Tpl2-deficient MDCK cell strain as well as construction method and application thereof

A cell line, defective technology, applied in animal cells, vertebrate cells, urinary tract/kidney cells, etc., can solve the problem of inability to obtain stable and safe high-producing avian influenza virus cell lines, etc., and achieve supernatant blood coagulation efficiency. Increase in valence, overcome genetic instability or insecurity, increase productivity

Inactive Publication Date: 2020-07-28
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the problem that the transformation of existing MDCK cells cannot obtain a stable and safe high-yield cell line of avian influenza virus, the present invention provides a Tpl2-deficient MDCK cell strain and a construction method thereof. The Tpl2-deficient MDCK cell strain is obtained by transplantation The Tpl2 gene is knocked out due to the deletion of 5bp bases by the code mutation, which can be inherited stably, does not have tumorigenicity, and is safe and reliable

Method used

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  • Tpl2-deficient MDCK cell strain as well as construction method and application thereof
  • Tpl2-deficient MDCK cell strain as well as construction method and application thereof
  • Tpl2-deficient MDCK cell strain as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Constructing the MDCK cell line that stably knocks out TPL2

[0067] (1) Design of sgRNA and synthesis of oligonucleotide chain: design two pairs of guide RNA (sgRNA) at the first exon of Tpl2 gene, add CCGG to the 5' end of the sense strand template, which can be digested with Bbs I The formed cohesive ends are complementary; AAAC is added to the 5' end of the antisense strand template, which is complementary to the cohesive ends formed after Bbs I digestion.

[0068] Table 1 TPL2-sgRNA oligonucleotide sequence

[0069]

[0070] (2) Construction of pUC19-sgRNA vector:

[0071] The single strand of the synthesized oligonucleotide was first annealed to form a double strand, and then constructed and inserted into the pUC19-sgRNA plasmid vector through the Bbs I restriction site, and the constructed plasmid was sequenced, and the sequencing results were as follows: figure 2 As shown, it proves that the pUC19-sgRNA plasmid vector was constructed successfully...

Embodiment 2

[0081] 1. Comparison of growth kinetics between Tpl2 knockout MDCK cells and parental cells

[0082] Spread the wild-type and Tpl2-deficient MDCK cell lines constructed in Example 1 in a 96-well plate at 2000 cells / well, count 3 wells each time, and set up 2 wells that only add medium as control wells, and then place them in 37 °C, 5% CO 2 cultivated under conditions. Using the MTS kit, with time as the horizontal axis and absorbance value (the actual absorbance of the cells = the absorbance of the cell well - the absorbance of the control blank) as the vertical axis, the growth curve was drawn using GraphPad Prisms software. The proliferation activity of the two kinds of cells was detected by the MTS method, and it can be seen from the drawn growth curve that there was no significant difference in the growth rate between the Tpl2 knockout MDCK cells and the parental cells ( Figure 4 ).

[0083] 2. Study on the quality control of cell matrix Tpl2-deficient MDCK cell line f...

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Abstract

The invention provides a Tpl2-deficient MDCK cell strain as well as a construction method and application thereof, and relates to the technical field of biology. In the MDCK cell strain, 239th to 243rd bases of a first exon of a Tpl2 gene are deleted. The Tpl2 gene in the MDCK cell strain is knocked out through frame shift mutation, but the mutation has no influence on the growth rate and cell morphology of an MDCK cell after passage for 10 generations; tests show that the Tpl2-deficient MDCK cell strain provided by the invention can be stably inherited, is safe and reliable, and solves the problem that an existing mutant MDCK cell is unstable or unsafe in heredity. The Tpl2-deficient MDCK cell strain disclosed by the invention can be used for improving the flu virus yield after being inoculated with flu viruses, and compared with conventional MDCK cell culture, the supernatant hemagglutination titer and the supernatant virus TCID50 titer are obviously improved, and the problem of insufficient flu virus yield in the existing MDCK cell culture is solved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Tpl2-deficient MDCK cell line and its construction method and application. Background technique [0002] Influenza virus (AIV) belongs to the Orthomyxoviridae family and the genus Influenzavirus. It is a zoonotic RNA virus with 16 and 9 species of surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). kind. It has caused huge economic losses to the breeding industry worldwide, and at the same time brought serious threats to human health. Vaccination is one of the most effective strategies for preventing viral infections. Traditional influenza vaccines are produced by inoculating chicken embryos and have been widely used around the world, and their safety has been recognized by the public. However, this technology still has certain limitations, such as the long preparation period of chicken embryos, it is difficult to produce the required vaccine in a short time with ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N7/00
CPCC07K14/47C12N5/0686C12N7/00C12N15/85C12N2510/02C12N2760/16151C12N2760/16251C12N2760/16351
Inventor 孟庆文王伟陈洪岩
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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