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A kind of ev71-vp1 hand, foot and mouth disease polypeptide vaccine and its preparation method and application

A technology of EV71-VP1, hand, foot and mouth disease, applied in the field of immune biomedicine, can solve problems such as harmful effects on the human body

Active Publication Date: 2020-01-07
ZUNYI NO 1 PEOPLES HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The whole virus inactivated vaccine of EV71 virus uses the whole virus as the antigen, but from a safety point of view, especially in individuals with poor immunity, it may have harmful effects on the human body. Therefore, subunit vaccines have become the focus of research in recent years

Method used

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  • A kind of ev71-vp1 hand, foot and mouth disease polypeptide vaccine and its preparation method and application
  • A kind of ev71-vp1 hand, foot and mouth disease polypeptide vaccine and its preparation method and application
  • A kind of ev71-vp1 hand, foot and mouth disease polypeptide vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Detection of Serum Antibodies in Rehabilitated Children

[0027] Collect serum sample information of clinical children: Liu Zhehan, Liu Yueyue, Wu Linman, Gao Junhao, Chen Yixuan, Zhou Yutong. Use human enterovirus EV71 IgG ELISA kit to detect serum antibody concentration, the specific operation method is as follows:

[0028] Steps:

[0029] 1. Dilution and sample addition of the standard: Set 10 wells of the standard on the enzyme-labeled plate, add 100ul of the standard to the first and second wells, and then add the standard to the first and second wells Dilute 50ul, mix well; then take 100ul from the first well and the second well, add to the third well and the fourth hole respectively, then add 50ul standard diluent to the third and fourth well, mix well; Then take 50ul from the third hole and the fourth hole and discard, then add 50ul to the fifth and sixth holes respectively, then add 50ul of the standard dilution solution to the fifth and sixth holes...

Embodiment 2

[0041] Example 2 Synthetic polypeptide coated ELISA plate and evaluation and screening of polypeptide immune effect

[0042] 1. The buffer solution is prepared as follows:

[0043] ①Coating solution: 0.05mol / L carbonate buffer solution (pH9.6)

[0044] 0.75g sodium carbonate, 1.46g sodium bicarbonate, add deionized water to make the volume to 500ml.

[0045] ②0.02mol / L phosphate buffer (pH7.4)

[0046] 0.2g potassium dihydrogen phosphate, 2.90g disodium hydrogen phosphate, 8g sodium chloride, add deionized water to make up to 1000ml.

[0047] ③Antibody diluent: 0.02mol / LPBS(pH7.4)+0.2%BSA

[0048] Add 0.2gBSA to the prepared 0.02mol / L phosphate buffer solution to dissolve and quantify to 100g.

[0049] ④Blocking solution: 0.05mol / L carbonate buffer (pH9.6)+2.0%BSA

[0050] Add 2.0g of BSA to the prepared 0.05mol / L carbonate buffer solution to dissolve and quantify to 100g.

[0051] ⑤Washing solution: 0.02mol / LPBS(pH7.4)+0.05%Tween-20

[0052] Dissolve 50ul of Tween-20 i...

Embodiment 3

[0078] Example 3 In vivo experiments to further verify and evaluate the immune effect of polypeptides

[0079] 1. Model construction of immune mice (Shanghai Experimental Animal Center, Chinese Academy of Sciences)

[0080] Dissolve the obtained No. 2 polypeptide, No. 4 polypeptide, and No. 8 polypeptide in 0.9% sodium chloride solution to make the final concentration 1 mg / ml, and mix with the same volume of Freund's complete adjuvant (purchased from Roche Pharmaceuticals) , to make a polypeptide vaccine. Balb / c mice (purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences) used 12 6-8 week old female Balb / c healthy mice, divided into 4 groups (No. 2 polypeptide group, No. 4 polypeptide group, and No. 8 polypeptide group) Group) Inject 0.1ml of the above polypeptide solution into the abdominal cavity, and repeat the immunization one week later. The heat-inactivated virus is used as a positive control, and the hepatitis B virus surface antigen is used a...

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Abstract

The invention discloses an EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, and a preparation method and application thereof. A hand-foot-and-mouth disease virus is separated, whole genome sequencing is conducted to obtain EV71 virus VP1 structural protein, and the EV71 virus VP1 structural protein is divided into 20 groups to be synthesized into polypeptide segments. Serum containing an EV71 virus antibody is screened out, and No.2, No.4 and No.8 polypeptides with higher immune effects are initially screened out through an enzyme linked immune absorbent assay. In order to further verify whether the initially screened polypeptides have the immune effect in a mouse model, the invention reasonably designs a Balb / c mouse test model, the Balb / c mouse test model is subjected to pathological section HE dyeing pathological analysis, the No.8 polypeptide has the higher immune effect on the small intestine, the skeletal muscle and the brain of the model Balb / c mouse, and the No.2 polypeptide has the higher immune effect on the skeletal muscle of the model Balb / c mouse. Through the enzyme-linked immunosorbent assay and the pathological examination comprehensive analysis and verification of the model Balb / c mouse, a result indicates that the optimized No.2 polypeptide and No.8 polypeptide have huge potential in development and application of the EV71 virus vaccine.

Description

technical field [0001] The invention belongs to the field of immune biomedicine, and in particular relates to an EV71-VP1 hand, foot and mouth disease polypeptide vaccine and its preparation method and application. Background technique [0002] Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease in infants and young children. At present, there is no antiviral drug against EV71, and the main treatment is symptomatic. In terms of antiviral drug research, in vitro experiments have proved that pyridyl imidazoline dione derivatives can effectively inhibit the replication of EV71, and are currently more promising drug candidates. The whole virus inactivated vaccine of EV71 virus uses the whole virus as the antigen, but from a safety point of view, especially in individuals with poor immunity, it may have harmful effects on the human body. Therefore, subunit vaccines have become the focus of research in recent years. [0003] Polypeptide vaccine...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/125A61P31/14
CPCA61K39/12A61K2039/55C12N2770/32334
Inventor 黄波李青顾胜利汤正珍韩允李颖
Owner ZUNYI NO 1 PEOPLES HOSPITAL