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Screening method of cell line capable of stably expressing complete structural protein CHO-K1 of hog cholera virus

A CHO-K1, swine fever virus technology, applied in the field of biomedicine

Pending Publication Date: 2021-07-27
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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Problems solved by technology

Despite the presence of viral reservoirs and disease transmission, CSF eradication remains challenging, especially in countries where breeding is common

Method used

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  • Screening method of cell line capable of stably expressing complete structural protein CHO-K1 of hog cholera virus
  • Screening method of cell line capable of stably expressing complete structural protein CHO-K1 of hog cholera virus
  • Screening method of cell line capable of stably expressing complete structural protein CHO-K1 of hog cholera virus

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Embodiment Construction

[0028] The present invention and its effects will be described in detail below through a specific implementation process.

[0029] The detailed realization process of a kind of screening method of stably expressing the complete structural protein of classical swine fever virus (E0-E1-E2) CHO-K1 cell line of the present invention is as follows:

[0030] 1 Materials and methods

[0031] 1.1 Vectors, cells and strains

[0032] The pCDNA3.4 vector, CHO-K1 cells, and HEK-293 cells were all preserved by the laboratory of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; DH5α competent cells were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Shimen strain genome was used for this experiment Room preservation.

[0033] 1.2 Main reagents

[0034]Xho I and BamH I restriction endonucleases are products of NEB Company in the United States; homologous recombination kits are products of Nanjing Novizan Co., Ltd.; antibiotics G418, Lipofectamine 30...

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Abstract

The invention discloses a screening method of a cell line capable of stably expressing a complete structural protein CHO-K1 of a hog cholera virus, which mainly comprises the following steps: amplifying a complete structural protein (ShmEs) gene of the hog cholera virus (a rock gate strain) by utilizing a PCR (Polymerase Chain Reaction) method, and inserting the gene into an eukaryotic expression vector pcDNA3.4 to construct a recombinant expression plasmid pcDNA3.4-ShmEs. A transient transfection method is used for transferring the recombinant plasmid into CHO-K1 cells, and antibiotic G418 is used for screening. Test results show that the ShmEs gene is expressed in CHO-K1 cells and can be stably transmitted, and antigen immunized rabbit bodies produced by the stable cell line can stimulate the rabbit bodies to generate swine fever specific antibodies, and the swine fever specific antibodies can be maintained for at least 42 days. The successful expression of the ShmEs gene and the establishment of a stable cell line of the ShmEs gene lay a foundation for the development of novel genetic recombinant vaccines for swine fever.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a screening method for a CHO-K1 cell line stably expressing the complete structural protein (E0-E1-E2) of classical swine fever virus. Background technique [0002] Classical swine fever virus (CSFV) is a highly virulent virus that can cause a highly contagious disease of pigs called classical swine fever (CSF). CSFV belongs to the family Flaviviridae, a member of the genus Pestvirus, which also includes bovine viral diarrhea virus (BVDV) and sheep border disease virus (BDV). The clinical signs and severity of CSF range from the most acute, acute, and chronic to late onset or clinical signs of disease, depending on the strain of virus infecting, the age of the host, and the immune status of the herd [1,2] . Despite the presence of viral reservoirs and disease transmission, CSF eradication remains challenging, especially in countries where breeding is common. Pestiviruses s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/40C12R1/91
CPCC07K14/005C12N5/0682C12N15/85C12N2510/02C12N2770/24322C12N2770/24351C12N2800/107
Inventor 尹双辉郭慧琛杨顺利孙世琪尚佑军卢曾军李坤曹钰莹刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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