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A human rotavirus vp8 recombinant protein and a human rotavirus vaccine using the vp8 recombinant protein

A rotavirus and recombinant protein technology, applied in the field of vaccines, can solve the problem of low immunogenicity of protein fragments

Active Publication Date: 2021-09-17
CHENGDU MAXVAX BIOTECHNOLOGY LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its yield can reach 40-70 mg per liter (Wen X, Cao D, Jones RW, Li J, Szu S, Hoshino Y. Construction and characterization of human rotavirus recombinant VP8*subunitparenteral vaccine candidates. Vaccine. 2012 Sep 21; 30( 43):6121-6), but the immunogenicity of the obtained VP8 protein fragment is relatively low, and a tetanus toxin polypeptide needs to be added to improve its immunogenicity (WenX, Wen K, Cao D, Li G, Jones RW, Li J, Szu S, Hoshino Y, Yuan L. Inclusion of auniversal tetanus toxoid CD4(+)T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirus ΔVP8*subunit parenteral vaccines. Vaccine.2014 Jul 31;32(35):442 -7.)

Method used

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  • A human rotavirus vp8 recombinant protein and a human rotavirus vaccine using the vp8 recombinant protein
  • A human rotavirus vp8 recombinant protein and a human rotavirus vaccine using the vp8 recombinant protein
  • A human rotavirus vp8 recombinant protein and a human rotavirus vaccine using the vp8 recombinant protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Screen foreign peptides and fusion proteins

[0053] Tetanus toxin and diphtheria toxin have been used as human vaccines for nearly 100 years. It is safe to use its fragment polypeptide as a rotavirus vaccine. By analyzing the primary structure of tetanus toxin and diphtheria toxin protein with SwissInstitute Bioinformatics software (http: / / web.expasy.org / compute_pi / ), four polypeptide fragments were screened out (as shown in Table 1). One was from tetanus toxin and three were from diphtheria toxin. These peptide fragments contain 1 to 3 negatively charged amino acids (aspartic acid or glutamic acid) and little or no positively charged amino acids (arginine and lysine), so the isoelectric point of these peptides Both are very low. In this embodiment, the DT4 polypeptide is used as a control. The sequence of the DT4 polypeptide is Gly Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys ThrHis Ile, and its isoelectric point is 9.7, which contains three posi...

Embodiment 2

[0074] Construction of expression system and protein purification

[0075] VP8 protein is a non-glycosylated protein, therefore, the Escherichia coli expression system purchased from Merck Mllipore was selected . The plasmid used was pET30a ; host cell E. coli BL21(DE3). Firstly, codon optimization was carried out on the DNA sequences of the above six proteins, and then their artificially synthesized nucleotide sequences ( http: / / www.blueheronbio.com / ), and cloned into pET30a plasmid expression system. Then transform the plasmid with the target gene into BL21(DE3) competent bacteria for antibiotic selection. Confirm that the well-grown colonies were picked and cultured in a shaker flask at 37°C until the turbidity was 1-1.5 (OD600), then the culture temperature was lowered to 20°C, and 2mM IPTG was added to induce expression for 4-6 hours. Then the bacterial cells were collected, lysed by high pressure (ATS lyser), and centrifuged. Samples were taken before and after c...

Embodiment 3

[0081]Production of monoclonal antibodies. In this example, VP8P[8] protein was used to immunize mice (BALB / c) to produce monoclonal antibodies. 20 micrograms of VP8P[8] adsorbed on 100 micrograms of aluminum hydroxide were used to subcutaneously inject mice 3 times with an interval of 7 days. Blood was collected on the 21st day, and an enzyme-linked immunoassay test found that there was a high titer of anti-VP8P[8] protein antibody in the serum (1:105 dilution positive). Splenocytes from a mouse were cultured in fusion with myeloma cells, and 22 monoclonal antibodies were screened by VP8P[8] protein-coated ELISA. Then use the fluorescent labeling method to further detect whether these monoclonal antibodies can bind to the VP8 protein on the surface of the virus. In a 6-well cell culture dish, grown on top of a 100% MA104 cell monolayer, approximately 100x CC ID50 of JX406750 virus was added. After culturing at 37 degrees for 18 hours, add monoclonal antibody culture soluti...

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Abstract

The present invention provides a human rotavirus VP8 recombinant protein, the amino acid sequence of the VP8 recombinant protein includes the amino acid sequence of the human rotavirus VP8 protein (hereinafter referred to as VP8 protein) and the amino acid sequence of an exogenous polypeptide, the exogenous polypeptide The isoelectric point of is less than or equal to 5. The invention also provides a human rotavirus vaccine prepared based on the VP8 recombinant protein. Compared with the prior art, the VP8 recombinant protein provided by the application is expressed through the fusion expression of the VP8 protein and the polypeptide fragment containing negatively charged amino acids in the tetanus toxin and / or diphtheria toxin protein, which improves the expression of the rotavirus VP8 protein expressed by Escherichia coli. Water solubility and yield, and the obtained fusion protein has good immunity, and the prepared vaccine has good effect.

Description

technical field [0001] The invention relates to the field of vaccines, in particular to a human rotavirus vaccine and the rotavirus recombinant protein involved in the vaccine. [0002] Background of the invention [0003] Rotavirus is the most common cause of severe dehydrating diarrhea, causing huge economic losses. Almost every child is infected with rotavirus before the age of 5. At present, nearly 200,000 children under 5 years old die from rotavirus diarrhea every year in the world, including about 20,000 children in China. Vaccination is the most economical and effective way to prevent rotavirus diarrhea. [0004] Back in 1998, Wyeth launched an oral rotavirus vaccine - Rotashield. During one year of use, an increased risk of intussusception was found in vaccinated children, and the product had to be withdrawn from the market. Later, Merck and GSK respectively launched live oral rotavirus vaccines. These two products are widely used in more than 80 countries around...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/14A61K39/15A61P31/14
CPCA61K39/12A61P31/14C07K14/005C12N2720/12322C12N2720/12334A61K39/39
Inventor 陈德祥董丽春
Owner CHENGDU MAXVAX BIOTECHNOLOGY LLC
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