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Establishment of MDCK cell line stably expressing beta-galactoside alpha-2, 3-sialytransferase I(ST3GAL I)

A technology of MDCK-ST3GALI and cell lines, which is applied in the field of invention related to biotechnology, and can solve the problems of low virus titer, difficulty in developing vaccines and antiviral drugs, low receptor abundance, etc.

Inactive Publication Date: 2009-12-30
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some strains have the ability to replicate and proliferate in mammalian cells such as MDCK, due to the low abundance of receptors, the virus titer is low and the ability to form plaques is weak, which poses great challenges for further research and even the development of vaccines and antiviral drugs. great difficulty

Method used

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  • Establishment of MDCK cell line stably expressing beta-galactoside alpha-2, 3-sialytransferase I(ST3GAL I)
  • Establishment of MDCK cell line stably expressing beta-galactoside alpha-2, 3-sialytransferase I(ST3GAL I)
  • Establishment of MDCK cell line stably expressing beta-galactoside alpha-2, 3-sialytransferase I(ST3GAL I)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction of eukaryotic expression plasmid pIRES2-eGFP-ST3GAL I

[0070] Materials and Methods

[0071] Reagents and Instruments

[0072] Trizol and reverse transcriptase M-MLV were purchased from Invitrogen; pMD18-T vector, oligodT, various restriction enzymes and Taq enzymes were purchased from TaKaRa; plasmid pIRES2-eGFP was purchased from BD Biosciences Clontech; T4 DNA ligase Purchased from MBI Company; gel recovery kit was purchased from Shanghai Huashun Company, plasmid medium extraction kit was purchased from Qiagen Company; CEQ8000 automatic sequencer was a product of Beckman Company.

[0073] Cloning of chicken beta-galactoside alpha-2, 3-sialyltransferase I (ST3GAL I) gene

[0074] Chicken beta-galactoside alpha-2, 3-sialyltransferase I (ST3GAL I) gene (Accession NO.NM_205217) with a full-length 1029bp open reading frame (ORF) was synthesized by a biological company, and primers were designed with oligo 6 software:

[0075] pf:ta gagctc gccg...

Embodiment 2

[0083] Construction of embodiment 2MDCK-ST3GAL I cell line

[0084] Materials and Methods

[0085] Reagents and Instruments

[0086]G418, DMEM medium, transfection reagent Lipofectamine 2000Regeant were purchased from Invitrogen; fetal bovine serum was purchased from GIBCO; MDCK cells were cultured in DMEM medium containing 5% fetal bovine serum (purchased from gibco). The inverted microscope is a product of Olympus, and the fluorescent inverted microscope is a product of ZEISS. DIG Glyan Differentiation Kit is a Roche product.

[0087] Determining the appropriate concentration of G418 in the selection medium

[0088] MDCK cells were inoculated into 24-well cell culture plates, and when they grew to 80% full, G418 was added to make the concentrations 200, 400, 600, 800, 1000, 1200 μg / ml, and placed at 37°C, 5% CO 2 Culture in an incubator, observe the cell growth every day, and record the cell death. After two weeks of culture, the cell growth curve was drawn to determine...

Embodiment 3

[0106] Embodiment 3 flow cytometry detects the expression of ST3GAL I

[0107] Materials and Methods

[0108] Reagents and Instruments

[0109] The flow cytometer is a Beckman product. DIG Glyan Differentiation Kit (CatNo. 11210238001) and Anti-digoxigenin-rhodamine were purchased from Roche Company.

[0110] Expression of ST3GAL I detected by flow cytometry

[0111]MDCK and MDCK-ST3GAL I were placed in three wells of a six-well plate, respectively. When the cell density is about 90% (about 1.5×106 cells per well), digest with trypsin, collect the cells in each well, add a small amount of serum-containing culture medium to neutralize the activity of trypsin, and collect the cell pellet. Wash twice with PBS containing 10mM Glycine, and then wash with Buffer 1 (50mM Tris-HCl, 0.15M NaCl, 1mM MgCl 2 , 1mM MnCl 2 , 1mM CaCl 2 , PH 7.5) wash once. Evenly resuspend the cell pellet with the prepared blocking solution Blocking Solution (10-fold dilution of Blocking Solution in...

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Abstract

The invention provides an MDCK-ST3GAL I cell line, concretely an MDCK cell line stably expressing beta-galactoside alpha-2, 3-sialytransferase I(ST3GAL I) with the preservation number of CGMCC No. 2620; the invention further provides a method for establishing the MDCK-ST3GAL I cell line and a method for improving the growth titre of avian influenza virus and the forming capability of plaque. The invention further provides an application of the MDCK-ST3GAL I cell line in researching avian influenza virus 2, 3 linked receptor tendency; and the invention is used for applications in monitoring the avian influenza virus receptor associative property variant strain, screening medicines resisting avian influenza virus, measuring the neutralizing antibody and mass producing cell culture vaccine.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to an engineered cell line clone MDCK-ST3GAL I and a construction method thereof, and more specifically relates to an engineered cell line clone MDCK-ST3GAL I, whose preservation number is: CGMCC No.2620, And the cell line can improve the growth titer and plaque formation ability of isolated strains of avian influenza virus, monitor the variant strains of receptor binding characteristics of avian influenza virus, screen anti-avian influenza virus drugs, measure neutralizing antibodies and even large-scale Production of cell culture applications in vaccine production. Background technique [0002] The hemagglutinin (HA) on the surface of the influenza virus binds to the sialooligosaccharide receptor on the cell surface to infect the host cell, and the avian influenza virus tends to recognize the α-2,3-linked receptor (NeuAc α-2,3-Gal), Human influen...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/54C12N9/10C12N15/85C12N7/00C12Q1/02G01N33/53G01N33/15
Inventor 步志高陈伟业陈化兰
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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