Preparation and application of a dengue virus universal ctl epitope DNA vaccine

A DNA vaccine and dengue virus technology, applied in the field of medicine and biology, can solve the problems of lack of effective methods for preventing and treating DENV infection

Inactive Publication Date: 2018-08-21
WENZHOU MEDICAL UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since there is still a lack of specific methods to prevent and treat DENV infection, it is urgent to develop new vaccines that can be used to prevent and treat DENV infection, especially CTL epitope DNA vaccines based on CTL epitopes

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
  • Preparation and application of a dengue virus universal ctl epitope DNA vaccine
  • Preparation and application of a dengue virus universal ctl epitope DNA vaccine
  • Preparation and application of a dengue virus universal ctl epitope DNA vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the design of dengue virus multi-epitope gene

[0034] The 15 epitopes involved in the present invention are all DENV-1 specific dominant epitopes that we have identified, and are connected with Igκ chain signal sequence, flexible small molecule connecting bridge (Linker) and PADRE epitope between each epitope, make 15 Each CTL epitope is spatially independent and plays a role respectively, finally designed and synthesized the multi-epitope gene of dengue virus (through mouse codon optimization, and the restriction endonuclease XbaI and HandIII restriction endonuclease restriction endonuclease XbaI and HandIII respectively at both ends point).

[0035] Results: The following table shows the 15 CTL epitope sequences derived from DENV-1. The sequence of the nucleotide sequence and its encoded CTL epitope is as follows figure 1 shown. The 15 DENV-1 CTL epitope sequences restricted by HLA-A*0201, HLA-A*1101, and HLA-A*2402 are separated by Igκ chain signal ...

Embodiment 2

[0038] Embodiment 2, construction and plasmid purification of eukaryotic recombinant expression vector pcDNA3.1 (-)

[0039] The nucleotide sequence synthesized above was digested with Xba I and Hind III, and then ligated with pcDNA3.1(-) digested with Xba I and Hind III under the action of T4 DNA ligase. The ligation product was transformed into competent Escherichia coli DH5α, the transformed colony was screened with ampicillin, the plasmid was extracted, and the dengue virus multi-epitope recombinant expression plasmid was identified by agarose electrophoresis after sequencing and double enzyme digestion. The Escherichia coli containing the recombinant plasmid was shaken and cultured in LB liquid medium for 15 hours, the bacteria were collected, and the purified recombinant plasmid was extracted with a plasmid extraction kit, which is the dengue virus universal CTL epitope DNA vaccine.

[0040] Results: The sequence diagram of the nucleotide sequence inserted in the recombi...

Embodiment 3

[0041] Example 3, dengue virus universal CTL epitope DNA vaccine immunization of transgenic mice

[0042] The recombinant pcDNA3.1(-) plasmid obtained in Example 2 was dissolved in sterile PBS buffer to a final concentration of 2 μg / μl. Six to eight-week-old female HLA-A*0201 transgenic mice, six HLA-A*1101 transgenic mice and six HLA-A*2402 mice were injected with 50 μg recombinant pcDNA3.1(- ) plasmid to immunize each mouse, and booster immunization twice a week later (one week apart).

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 discloses a DNA vaccine for dengue virus universal CTL epitopes. The DNA vaccine takes eukaryotic expression plasmids pcDNA3.1(-) as a vector backbone, takes Igk chain signal peptides as guide sequences, and contains 15 highly-conserved immunodominance CTL epitopes and one universal helper T cell epitope in dengue virus type 1, wherein the various epitopes are connected by Linker composed of 1-4 amino acids respectively. The DNA vaccine is capable of inducing CTLs with the CTL epitopes specifically secreting IFN-gamma after immunizing HLA-A*0201 transgenic mice, HLA-A*1101 transgenic mice and HLA-A*2402 transgenic mice, and the CTLs are capable of killing mouse spleen cells loading the CTL epitopes and the mouse spleen mononuclear cells infected by the dengue virus type 1. The DNA vaccine has a (universal) wide people coverage rate, and a great potential value in the aspect of immunotherapy and prevention for dengue virus.

Description

technical field [0001] The invention belongs to the field of medical biotechnology; more specifically, the invention relates to the construction and application of dengue virus universal CTL epitope DNA vaccine. Background technique [0002] Dengue virus (DENV) is a mosquito-borne virus of the flavivirus genus that causes dengue fever (DF) as well as fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Four different serotypes of DENV (DENV-1, 2, 3, 4) can be transmitted to humans through the bites of Aedes aegypti and Aedes albopictus. DENV has been prevalent in more than 100 countries and regions in the tropics and subtropics, and there are about 50-100 million DF cases and 250,000-500,000 DHF / DSS cases each year. Currently, dengue virus type 1 (DENV-1) is the main serotype circulating in the world. Since the reappearance of DENV in Guangdong, China in 1978, DENV epidemics and outbreaks have occurred every few years in Guangdong, Hainan, Taiwan, Yunnan, ...

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 & AuthorityPatents(China)
IPC IPC(8): A61K48/00A61K39/12A61P31/14C12N15/79C12N15/40C07K14/18
CPCY02A50/30
Inventor文金生段志良王思娜黄曦李静刘慧芳杨金霖
OwnerWENZHOU MEDICAL UNIV