Recombination broad-spectrum vaccine specific to Human enterovirus 71

A human enterovirus, virus-like technology, applied in the fields of biomedicine and biology, can solve the problems of high cost and weak immunogenicity of synthetic peptides

Active Publication Date: 2012-05-23
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the immunogenicity of SP55 and SP70 peptides is relatively weak, and a large dose of immunization is required to induce the desired immune effect. Moreover, the cost of synthesizing peptides is very high, and they are not suitable for direct use as vaccines.

Method used

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  • Recombination broad-spectrum vaccine specific to Human enterovirus 71
  • Recombination broad-spectrum vaccine specific to Human enterovirus 71
  • Recombination broad-spectrum vaccine specific to Human enterovirus 71

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Construction of expression plasmid

[0096] Prepare primers as follows:

[0097] SP55-Xba I-F (SEQ ID NO: 4):

[0098] 5’-CTAGAGCCAGATTCTAGGGAATCTCTTGCATGGCAAACTGCTACTAACCCTGGA-3’,

[0099] SP55-BglII-R (SEQ ID NO: 5):

[0100] 5’-GATCTCCAGGGTTAGTAGCAGTTTGCCATGCAAGAGATTCCCTAGAATCTGGCT-3’,

[0101] SP70-Xba I-F (SEQ ID NO: 6):

[0102] 5’-CTAGAGTACCCAACATTCGGAGAACATAAACAGGAGAAAGACCTTGAATATGGA-3’,

[0103] SP70-BglII-R (SEQ ID NO: 7):

[0104] 5’-GATCTCCATATTCAAGGTCTTTCTCCTGTTTATGTTCTCCGAATGTTGGGTACT-3’,

[0105] HBc-F-NcoI (SEQ ID NO: 8):

[0106] 5’-CTGCCATGGACATTGACCCTTACAAAG-3’,

[0107] HBc-R-XhoI (SEQ ID NO: 9):

[0108] 5’-GGCCTCGAGACATTGAGATTCCCTAGA-3’.

[0109] Take an equal volume of 10uM primers SP55-Xba I-F and SP55-BglII-R, SP70-Xba I-F and SP70-BglII-R and mix them, then denature at 94°C for 4 minutes, and then ice bath for 10 minutes. The product was ligated with pIBT-HBc digested with restriction enzymes Xba I and BglII, and ligated with T4 DNA ligase. The ligati...

Embodiment 2

[0112] Example 2. Expression and purification of fusion protein

[0113] Transform the recombinant plasmids pET28b-HBc, pET28b-HBc-SP55, and pET28b-HBc-SP70 into Escherichia coli BL21, inoculate them on LB agar plates (containing kanamycin 50mg / L), and cultivate them at 37°C. After a single bacteria grows, Pick a single colony and inoculate it in LB culture medium (containing kanamycin 50mg / L), shake culture overnight at 37°C, inoculate it in 500 mL of the same culture medium at a ratio of 1:100 the next day, and continue shaking culture until the OD value of the culture reaches IPTG (final concentration 0.8mmoL / L) was added at 0.6 for induction. After 5h at 37℃, the cells were harvested by centrifugation at 12000rpm for 10min, and the cells were resuspended in 50mL buffer I (0.5M NaCl, 20mM Tris, 10mM imidazole, pH7.9) , Ultrasonic crushing for 5min. Centrifuge at 12000 rpm for 10 min, discard the supernatant, and dissolve the inclusion body pellet with 30 mL buffer II (0.5M Na...

Embodiment 3

[0126] Example 3. Mouse immune program and antibody titer detection

[0127] The purified virus-like particles were diluted with PBS to 0.1 μg / μL, and 100 μL of each was mixed with an equal volume of aluminum adjuvant by shaking for 1 hour. Three experimental groups, each with 6 mice, were injected with 200μL / mouse of BALb / c mice intraperitoneally, and immunized once at 0, 3, and 5 weeks. At the same time, a PBS control group was set up, and PBS and aluminum adjuvant were intraperitoneally administered The mixture is 200μL / only×6. Blood was collected from the orbital vein before and two weeks after immunization, and all serum was stored at -20°C.

[0128] Take the three immunizations (0, 3, and 5 weeks each immunization) two weeks (ie, the 7th week) of the serum for ELISA detection, respectively coat 96-well plate with HBc, SP55, SP70, 100ng / well, 37℃ coating 2h; 2h after sealing with 5% milk. Dilute the tested serum, use the mouse serum of the PBS control group as a negative co...

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Abstract

The invention relates to a recombination broad-spectrum vaccine specific to Human enterovirus 71. The invention constructs a fusion protein which comprises at least one copied peptide SP55 and/or SP70 of Human enterovirus 71 VP1 protein and a hepatitis B virus core antigen. The fusion protein can renature and assemble by self in a solubility manner after being expressed, denatured and purified, thereby forming virus-like particles with strong immunogenicity; and the virus-like particles can induce the generation of a broad-spectrum neutralizing antibody with high valence in vivo, thereby being capable of serving as the vaccine for preventing diseases related to Human enterovirus 71 infection. According to the invention, suitable carriers for forming the virus-like particles are found for antigens deriving from the enterovirus 71 VP1 protein, thereby the formed virus-like particles properly expose the antigens and increase the immunogenicity of the antigens.

Description

Technical field [0001] The present invention belongs to the fields of biomedicine and biotechnology; more specifically, the present invention relates to a recombinant broad-spectrum vaccine against human enterovirus 71. Background technique [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus, which is mainly transmitted through close contact or the digestive tract. It mostly occurs in infants under 10 years of age. Skin rashes, herpes, and ulcers on the skin and mucous membranes of the mouth and other parts are typical manifestations. Patients with severe conditions can cause complications such as myocarditis, pulmonary edema, aseptic meningoencephalitis, and polio. The disease is highly contagious and has a high mortality rate, which can easily cause outbreaks or epidemics. In the past few years, hand, foot and mouth disease has been prevalent in many countries and regions in the Far East, including China, Hong Kong, Japan, Singapor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K39/295C12N15/62C12N15/63C12P21/02A61P31/14
Inventor 黄忠王波
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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