Porcine epidemic diarrhea virus-porcine rotavirus bivalent subunit vaccine as well as preparation method and application thereof

By optimizing the structure of PEDV S protein and PoRV VP4 protein and using CHO cell and Escherichia coli expression systems, the problems of low expression level and difficult purification were solved, and an efficient and safe bivalent subunit vaccine was prepared, which significantly improved the immunogenicity and protective effect and is suitable for industrial production.

CN120757664APending Publication Date: 2025-10-10CHENGDU YISIKANG PHARM TECH CO LTD +1
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Patent Information

Application Number
CN202510936248.7
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-07-08
Publication Date
2025-10-10

AI Technical Summary

Technical Problem

Existing porcine epidemic diarrhea virus and porcine rotavirus vaccines have problems such as low expression level, difficult purification, and insufficient immunogenicity. In particular, the prevention and control of mixed infection of PEDV and PoRV increases the difficulty, and the protective efficacy of existing vaccines decreases.

Method used

By optimizing the structures of PEDV S protein and PoRV VP4 protein, designing PEDV S mutant protein and PoRV VP4 optimized protein, using CHO cells and Escherichia coli expression system, optimizing signal peptidase cleavage, forming disulfide bonds to stabilize trimers, adding His tags to increase expression and purification efficiency, a bivalent subunit vaccine was prepared.

Benefits of technology

An efficient, safe and low-cost bivalent subunit vaccine has been achieved, which can effectively induce high-titer neutralizing antibodies and cytokine responses with a protection rate of 100%, reducing production costs and enhancing immunogenicity.

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Abstract

The invention discloses a porcine epidemic diarrhea virus PEDV S mutant protein, a porcine rotavirus PoRV VP4 optimized protein and a preparation method of a bivalent subunit vaccine of the porcine epidemic diarrhea virus PEDV S mutant protein and the porcine rotavirus PoRV VP4 optimized protein. Aiming at PEDV S protein, 17th phenylalanine of a signal peptide is mutated into alanine, 1097th phenylalanine of HR1 and 1263rd threonine of HR2 in a heptapeptide repeat region are respectively mutated into cysteine to form a disulfide bond, the protein expression quantity and the trimer proportion are obviously improved, the expression quantity in CHO cells reaches 1530mu g / mL, and the trimer proportion reaches 85%. For PoRV VP4 protein, a flexible peptide GSSG is inserted in front of a C-terminal 6 * His tag, the problem that a nickel column cannot be combined due to C-terminal negative charge interference is solved, and efficient purification is achieved. The bivalent vaccine is prepared by equivalently mixing the two proteins and adding an M108L adjuvant, animal experiments show that the bivalent vaccine can induce piglets to generate high-titer neutralizing antibodies, the protection rates for PEDV and PoRV challenge both reach 100%, and the bivalent vaccine is good in safety. The invention provides an efficient and safe novel vaccine for prevention and control of porcine viral diarrhea, and the application value is remarkable.
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