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Genetic engineering protein vaccine for preventing EV71, and preparation method thereof

A genetic engineering and protein technology, applied in the field of prevention of EV71 genetic engineering protein vaccine and its preparation, can solve the problems of no reports, etc., and achieve the effects of convenient transportation and storage, good safety and low production cost

Active Publication Date: 2013-09-25
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the EV71-P1 protein has not been reported as the main component of the EV71 vaccine in the prior art.

Method used

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  • Genetic engineering protein vaccine for preventing EV71, and preparation method thereof
  • Genetic engineering protein vaccine for preventing EV71, and preparation method thereof
  • Genetic engineering protein vaccine for preventing EV71, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A preparation method for preventing EV71 genetic engineering protein vaccine, comprising the following steps:

[0040] (1) Transformation using the Henan strain as a design template to remove amino acids 189-309 and 440-543 in the P1 protein, and at the same time optimize the P1 gene for Pichia pastoris codons, and obtain the gene described in claim 1 after transformation The sequence is P1', and the original virus strain of the P1 genetic modification design reference is Human enterovirus71strain Henan10-08-China (gene bank GU366191.1GI:285013169);

[0041](2) Cloning the P1' gene into the eukaryotic expression vector pPIC9K, and then electroporating into Pichia pastoris GS115;

[0042] (3) For strains screened on MM and MD plates, high-copy strains were screened on G418 plates with different gradient concentrations;

[0043] (4) Determine high-copy strains by fluorescent quantitative real-time PCR;

[0044] (5) Through the methanol utilization test experiment, test ...

Embodiment 2

[0059] The vaccine that the present embodiment 1 prepares carries out serum antibody titer neutralization test, and its effect is as follows figure 2 shown. Antibody neutralization experiments showed that the highest antibody dilution in the 2ugEV71-P1 protein dose immunized rabbit experimental group could reach 1:32, and a relatively high level of dilution could be maintained within 20 weeks of detection. Compared with the same dose of inactivated EV71 immunization group, Although a 1:32 dilution ratio can also be achieved, the antibody titer drops very quickly.

Embodiment 3

[0061] The vaccine prepared in Example 1 was used for challenge experiments: Balc mice (female) were immunized with 2ug of EV71-P1 protein for 3 times, and the same dose of inactivated virus (C4 subtype) was injected into female mice at the same time (14, 28, 60 days) After pregnancy, newborn mice were challenged within 8 hours after birth. The experimental results are as follows:

[0062] EV71-P1 immune group:

[0063] Newborn mice can simultaneously resist B (BrCr) type, C2, C4 subtype EV71 virus infection, and can form cross immune protection against different subtype virus strains

[0064] Newborn rats are challenged with 1x10 4 LD50C2, C4 subtype EV71 virus infection, 5x10 3 100% protection against LD50B (BrCr) type EV71 virus infection

[0065] Newborn rats have an anti-challenge dose of 5x10 4 75% protection against LD50C2, C4 subtype EV71 infection, 1x10 4 80% protection against LD50B (BrCr) type EV71 virus infection

[0066] Inactivated EV71 experimental group: ...

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Abstract

The invention discloses a genetic engineering protein vaccine for preventing EV71 and a preparation method thereof, and belongs to the technical field of protein engineering vaccines. The vaccine consists of an EV71-preventing gene represented by SEQ ID NO: 1 and a eukaryotic expression vector. The vaccine is good in security, low in production cost and obvious in EV71 prevention effect.

Description

technical field [0001] The invention belongs to the technical field of protein engineering vaccines, and in particular relates to a genetic engineering protein vaccine for preventing EV71 and a preparation method thereof. Background technique [0002] Since enterovirus type 71 (Enterovirus type71; EV71) was isolated and reported for the first time in 1974, EV71 has been prevalent in many countries and regions. EV71 can invade multiple organs and cause a variety of clinical syndromes, including aseptic meningitis, encephalitis, brainstem encephalitis, and polio-like paralytic diseases in addition to hand, foot and mouth disease. System-related diseases seriously endanger children's health, can cause public uneasiness and panic, and cause great losses to the social economy. In recent years, the proportion of EV71 in HFMD prevalent in the Asia-Pacific region has been on the rise, leading to increasingly severe central nervous system symptoms in HFMD patients in this region. R...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/81C07K14/085C07K1/18A61K39/125A61P31/14C12R1/84
Inventor 韩雪周世力应晓玲韩涛孙宪迅
Owner JIANGHAN UNIVERSITY
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