A monoclonal cell line stably expressing serine protease, its preparation method, a kit containing the cell line and its application

A serine protease and stable expression technology, applied in the field of monoclonal cell lines and their preparation, and kits containing the cell lines, can solve the problems of poor repeatability, expensive detection, and the need for fluorescent microscopes, etc., and achieve simple and accurate results. Good experimental repeatability

Active Publication Date: 2021-03-30
BEIJING VBIOSCI INC +1
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Problems solved by technology

[0004] After swine fever virus infects porcine testicular cells, it cannot cause obvious CPE phenomenon, so the TCID50 value of swine fever virus can only be detected by immunofluorescence detection
However, the experimental method using immunofluorescence to detect the titer of CSFV has the disadvantages of expensive detection, poor repeatability, and the need for a fluorescent microscope. Therefore, a simple and effective detection method is urgently needed

Method used

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  • A monoclonal cell line stably expressing serine protease, its preparation method, a kit containing the cell line and its application
  • A monoclonal cell line stably expressing serine protease, its preparation method, a kit containing the cell line and its application
  • A monoclonal cell line stably expressing serine protease, its preparation method, a kit containing the cell line and its application

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Embodiment 1

[0027] 1. Materials

[0028] ST cells were purchased from ATCC Cell Preservation Center, MEM Alph medium and 0.25% trypsin were purchased from Gibco Company, newborn bovine serum (NBS) was purchased from Wuhan Sanli Company, Puromycin was purchased from Suleibao Company, The 96-well cell culture plate was purchased from Corning Company, the classical swine fever E2 antibody was purchased from MEDLAN Company, the goat anti-mouse FITC secondary antibody was purchased from Invitrogen Company, and the plasmid PLJM1 RagB 99L and CSFV-DNS3 were donated by Ma Ningning Laboratory of Shenyang Pharmaceutical University. Primer synthesis and sequencing services were completed at Thermo. ;EasyPfu DNA polymerase, dNTP and nucleic acid electrophoresis Marker were purchased from Quanshijin Biotechnology Co., Ltd. Competent cells DH5á were purchased from Bomed Biotechnology Co., Ltd.; plasmid small extraction kit, plasmid large extraction kit, agarose gel Gel DNA recovery kit and erythrocyte...

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Abstract

The invention discloses a monoclonal cell strain stably expressing a serine protease gene, a preparation method of the monoclonal cell strain, a kit containing the monoclonal cell strain, and application of the monoclonal cell strain. The monoclonal cell strain stably expressing the serine protease gene is cultured in a culture plate, and cell culture liquid is removed; classical swine fever virusraw liquid is subjected to doubling dilution, and the diluted virus liquid is added into the culture plate with the cell culture liquid being removed; and culture continues, a classical swine fever virus TCID50 is judged through a CPE phenomenon, and the TCID50 is calculated through a Reed-Muench method. The advantages that the monoclonal cell strain is used for visual detection of the classicalswine fever virus, the classical swine fever virus TCID50 is detected through the detection kit, a fluorescent microscope and a classical swine fever detection antibody are not needed, result judgement of the classical swine fever virus TCID50 is simple, and experimental repeatability is good are achieved.

Description

technical field [0001] The invention belongs to the technical field of virus detection. Specifically, the invention relates to a monoclonal cell strain stably expressing serine protease, a preparation method thereof and a kit containing the cell strain. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease caused by classical swine fever virus (CSFV). Wild boars and domestic pigs are the only natural hosts of CSFV. It is an important disease that threatens the pig industry. Therefore, the prevention and control of swine fever is in a severe situation, and research on the development of swine fever virus and its vaccine is still very urgent. Therefore, the development of a detection kit that can easily and economically detect the titer of swine fever virus is of great importance for the prevention and control of swine fever disease. significance. [0003] Classical swine fever virus belongs to Flaviviridae and Pestivirus genus. Virus parti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/85C12N15/57C12Q1/70C12Q1/02
CPCC12N5/0683C12N9/50C12N15/85C12N2510/00C12Q1/02C12Q1/70C12Y304/21106
Inventor 周佳彬郑杰米梦柔候野张凌云陈晓娟
Owner BEIJING VBIOSCI INC
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