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Enterovirus 71 epitope, antibody and its application and vaccine

An antigenic epitope, enterovirus technology, applied in the fields of molecular biology and immunology, to achieve the effect of high neutralization titer

Active Publication Date: 2018-05-08
CHANGCHUN BCHT BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, the currently screened EV71 virus epitopes are mainly concentrated on VP1 and VP2, while VP3 epitopes and spatial conformational epitopes are rarely reported.

Method used

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  • Enterovirus 71 epitope, antibody and its application and vaccine
  • Enterovirus 71 epitope, antibody and its application and vaccine
  • Enterovirus 71 epitope, antibody and its application and vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] A fragment of the nucleotide sequence shown in SEQ ID NO: 2 is constructed into the Noru P particle plasmid expression vector PET28a-Pdomain

[0064] Primers were designed as follows:

[0065]

[0066] GCGGCCGC is the Not I restriction site, CCATGG is the Nco I restriction site, CTCGAG is the Xho I restriction site, and additional GCs are added to avoid frameshift mutations. Obtain the PCR product fragment of the target gene by overlapping PCR, recover the PCR product fragment of the target gene and connect it to the amplified plasmid Peasy-Blunt, transform Escherichia coli, screen positive transformants, extract the plasmid after culture, and treat the plasmid with NcoI and XhoI double enzymes. The original plasmid PET28a-P domain that had been digested with NcoI and XhoI was ligated with T4 ligase to obtain the expression plasmid. The expression plasmid construction process is as follows figure 1 shown.

[0067] The expression plasmid was transformed into expres...

Embodiment 2

[0071] Take 1 L of antigen-presenting cells induced in Example 1, blow evenly with 80ml of equilibrium solution (20mM Tris, 0.5M NaCl, [pH8.0]), sonicate at 4°C for 40min, sonicate for 5s and stop for 5s, and keep the precipitate at 16000rpm for 30min, 40ml Treatment with urea balance solution (8M urea, 20mM Tris, 0.5M NaCl, [pH 8.0]). Stir overnight at 4°C, then keep the supernatant at 10,000rpm for 30min the next day, and present it to the nickel ion affinity layer suction column. The eluent is urea equilibrium solution containing imidazole, and the final elution gradient of the target protein is 200mM imidazole to obtain the fusion protein. Use the same method to purify the control strain expressing the control protein.

[0072] respectively by SDS-PAGE ( Figure 2-a ), western blot( Figure 2-b ), analysis of two-color infrared laser imaging system ( Figure 2-c ) to identify the fusion protein and the control protein, and the two-color infrared laser imaging system qua...

Embodiment 3

[0075] The fusion protein that embodiment 2 makes and AL (OH) 3 The adjuvant is mixed, and the mass ratio of the fusion protein to AL(OH)3 is 25 μg:200 μg / dose to prepare a vaccine.

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Abstract

The invention relates to the technical fields of molecular biology and immunology, in particular to an enterovirus type 71 epitope, an antibody, its application and a vaccine. The antigenic epitope of the EV71 virus provided by the present invention is located on the coat protein VP3 of the EV71 virus, the epitope can specifically recognize the EV71 virus, and the epitope can cause a higher neutralization titer after being presented by the PP carrier, which The serum produced after immunization can effectively protect young mice against EV71 virus infection. However, the neutralization test results of the polyclonal antibodies provided by the present invention to various subtypes of EV71 pseudoviruses show that the polyclonal antibodies provided by the present invention can exhibit high and broad-spectrum neutralization titers. Moreover, it is confirmed by passive protection and maternal vertical protection experiments that the polyclonal antibody provided by the present invention can completely protect young mice against EV71 infection.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and immunology, in particular to an enterovirus type 71 epitope, an antibody, its application and a vaccine. Background technique [0002] Hand, foot and mouth disease (HFMD) is a global infectious disease, enterovirus 71 (Enterovirus 71, EV71) and Coxsackievirus A16 (Coxsackievirus A16, CA16) are hand, foot and mouth disease (Hand, Foot and mouth disease (HFMD) is the main pathogen. Since EV71 was first isolated in the United States in 1969, the prevalence of HFMD caused by EV71 infection has been reported in Australia, Japan, Brazil, the Netherlands, Malaysia, and Taiwan. On May 2, 2008, the Chinese Ministry of Health listed HFMD as a Class C infectious disease. Virus particles of natural enteroviruses are spherical structures with icosahedral stereosymmetry, no envelope and protrusions, and the diameter is about 24-30 nm. Enteroviruses mainly infect children under the age of 5. Th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K19/00C07K16/10C12N15/41C12N15/70C12N1/21G01N33/68G01N33/569A61K39/125A61K39/42A61P31/14
Inventor 姜春来孔维蒋丽萍孙世洋
Owner CHANGCHUN BCHT BIOTECH
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