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Truncated rotavirus vp8 protein and use thereof

A rotavirus and protein technology, applied in the direction of viral peptides, antiviral agents, viral antigen components, etc., can solve problems such as inappropriate and efficient anti-rotavirus vaccines

Active Publication Date: 2019-05-14
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This makes △VP8* unsuitable for use as a highly effective anti-rotavirus vaccine

Method used

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  • Truncated rotavirus vp8 protein and use thereof
  • Truncated rotavirus vp8 protein and use thereof
  • Truncated rotavirus vp8 protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0177] Example 1: Construction of an expression vector encoding a truncated rotavirus VP8 protein

[0178] The rotavirus LLR strain was cultured with Rhesus monkey embryonic kidney cell line (MA-104). The medium used was DMEM supplemented with 2 μg / ml trypsin, 0.5 mg / ml ampicillin and 0.4 mg / ml streptomycin, 3.7 mg / ml sodium bicarbonate, 0.34 mg / ml L-glucose Aminoamide.

[0179] According to the manufacturer's instructions, the genomic RNA of rotavirus was extracted using the viral DNA / RNA extraction kit produced by Beijing Kingmag Biotechnology Co., Ltd., and the cDNA encoding the VP4 protein was obtained by reverse transcription. The obtained cDNA is used as a template, and the gene fragment encoding the truncated rotavirus VP8 protein is amplified by PCR reaction.

[0180] The primers used were as follows:

[0181] Upstream primers:

[0182] 5'- GGATCCCATATG ATACAGTTAATTGGATCAGAAAA-3' (SEQ ID NO: 17)

[0183] 5'- GGATCCCATATG GGATCAGAAAAAACGCAG-3' (SEQ ID NO: 18) ...

Embodiment 2

[0207] Example 2: Expression of truncated rotavirus VP8 protein

[0208] Take out the recombinant plasmids pTO-T7-VP8-5A, pTO-T7-VP8-5, pTO-T7-VP8-6, pTO-T7-VP8-8, pTO-T7-VP8 prepared in Example 1 from -70°C -9, pTO-T7-VP8-10, pTO-T7-VP8-11 or pTO-T7-VP8-12 Escherichia coli bacterial liquid, inoculate it into 50ml LB liquid medium containing kanamycin, in Cultivate at 180rpm at 37°C for about 4 hours; then transfer to 10 bottles of 500ml LB medium containing kanamycin (500ul bacteria solution in each bottle). When the absorbance value of the culture at 600 nm reached 0.5, IPTG was added to a final concentration of 1 mM, and culture was continued for 6 hours at 180 rpm at 25°C.

[0209] In addition, recombinant proteins PTO-T7-VP8, PTO-T7-ΔVP8* were also expressed using Escherichia coli by a method similar to the above.

Embodiment 3

[0210] Example 3: Purification and Characterization of Truncated Rotavirus VP8 Protein

[0211] Under the condition of Tris-HCl 8.0 buffer solution, at 4°C, use a sonicator (Thermo) according to the condition of 4min / 1g wet bacteria, destroy the cell wall of E.coli cells, collect the soluble fraction, and use the following scheme to analyze The recombinant protein expressed by E.coli cells was purified.

[0212] Instrument system: AKTAexplorer 100 preparative liquid chromatography system produced by GE Healthcare (formerly Amershan Pharmacia).

[0213] Chromatography medium: Q-sepharose-HP (GE Healthcare).

[0214] Column volume: 5.5cm*20cm.

[0215] Buffer: 50mM Tris-HCl pH 8.0

[0216] 50mM Tris-HCl pH 8.0, 2M NaCl

[0217] Flow rate: 25mL / min.

[0218] Detector wavelength: 280nm.

[0219] The sample was the soluble fraction of E. coli lysate prepared previously containing recombinantly expressed VP8-5A, VP8-5, VP8-6, VP8-8, VP8-9, VP8-10, VP8-11 or VP8-12.

[0220] T...

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Abstract

Disclosed are a truncated rotavirus VP8 protein, fusion protein containing the truncated protein, conjugate containing the truncated protein, coding sequences of the truncated protein and the fusion protein and preparation methods therefor, and pharmaceutical compositions and vaccines containing the truncated protein, the fusion protein or the conjugate. The truncated protein, the fusion protein, the conjugate, the pharmaceutical compositions and the vaccines can be used for preventing, relieving or treating rotavirus infection and diseases caused by rotavirus infection, for example, rotavirus gastroenteritis and diarrhea. Also disclosed are uses of the truncated protein, the fusion protein, and the conjugate for preparing pharmaceutical compositions or vaccines.

Description

technical field [0001] The present invention relates to the fields of biochemistry, molecular biology, molecular virology and immunology. Specifically, the present invention relates to a truncated rotavirus VP8 protein, a fusion protein comprising the truncated protein, a conjugate comprising the truncated protein, the coding sequence of the truncated protein and the fusion protein and Preparation method, pharmaceutical composition and vaccine comprising the truncated protein, fusion protein or conjugate, the truncated protein, fusion protein, conjugate, pharmaceutical composition and vaccine can be used to prevent, alleviate or treat rotavirus Viral infection and diseases caused by rotavirus infection, such as rotavirus gastroenteritis and diarrhea. The present invention also relates to the use of the above-mentioned truncated protein, fusion protein, and conjugate for preparing a pharmaceutical composition or vaccine, which is used for preventing, alleviating or treating ro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/14C07K19/00C12N15/46C12N15/62A61K39/15A61K39/39A61P31/14C12N15/70
CPCA61K39/15C07K14/14C12N15/63
Inventor 葛胜祥薛淼舸俞林岐车耀健李廷栋张军夏宁邵
Owner XIAMEN UNIV
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