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Recombinant adenovirus expressing human enterovirus 71 capsid protein and vaccine prepared from same and application thereof

A technology of human enterovirus and recombinant adenovirus, applied in the field of recombinant adenovirus, can solve the problems of unreasonable construction strategy, no application prospect, low expression of EV71 virus capsid protein, etc., and achieve the effect of good application prospect

Active Publication Date: 2017-01-11
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Some researchers tried to develop a new vaccine using adenovirus as a vector to express the EV71 virus capsid protein, but due to factors such as unreasonable construction strategy and the instability of the obtained recombinant adenovirus, the expression level of the EV71 virus capsid protein was extremely low and it was not applied. Prospect (Ge Xiaoqin et al., Chinese Journal of Biological Products, 2010)

Method used

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  • Recombinant adenovirus expressing human enterovirus 71 capsid protein and vaccine prepared from same and application thereof
  • Recombinant adenovirus expressing human enterovirus 71 capsid protein and vaccine prepared from same and application thereof
  • Recombinant adenovirus expressing human enterovirus 71 capsid protein and vaccine prepared from same and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] Example 1 Construction of recombinant adenovirus expressing human enterovirus type 71 capsid protein

[0052] 1. Experimental method

[0053] 1.1 Primer design and synthesis

[0054]According to the C4 subtype EV71 / Fuyang.Anhui.CHN / 1708 / 1 strain genome sequence registered in GeneBank, the EV71P1-F2A-3CD subgenome was artificially synthesized, cloned into the pUC57 vector, and named pUC57-A. The primers used to construct the EV71P1-IRES-3CD subgenome are listed in Table 1. Primers were designed using Oligo 6.0 software, and the primers and EV71P1-F2A-3CD gene were synthesized by Shanghai Bioengineering Co., Ltd.

[0055] The nucleotide sequence of the P1-F2A-3CD subgenome is shown in SEQ ID NO.1; the nucleotide sequence of the P1-IRES-3CD subgenome is shown in SEQ ID NO.2.

[0056] Table 1 is used to construct the primers of EV71P1-IRES-3CD subgenome

[0057]

[0058] 1.2 Construction of recombinant adenovirus shuttle vector

[0059] Using the plasmids pUC57-A an...

experiment example 1

[0069] Experimental example 1 Identification of recombinant adenovirus expressing capsid protein of human enterovirus type 71

[0070] 1. Experimental method

[0071] 1.1 PCR identification of recombinant adenovirus

[0072] The recombinant adenoviruses Ad5-A, Ad5-A13, Ad5-B, and Ad5-B17 obtained in Example 1 were inoculated into HEK-293 cells for 10 consecutive generations; the 3rd and 10th generation viruses were used for DNA extraction reagents After extracting DNA from the cassette, use EV71VP1 gene-specific primers (VP1-U: 5'GGGGAT AGA GTG GCA GAT GTG AT 3', VP1-L: 5'AAG AGT AGT GATGGC ATT GCG AC 3') for PCR on the target gene VP1 Amplification detection.

[0073] 1.2Western blot

[0074] HEK-293 cells infected with the 3rd and 10th generation recombinant adenoviruses were analyzed by SDS-PAGE, and HEK-293 cells infected with wild-type adenovirus (Ad5-WT) and HEK-293 cells were respectively set up as negative controls to MAb 22A12 (diluted 1:1000) was the primary anti...

experiment example 2

[0085] Experimental example 2 Immunization experiment of recombinant adenovirus expressing human enterovirus type 71 capsid protein

[0086] 1. Experimental method

[0087] 1.1 Immunization of BALB / c mice with recombinant adenovirus

[0088] A total of 140 6-week-old SPF grade BALB / c female mice were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Randomly divided into 4 groups, 35 in each group, wherein the first group was immunized with recombinant adenovirus Ad5-A (microorganism deposit number: CGMCC No. 10889); the second group was immunized with recombinant adenovirus Ad5-B (microorganism deposit number: CGMCC No.10890); the third group was the negative control group, which was immunized with recombinant adenovirus Ad5-WT; the fourth group was the positive control group, which was immunized with inactivated EV71 whole virus purified antigen 0.25 μg / time / body. The inoculum dose of recombinan...

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Abstract

The invention discloses a recombinant adenovirus expressing human enterovirus 71 (EV71) capsid protein and a vaccine prepared from same and an application of the recombinant adenovirus. In the invention, two groups of EV71 sub-genome containing the P1 gene and 3CD gene of EV71 capsid protein are constructed from foot and mouth disease virus 2A gene fused with an alkaline protease cleavage site and a foot and mouth disease virus IRES element as Linker; in the invention, the sub-genome is recombined into an adenovirus expression vector, and a recombinant adenovirus with good replication capacity, genetic stability and high protein expression is obtained by screening and can efficiently express P1 and 3CD proteins in the infected cells while the 3CD can correctly cut the P1; and the recombinant adenovirus can induce a high-level EV71 specific broad-spectrum neutralizing antibody in a mouse, has remarkably better ability of inducing anti-EV71 specific cellullar immunologic response than an inactivated whole-virus antigen, and can be used for preventing and treating human hand-foot-and-mouth disease caused by EV71.

Description

technical field [0001] The present invention relates to a recombinant adenovirus, in particular to a recombinant adenovirus expressing human enterovirus type 71 capsid protein. The present invention also relates to the application of said recombinant adenovirus in the preparation of a vaccine for the prevention or treatment of hand, foot and mouth disease, which belongs to recombinant The construction and application fields of adenovirus. Background technique [0002] Enterovirus 71 (EV71) is the main pathogen causing human Hand, Foot and Mouth Disease (HFMD). The disease mainly occurs in children under the age of 5. In addition to herpes on the hands, feet, and mouth, the children also manifest clinically as herpetic angina, aseptic meningitis, and polio-like paralysis. May cause death. At present, there are no effective measures to prevent the spread of EV71 disease, and the only way to control the spread of EV71 during the outbreak is public health supervision and isola...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/861C12N7/01A61K39/125A61P31/14C12R1/93
Inventor 于力杨德成梁争论毛群颖
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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