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Fusion protein of bovine rotavirus and multiple vaccine against calf diarrhea

A technology of bovine rotavirus and fusion protein, which is applied in the field of bovine rotavirus fusion protein and calf diarrhea multiple vaccine to achieve the effects of strong immune protection, labor saving and good immunogenicity

Active Publication Date: 2020-09-29
TIAN KANG ZHI YAO GU FEN YOU XIAN GONG SI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are currently no commercially available vaccines for the prevention and control of calf diarrhea caused by multiple pathogens

Method used

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  • Fusion protein of bovine rotavirus and multiple vaccine against calf diarrhea
  • Fusion protein of bovine rotavirus and multiple vaccine against calf diarrhea
  • Fusion protein of bovine rotavirus and multiple vaccine against calf diarrhea

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preparation example Construction

[0064] In some optional embodiments, taking yeast as an example of a host cell, the preparation method includes the steps of gene preparation, recombinant vector construction, cell transfection, screening of high-efficiency expression strains, and recombinant protein acquisition, specifically including: first After linearizing the obtained vector, electrotransfer yeast competent cells, express the fusion protein, and purify the obtained fusion protein, then select an appropriate buffer to assemble the purified fusion protein with virus-like particles, and use electron microscopy to identify the assembly effect. As the vector, pPIC3.5K vector is preferably used. After the linearized vector is electrotransferred to yeast competent cells, it is preferably directly spread on a plate with high antibiotic concentration, and a high-expression monoclonal strain is selected by using a higher selection pressure. Electroporation is preferably performed multiple times, that is, on the bas...

Embodiment 1

[0070]Expression of fusion protein by Pichia pastoris expression system:

[0071] By means of structural biology, the structure of bovine rotavirus VP6 protein was analyzed, and the epitopes of bovine coronavirus and pathogenic Escherichia coli were chimerized onto the backbone of bovine rotavirus VP6 protein, and the sequence was named VP6-S -K88 / K99, the specific amino acid sequence is shown in SEQ ID NO.8;

[0072] The fusion protein VP6-S-K88 / K99 is codon-optimized in the expression system of Pichia pastoris, and the nucleotide sequence is shown in SEQ ID NO.9.

[0073] And by means of genetic engineering, the gene encoding VP6-S-K88 / K99 fusion protein was synthesized, and the target gene and pPIC3.5k plasmid were digested by Bam HI and AgeI respectively (enzyme digestion system: 1 μg plasmid, 1 μL Bam HI, 1 μL Age I, 5 μL CutSmart, ddH 2 O make up to 50 μL; incubate at 37°C for 1 h), recover the target band through 1% agarose gel (Axyprep DNA Gel Recovery Kit AP-GX-250)...

Embodiment 2

[0079] Expression of multilayered chimeric VLP particles by the Pichia pastoris expression system:

[0080] By means of genetic engineering, the gene encoding VP2-N93 protein is synthesized, and the amino acid sequence is shown in SEQ ID NO.10. Digest the target gene and pPICZ A vector with Bst BI and Age I respectively (digestion system: 1 μg plasmid, 1 μL Bst BI, 1 μL Age I, 5 μL CutSmart, ddH 2 O make up to 50 μL; incubate at 37°C for 1 h), recover the target band through 1% agarose gel (Axyprep DNA Gel Recovery Kit AP-GX-250), measure the concentration respectively, and pass through T4 DNA ligase (NEB , M0202) to connect the vector to the gene, mix the connection system with E.Coli DH5α competent cells, incubate on ice for 30 minutes, place at 42°C for 90s, then immediately place it on ice for 2 minutes, and apply 0.1mg / mL ampicillin directly Plate, cultured at 37°C for 16 hours, picked a single clone and cultured it in 2 mL LB medium for 16 hours, extracted the plasmid w...

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Abstract

The invention provides a bovine rotavirus fusion protein and calf diarrhea multiple vaccine, and relates to the technical field of molecular biology. The bovine rotavirus fusion protein contains a VP6 fragment, and the VP6 fragment contains the amino acid sequence shown in SEQ ID NO.4, and at least one loop region in the following (a) to (c) is replaced with an antigenic expression derived from bovine coronavirus and / or antigenic epitopes derived from Escherichia coli: (a) amino acid residues 168‑177, amino acid sequence such as SEQ ID NO.1; (b) amino acid residues 194‑205, amino acid sequence such as SEQ ID NO.1 ID NO.2; (c) 296‑316th amino acid residue, the amino acid sequence is as shown in SEQ ID NO.3. The bovine rotavirus fusion protein contains multiple antigenic epitopes, and after immunizing the host, the host can produce multiple antibodies.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a bovine rotavirus fusion protein and calf diarrhea multiple vaccine. Background technique [0002] Calf diarrhea is one of the most common clinical diseases in cattle farms. It is common in calves born 7-15 days old. It has high morbidity and mortality. It not only causes great economic losses to cattle farms, but also affects calves. growth and lactation performance in adulthood. Among the pathogenic factors, bovine rotavirus (Bovine rotavirus, BRV), bovine coronavirus and toxin-producing Escherichia coli are the three most important pathogens causing calf diarrhea, among which BRV has the highest incidence of calf diarrhea, accounting for about 46% of calf diarrhea cases. [0003] After bovine rotavirus infection, it is clinically characterized by wilting, anorexia, vomiting, diarrhea, dehydration, and weight loss. Bovine coronavirus is one of the other main patho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62A61K39/295A61K39/15A61K39/215A61K39/108A61P31/14A61P31/04A61P1/12
CPCC07K14/005C07K14/245A61K39/12A61K39/0258A61P31/14A61P31/04A61P1/12C12N2720/12322C12N2720/12323C12N2720/12352C12N2720/12334C12N2770/20022C12N2770/20034C12N2770/20023C12N2770/20052C07K2319/40A61K2039/5258A61K2039/552A61K2039/70A61K2039/55566C07K2319/00C12R2001/19Y02A50/30A61K39/15C12N7/00
Inventor 贺笋张国庆闫鹏先席娜郭苗苗肖升东聂祥祥孔楚心李延涛潘毅平
Owner TIAN KANG ZHI YAO GU FEN YOU XIAN GONG SI
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