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Recombinant adenovirus capable of displaying neutralization antigenic epitope of EV71 virus and CVA16 virus simultaneously

A technology of recombinant adenovirus and CVA16, applied in the field of genetic engineering to achieve the effect of preventing hand, foot and mouth disease

Pending Publication Date: 2019-09-27
GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The EV71 virus inactivated vaccine has been approved by the China Food and Drug Administration in 2015, but the CVA16 virus vaccine is still in the clinical research stage

Method used

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  • Recombinant adenovirus capable of displaying neutralization antigenic epitope of EV71 virus and CVA16 virus simultaneously
  • Recombinant adenovirus capable of displaying neutralization antigenic epitope of EV71 virus and CVA16 virus simultaneously
  • Recombinant adenovirus capable of displaying neutralization antigenic epitope of EV71 virus and CVA16 virus simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of recombinant adenovirus

[0026] First, the human type 3 adenovirus backbone plasmid pBRAd△E3GFP (constructed by the laboratory according to the conventional genetic engineering technology in this field, the E3 region nt27 ,737–30,900 (SEQ ID NO: 10) was knocked out and replaced with the CMV-EGFP gene (SEQ ID NO: 11)) as a template, and the primer pairs HexU / hr1-sp70R and HexD / hr1-sp70U were used for PCR amplification (General reaction conditions) 1L fragment (nt16,764-21,140) (SEQ ID NO: 12) and 1R fragment (nt 21125-24569) (SEQ ID NO: 13) of pBRAd△E3GFP were obtained. After gel recovery and purification, 1L and 1R 50 ng of each mixture was used as a template, and Hexon upstream and downstream primers were added for PCR amplification of HexU and HexD (conventional reaction conditions) to obtain the hexon fragment R1SP70 in which the SP70 sequence was embedded in the HVR1 region.

[0027] Use the same method to insert the PEP55 antigen epitope...

Embodiment 2

[0034] Example 2: Biological Characteristic Test of Recombinant Adenovirus

[0035] 2.1 Thermal stability test of recombinant adenovirus.

[0036] Place the recombinant adenovirus in a 45°C water bath for 5, 10, 20, and 40 minutes respectively, and then inoculate the recombinant adenovirus with different heat exposure times into 293 cells. After 48 hours of virus infection, take the virus supernatant to test the TCID50 data , comparing the thermal stability curves of recombinant adenovirus with non-recombined type 3 adenovirus as a control. Thermal stability curve see image 3 .

[0037] It was found through experiments that the difference between the recombinant adenovirus and type 3 adenovirus was not significant, indicating that the chimeric neutralizing antigen gene fragments of EV71 virus and CVA16 virus had no effect on the heat stability of the recombinant adenovirus.

[0038] 2.2 ELISA data test of neutralizing epitope of recombinant adenovirus.

[0039] The recomb...

Embodiment 3

[0045] Example 3: Neutralizing antibody test of recombinant adenovirus

[0046] The above-mentioned recombinant adenovirus is purified by ion exchange adsorption.

[0047] The operation steps are as follows: repeatedly freeze-thaw the culture of the recombinant adenovirus three times, put the freeze-thaw product into a centrifuge tube, centrifuge at a high speed of 6000g, and centrifuge at 4°C for 30min. The centrifuged supernatant is concentrated by ultrafiltration, and a 500KD molecular weight cut-off ultrafiltration column (GE) is selected for ultrafiltration concentration of the adenovirus particle suspension. The concentrated recombinant adenovirus was purified with DEAE SepharoseFF (GE Company), the loading buffer was 10mM NaCl+10mM PBS (PH7.4), the elution buffer was 1.2MNaCl+10mM PBS (PH7.8), and the protein was collected The eluents with high concentration were mixed, and the protein concentration of the purified recombinant adenovirus was tested by BCA method, and s...

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Abstract

The invention provides recombinant adenovirus capable of displaying neutralization antigenic epitope of an EV71 virus and a CVA16 virus simultaneously. An SP70 neutralization antigenic epitope gene of the EV71 virus is inserted into a hexon HVR1 region of the recombinant adenovirus, and a PEP55 neutralization antigenic epitope gene of the CVA16 virus is inserted into a hexon HVR2 region of the recombinant adenovirus. The invention also provides a preparation method of the recombinant adenovirus. The recombinant adenovirus can induce organisms to produce effective neutralization antibodies of the EV71 virus and the CVA16 virus so as to prevent hand, foot and mouth diseases caused by the EV71 virus and the CVA virus.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a recombinant adenovirus vaccine displaying neutralizing epitopes of EV71 virus and CVA16 virus at the same time, and a preparation method of the recombinant adenovirus vaccine. Background technique [0002] EV71 virus (enterovirus type 71) and CVA16 virus (Coxsackie virus group A type 16) are the main pathogens that cause hand, foot, and mouth disease, which can cause hand, foot, and mouth disease epidemics every year, bringing a greater social impact to Southeast Asia. burden. Among them, the EV71 virus is the most harmful and can cause severe neurological diseases, such as aseptic meningitis, neurogenic pulmonary edema, poliomyelitis-like paralysis, brainstem encephalitis, etc., and can lead to death in severe cases. The CVA16 virus is also relatively harmful and often leads to severe hand, foot and mouth symptoms that require hospitalization. At present, there...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/41C12N15/63
CPCC12N7/00C07K14/005A61K39/12A61P31/14C12N2710/10021C12N2770/32322C12N2770/32022C12N2770/32334A61K2039/70
Inventor 王长兵田新贵许甜甜朱冰
Owner GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER