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Human rotavirus VP8 recombinant protein and human rotavirus vaccine using same

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: 2018-09-18
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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  • Human rotavirus VP8 recombinant protein and human rotavirus vaccine using same
  • Human rotavirus VP8 recombinant protein and human rotavirus vaccine using same
  • Human rotavirus VP8 recombinant protein and human rotavirus vaccine using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Screening of foreign polypeptides and fusion proteins

[0052]Tetanus and diphtheria toxins have been used as human vaccines for nearly 100 years. It is safe to use its fragment polypeptides as rotavirus vaccines. By analyzing the primary structure of tetanus toxin and diphtheria toxin protein with SwissInstitute Bioinformatics software (http: / / web.expasy.org / compute_pi / ), 4 polypeptide fragments were screened out (as shown in Table 1). One is from tetanus toxin and three are from diphtheria toxin. These polypeptide fragments contain 1 to 3 negatively charged amino acids (aspartic acid or glutamic acid) and few or no positively charged (arginine and lysine), so the isoelectric point of these polypeptides are very low. In this example, 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 positively charged ...

Embodiment 2

[0073] Construction of expression system and protein purification

[0074] VP8 protein is a non-glycosylated protein, therefore, the E. coli expression system purchased from Merck Mllipore was selected . The plasmid used was pET30a ; host cell E. coli BL21 (DE3). First, the DNA sequences of the above six proteins were codon-optimized, and then their nucleotide sequences were artificially synthesized ( http: / / www.blueheronbio.com / ) and cloned into the pET30a plasmid expression system. Then, the plasmid with the target gene was transformed into BL21(DE3) competent bacteria for antibiotic screening. Confirm that the grown colonies are picked and cultured in shake flasks at 37°C until the turbidity is 1-1.5 (OD600), then the culture temperature is lowered to 20°C, and 2mM IPTG is added to induce expression for 4-6 hours. The cells were then collected, lysed by high pressure (ATS lyser), and centrifuged. Samples were taken before and after centrifugation (sample 1 before ce...

Embodiment 3

[0080] Production of monoclonal antibodies. In this example, monoclonal antibodies were produced by immunizing mice (BALB / c) with VP8P[8] protein. The mice were injected subcutaneously with 20 μg of VP8P[8] adsorbed on 100 μg of aluminum hydroxide 3 times at 7-day intervals. Blood was collected on the 21st day, and high titer of anti-VP8P[8] protein antibody (positive at 1:105 dilution) was found in serum by enzyme-linked immunosorbent assay. The spleen cells of a mouse were fused and cultured with myeloma cells, and 22 monoclonal antibodies were screened by VP8P[8] protein-coated enzyme-linked immunosorbent assay. Then use 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, on top of a monolayer of grown 100% MA104 cells, approximately 100x CC ID50 of JX406750 virus was added. After 18 hours of incubation at 37 degrees, monoclonal antibody culture medium (1...

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Abstract

The invention provides a human rotavirus VP8 recombinant protein. The amino acid sequences of the VP8 recombinant protein include an amino acid sequence of a human rotavirus VP8 protein (hereinafter referred to as VP8 protein) and an amino acid sequence of exogenous polypeptide. The isoelectric point of the exogenous polypeptide 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 invention can be expressed by fusing the VP8 protein with negativecharge-containing amino acid polypeptide fragments in tetanus toxin and / or diphtheria toxin proteins, and the water solubility and yield of the rotavirus VP8 protein expressed by escherichia coli areimproved; and moreover, the obtained fused protein has good immune ability, and the prepared vaccine has good effect.

Description

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

Claims

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Application Information

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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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