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MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof

A MDCK-1F7, cell line technology, applied in the field of cloned cell lines, can solve problems such as restricting application, and achieve the effect of high application value

Active Publication Date: 2015-06-10
哈药集团生物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, the virus titer of influenza virus cultured in MDCK cells is still lower than that of cultured in chicken embryos, which restricts its application in the preparation of avian influenza vaccines.

Method used

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  • MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof
  • MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof
  • MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Cloning and Identification of Example 1 MDCK Clone Cell Strain

[0032] 1 Experimental method

[0033] 1.1 Cloning of MDCK clone cell line

[0034] (1) Select the kind of cell MDCK;

[0035] (2) Cell passage: first recover the seed cells selected in step (1) into a T25 cell flask, grow for about 48 hours, passage according to the ratio of 1:4, and continue to passage 20 times before cloning;

[0036] (3) MDCK single cell cloning was carried out in a 96-well plate by the limiting dilution method, and the digested MDCK cells were counted and diluted to 1 cell / ml, and the diluted cells were spread on a 96-well plate, 0.1ml / well, 37 ℃5%CO 2 Cultivate in the incubator and observe for 10 days;

[0037] (4) Select a single clone for further subcloning. After subcloning three times, select a single clone for expansion culture in DMEM (containing 10% FBS), and then further identify the cloned cells.

[0038] 1.2 Identification of MDCK clonal cell lines

[0039] 1.2.1 Morph...

experiment example 1

[0068] Experimental example 1 Optimizing experiment of cultivating avian influenza virus conditions using MDCK cloned cell line MDCK-1F7 or MDCK-2D6

[0069] 1. Experimental method

[0070] The MDCK clone cell line MDCK-1F7 or MDCK-2D6 cloned in Example 1 of the present invention is used to culture avian influenza virus and optimize the culture conditions.

[0071] Monoclonal cells MDCK-1F7 or MDCK-2D6 were seeded in cell culture flasks (T25Corning), 37°C, 5% CO 2 After culturing for 48 hours to form a single layer, discard the medium, wash twice with PBS (pH7.2), add avian influenza virus H5N1RE-6 strain according to a certain multiplicity of infection, 37 ° C, 5% CO 2 Adsorb in the incubator for 1h, add 9ml DMEM maintenance solution, 37°C, 5% CO 2 Continue to cultivate and observe day by day. When the lesions reached 80% (within 5 days), freeze-thaw was repeated twice, centrifuged at 2,000 rpm at 4° C. for 20 minutes, and the supernatant was harvested.

[0072] 1.1 Optim...

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Abstract

The invention discloses MDCK (Madin-Darby Canine Kidney) clone cell strains and application thereof, belonging to the field of MDCK clone cell strains. A limiting dilution process is adopted to perform MDCK unicell cloning, and subcloning is further performed to finally obtain 2 monoclonal cell strains with excellent performance. The two monoclonal cell strains have higher growth rate than the female parent, can well reproduce avian influenza virus, obviously enhances the duplication level of the virus in cells, and obtains the virus solution with higher potency. Compared with the virus by female parent cell culture, the HA potency titer is obviously enhanced. The microbe collection numbers of the two cell strains are respectively CGMCC No.10008 and CGMCC No.10009. The MDCK clone cell strains disclosed by the invention can efficiently culture viruses, obviously enhances the virus titer, and has high application value in influenza virus vaccine production.

Description

technical field [0001] The invention relates to a cloning cell strain, in particular to an MDCK cloning cell strain. The invention also relates to the application of the MDCK cloning cell strain in cultivating avian influenza virus, and belongs to the field of cloning and application of the MDCK cloning cell strain. Background technique [0002] In the past, China has always used chicken embryos to produce bird flu vaccines, which required a large amount of chicken embryos, a long production cycle, easy pollution, and difficult to control. This production method is inefficient and not conducive to responding to large-scale influenza outbreaks. The prevalence of bird flu in different regions of China is different, and bird flu spreads quickly between different regions; precise planning must be made in advance to produce bird flu vaccines with chicken embryos, but generally speaking, bird flu often occurs in outbreaks, and it is difficult to The millions of eggs needed to pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/93
Inventor 涂映霞李彦伟丁国杰毛春玲步帆卢爱国郑铁鑫王昊孙凯于洪涛李莉莉王丹丹
Owner 哈药集团生物疫苗有限公司
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