence is shown in SEQ ID NO:2.

[0092] All other reagents were purchased commercially.

[0093] 2. Preparation process:

[0094] (1) PF-127 (average molecular weight 12.6KDa) was dissolved in ultrapure water, and dissolved under magnetic stirring to obtain 0.3 mg / mL of PF-127.

[0095] (2) Disperse tannic acid (TA) in ultrapure water, and obtain a TA concentration of 0.2 mg / mL under magnetic stirring.

[0096] (3) EBNA1△GA protein was dissolved in ultrapure water, and the pH was adjusted to 4.1 with 1M hydrochloric acid to obtain a concentration of EBNA1△GA protein of 71.43 μg / mL.

[0097] (4) CpG ODN1826 was dissolved in ultra...

Embodiment 3

[0126] Example 3 Construction of murineized nasopharyngeal carcinoma cells stably expressing EBNA1 antigen

[0127] 1. Reagents: The vector plasmid pBABE-Puro, the packaging plasmid pVSVG, and phit60 are original in the laboratory. The transfection reagent lipo2000 and puromycin were purchased commercially.

[0128] 2. Construction process:

[0129] (1) The vector plasmid pBABE-Puro-EBNA1 containing the full-length sequence of EBNA1 and the vector plasmid pBABE-Puro-EBNA1-EGFP fused with green fluorescent protein were constructed using EcoRI and BamHI sites.

[0130] (2) Using the pBABE-Puro-EBNA1-EGFP plasmid, 293T cells were transiently transfected with lipo2000, and the expression of EGFP was observed by fluorescence microscopy after 24 hours.

[0131] (3) Use the vector plasmid pBABE-Puro-EBNA1 constructed in step 1, together with the packaging plasmid pVSVG, phit60. The mass ratio of pBABE-Puro-EBNA1 or pBABE-Puro-EBNA1-EGFP:pVSVG:phit60 is 1:1:1, 2:2:3 or 3:2:2. With t...

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
particle diameteraaaaaaaaaa
concentrationaaaaaaaaaa
Login to view more

Abstract

The invention discloses a subunit nano vaccine for preventing and treating nasopharyngeal carcinoma and a preparation method of the subunit nano vaccine. A nanoparticle is specifically disclosed, which comprises an EBNA1 [delta]GA protein, an immunoadjuvant, poloxamer and a polyphenol substance, and the EBNA1 [delta]GA protein is a recombinant protein obtained by deleting a repeat sequence of glycine-alanine in an EBNA1 protein. The invention also discloses the method for preparing the nanoparticle. The nanoparticle prepared by the invention has regular morphology, a round shape, a smooth surface, and good dispersibility, and does not have the phenomena of obvious adhesion, breakage, collapse, etc.; the EBNA1 [delta]GA protein and the immunoadjuvant in the nanoparticle have a high encapsulation rate; after applied to annimals, the nanoparticle can produce strong humoral immunity, significantly enhance cellular immunity, and has better prevention and treatment effects than a free-form antigen / adjuvant mixed injection and an existing vaccine containing an aluminum adjuvant in a mouse subcutaneous tumor model; and the subunit nano vaccine has great application prospects.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, and more specifically, relates to a subunit nano-vaccine for preventing and treating nasopharyngeal carcinoma, which comprises EBV nuclear antigen EBNA1 protein, immune adjuvants, pharmaceutical adjuvant polymers and polyphenols. Adjuvants are selected from CpG or IFN-α. The present invention also relates to a method for constructing murineized nasopharyngeal carcinoma cells stably expressing specific antigens, and a method for preparing the nano-vaccine, which is used to prevent or treat immunity to the nano-vaccine in a mouse model in mice answer. Background technique [0002] Epstein-Barr virus (EBV) belongs to the human gamma herpes virus, which spreads widely in humans, and about 95% of adults carry EBV throughout their lives. The recessive infection of Epstein-Barr virus is closely related to a variety of human malignant tumors, such as African children Burkitt's lymphoma (BL)...

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): A61K9/51A61K47/10A61K39/245A61P31/22A61P35/00
CPCA61K9/5146A61K39/12A61K2039/55522A61K2039/55561A61P31/22A61P35/00C12N2710/16234
Inventor 刘立新陈永明刘鸿
Owner SUN YAT SEN UNIV
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

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap