Porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine and preparation method thereof

A technology for porcine epidemic diarrhea and double inactivated vaccine, applied in the field of genetic engineering, can solve the problem of lack of immune effect, etc., and achieve the effects of good immunogenicity, high safety and high immune protection rate

Pending Publication Date: 2022-05-24
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to address the lack of porcine epidemic diarrhea and porcine rotavirus inactivated vaccines with good immune effect, safety and reliability in the ...

Method used

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  • Porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine and preparation method thereof
  • Porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Isolation, identification, cloning, purification and biological characteristics of porcine epidemic diarrhea virus

[0048] 1.1 Disease materials and cells

[0049] The disease materials were obtained from the small intestines of pigs with clinical symptoms of diarrhea in large-scale pig farms in my country, and Vero cells were kept and supplied by the laboratory of the R&D Center of Keqian Biological Co., Ltd. The porcine epidemic diarrhea virus used in this example is the porcine epidemic diarrhea virus 2aKQ01 strain recorded in the patent CN111041002B that has been deposited and the deposit number is: CCTCC NO: 202005.

[0050] 1.2 Isolation and identification of viruses

[0051] Use specific primers to amplify the PEDV M gene. The primer sequences are:

[0052] Upstream primer PEDV-MS: 5'-ATGTCTAACGGTTCTATTCCCGT-3'

[0053] Downstream primer PEDV-MA: 5'-CTTGGCGACTGTGACGAAAT-3'

[0054] The size of the amplified fragment was 519bp, and the primers were ...

Embodiment 2

[0094] Example 2 Isolation, identification, cloning, purification and biological characteristics of porcine rotavirus

[0095] 1.1 Disease materials and cells

[0096] The disease materials were obtained from the small intestines of pigs with clinical symptoms of diarrhea in large-scale pig farms in my country, and the MA104 cells were kept and supplied by the laboratory of the R&D Center of Keqian Biological Co., Ltd. Suspension PK-15 cells were kept and supplied by Wuhan Keqian Biological Co., Ltd. In the present invention, the medium for suspension culture of PK-15 cells (PK serum-free medium) was purchased from Zhejiang Yishengke Biotechnology Co., Ltd., item number ZJ02F1170.

[0097] 1.2 Isolation and identification of viruses

[0098] Use specific primers to amplify the PoRV VP6 gene. The primer sequences are:

[0099] Upstream primer PoRV-jdS: 5'-ATTTGATGGGAACTATGTGG-3'

[0100] Downstream primer PoRV-jdA: 5'-GGTGGACGAACTAATTGAAACG-3'

[0101] The size of the ampl...

Embodiment 3

[0188] Example 3 Preparation and inspection of porcine epidemic diarrhea and porcine rotavirus dual inactivated vaccine

[0189] Another object of the present invention is to provide a preparation method of porcine epidemic diarrhea and porcine rotavirus dual inactivated vaccine, comprising the following steps:

[0190] 1 Preparation of porcine epidemic diarrhea virus (porcine epidemic diarrhea virus 2A KQ01 strain) virus liquid

[0191] 1.1 Cell expansion culture in bioreactors

[0192] When the cells in the seed tank are cultured for 72h or 96h and the cell viability is above 95%, the cells are transferred to the bioreactor. Before inoculating cells, the dissolved oxygen (DO) electrode, pH electrode, and temperature electrode should be calibrated, and the tank should be autoclaved. Pump 20% of the total volume of the bioreactor with ST serum-free medium to 1 x 10 6 Cells were transferred to a bioreactor at a cell density of cells / ml, and the optimal culture conditions wer...

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Abstract

The invention provides a porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine and a preparation method thereof, and provides the porcine epidemic diarrhea and porcine rotavirus bivalent inactivated vaccine which is high in immune protection rate, good in stability, high in yield, safe and reliable and is used for preventing vaccination by adopting a cell continuous passage method and a cell suspension culture technology. And an effective means is provided for prevention and control of porcine epidemic diarrhea and porcine rotavirus.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a porcine epidemic diarrhea and porcine rotavirus dual inactivated vaccine and a preparation method thereof. Background technique [0002] Porcine epidemic diarrhea virus (PEDV) belongs to the genus Nidos and the genus Corona virus. The viral nucleic acid is an infectious linear single-stranded positive-segment non-segmented RNA. Due to the mutation deletion and insertion of PEDV gene bases, the diversity of PEDV gene is caused. At present, PEDV can be divided into classic GI-a, GI-b and variant strains GII-a, GII-b. The popular ones in our country's pig farms are variant strains GII-a and GII-b. GII-a and GII-b strains are highly pathogenic and can cross-protect, but studies have shown that GII-a strains can better regulate the body's immune protection against the infection of corresponding subtypes of PEDV than GII-b strains. At present, GII-a strains are mainly circulating...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/295A61K39/15A61K39/215A61K39/39A61P31/14
CPCC12N7/00A61K39/12A61K39/39A61P31/14A61K2039/5252A61K2039/552A61K2039/54A61K2039/70C12N2720/12321C12N2720/12363C12N2720/12334C12N2770/20034Y02A50/30
Inventor 徐高原罗修鑫张华伟周明光陈映瑾郝根喜朱娴静周飞燕刘慧
Owner WUHAN KEQIAN BIOLOGY CO LTD
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