PMKRN1 (porcine Makorin ring finger protein 1) gene-knockout porcine somatic cell, method for preparing same and application of pMKRN1 gene-knockout porcine somatic cell

A gene knockout and somatic cell technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve problems that have not been studied

Active Publication Date: 2018-08-07
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The effect of pMKRN1 binding on the main structural protein component of PCV2, that is, its capsid protein (Cap), has not been studied.

Method used

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  • PMKRN1 (porcine Makorin ring finger protein 1) gene-knockout porcine somatic cell, method for preparing same and application of pMKRN1 gene-knockout porcine somatic cell
  • PMKRN1 (porcine Makorin ring finger protein 1) gene-knockout porcine somatic cell, method for preparing same and application of pMKRN1 gene-knockout porcine somatic cell
  • PMKRN1 (porcine Makorin ring finger protein 1) gene-knockout porcine somatic cell, method for preparing same and application of pMKRN1 gene-knockout porcine somatic cell

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Embodiment Construction

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The examples are by way of illustration, not limitation, of the invention.

[0035] The present invention detects the effect of pMKRN1 on Cap degradation by co-expressing porcine MKRN1 (pMKRN1) and PCV2Cap; simultaneously constructs pMKRN1 overexpression cells and knockout cell lines to verify the effect of pMKRN1 on PCV2Cap degradation, and utilizes knockout cells to realize PCV2 virus in the host ( For example, high-speed replication in PK-15 cells).

[0036] (1) Construction of pMKRN1 and PCV2Cap eukaryotic expression vectors

[0037] Design the amplification primers for pMKRN1 and PCV2Cap:

[0038] pMKRN1 upstream primer P1: 5'-CCG CTCGAG ATGGATTACAAGGATGACGACGATAAGCATGGGGTTTGTAAGGAAG-3' (the underlined position indicates the Xhol restriction endonuclease recognition sequence, and the italicized position indicates the Flag tag);

[0039...

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Abstract

The invention provides a pMKRN1 (porcine Makorin ring finger protein 1) gene-knockout porcine somatic cell, a method for preparing the same and application of the pMKRN1 gene-knockout porcine somaticcell. The pMKRN1 gene-knockout porcine somatic cell, the method and the application have the advantages that mutant PK-15 (porcine kidney-15) cells can be obtained by the aid of gene knockout technologies, high-titer PCV2 (porcine circoviruses type 2) can be quickly replicated and obtained by the aid of the mutant PK-15 cells, and accordingly efficient virus amplification cells can be provided toresearch on PCV2 pathogenic mechanisms and preparation of PCV2 inactivated vaccine and attenuated vaccine.

Description

technical field [0001] The invention relates to the establishment of porcine circovirus type 2 (PCV2) strain rapidly amplifying cell strains, in particular to the construction of pMKRN1 knockout porcine somatic cells. Background technique [0002] Porcine circovirus associated disease (PCVAD) is caused by porcine circovirus type 2 (PCV2), mainly including porcine dermatitis and nephropathy syndrome (PDNS) , piglet congenital tremors (congenital tremors, CT) and postweaning multisystemic wasting syndrome (postweaning multisystemic wasting syndrome, PMWS) and other diseases. Although PCV2 can replicate in PK-15 cells, the replication efficiency is relatively low, which not only brings great difficulties to the study of PCV2 pathogenic mechanism, but also brings great difficulties to the production of PCV2 vaccine strains, resulting in high production costs of PCV2 vaccines. Therefore, obtaining a cell line that can significantly improve the replication ability of PCV2 is a k...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N9/22C12N7/00
CPCC12N7/00C12N9/22C12N15/86C12N2740/15043C12N2750/10051
Inventor 黄勇童德文王彤彤
Owner NORTHWEST A & F UNIV
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