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PK15 cell line with high infectivity against PCV2 and its preparation method and application

A cell line and infectious technology, applied in the field of PK15 cell line and its preparation, can solve problems such as unsatisfactory immune effect

Active Publication Date: 2020-07-31
XINXIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are currently vaccines targeting PCV2 on the market at home and abroad, the immune effect is still unsatisfactory

Method used

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  • PK15 cell line with high infectivity against PCV2 and its preparation method and application
  • PK15 cell line with high infectivity against PCV2 and its preparation method and application
  • PK15 cell line with high infectivity against PCV2 and its preparation method and application

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Experimental program
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Effect test

Embodiment Construction

[0041] The present invention will be described in detail below with reference to the accompanying drawings.

[0042] 1. Materials and methods

[0043] 1.1 Materials

[0044] 1.1.1 Cells and strains

[0045] PK15 cells were preserved in this experiment (Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences). PCV2-ZM field strain was isolated and preserved by our laboratory. PCV2 positive and negative sera were purchased from VMRD and do not cross-react with PCV1. FITC-rabbit anti-pig antibody was purchased from SIGMA Company.

[0046] 1.1.2 Main equipment and reagents

[0047] CO 2 The incubator was purchased from American Thermoelectricity, and the low-speed centrifuge was purchased from Beijing Rebel. Fluorescence microscope was purchased from Zeiss, Germany.

[0048] DMEM medium: take a pack of DMEM medium, weigh NaHCO 3 3.7g, dilute to 1000mL, mix well, filter and sterilize with 0.22μm pore size, divide each bottle into 450mL, and store at 4°C ...

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Abstract

The invention discloses a PK15 cell line for high infectivity of PCV2 (porcine circovirus 2), a method for preparing the PK15 cell line and application thereof. PK15 cells are cloned, and the method for the monoclonal cell line with high infectivity and high homogeneity is provided. The method includes 1, culturing non-homogeneity PK15 cell lines infected with the PCV2; 2, sufficiently diluting the PK15 cells by the aid of limiting dilution processes, adding the PK15 cells into 96-pore cell culture plates and allowing each pore to comprise 0.5 cell; 3, carrying out amplification culture; 4, detecting the PCV2 susceptibility of each monoclonal cell strain by the aid of IFA (indirect fluorescent antibodies); 5, stably culturing monoclonal cells. The PK15 cell line, the method and the application have the advantages that the PCV2 infection strength of a PK15-2G9 cell strain obtained by the aid of the method is far higher than the PCV2 infection strength of PK15 parent cells, and accordingly foundations can be laid for PCV2 vaccine and diagnostic research in the future.

Description

technical field [0001] The invention relates to biotechnology, in particular to a PK15 cell line with high infectivity for PCV2 and a preparation method and application thereof. Background technique [0002] Porcine circovirus type 2 (PCV-2) is the main pathogen causing postweaning multisystemic wasting syndrome (PMWS) in piglets. At present, the diagnostic criteria, pathogenic mechanism and Control research on the virus itself and PCVAD is not very clear. Although there are vaccines against PCV2 at home and abroad, the immune effect is still unsatisfactory. PCV2 vaccines are mainly PCV2 Cap protein vaccines or subunit vaccines, mainly because it is difficult to propagate PCV2 virions with high viral titers in vitro. Therefore, various laboratories use PCV2 virus-like particles (virus-like particles, VLPs) instead of PCV2 to study PCV2 infection. Therefore, in order to study PCV2 vaccine, diagnosis, treatment and PCV2-related diseases more directly and efficiently in vitr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00C12N7/04A61K39/12A61P31/20
CPCA61K39/12C12N5/0686C12N7/00C12N2750/10023C12N2750/10034C12N2750/10051
Inventor 李鹏张改平王选年王利平李青梅郭军庆金前跃邓瑞广万博刘肖
Owner XINXIANG UNIV
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