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Human laryngeal epithelium cancer cell as well as preparation method and application thereof

A cancer cell and epithelial technology, which is applied in the field of human laryngeal epithelial cancer cells and its preparation, can solve problems such as large gaps

Active Publication Date: 2011-08-31
STATE KEY LAB OF RESPIRATORY DISEASE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some large pediatric hospitals, although small-scale cell virus culture and isolation have been carried out, compared with the research and application of foreign clinical virus rooms, there is a big gap

Method used

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  • Human laryngeal epithelium cancer cell as well as preparation method and application thereof
  • Human laryngeal epithelium cancer cell as well as preparation method and application thereof
  • Human laryngeal epithelium cancer cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Method for constructing HEp-2 cell line expressing NS1 gene:

[0037] 1) HEp-2 cell culture

[0038] 2) NS1 gene primer design to amplify the NS1 gene

[0039] 3) Retroviral vector construction (pBabe-puro-NS1)

[0040] 4) Transformation and colony PCR to pick positive clones

[0041] 5) Sequencing identification

[0042] 6) Plasmid extraction

[0043] 7) Packaging toxin production (293FT cell culture, calcium phosphate co-transfection expression plasmid and packaging plasmid)

[0044] 8) Collecting viruses and preserving species

[0045] 9) Target cell culture (HEp-2 cells before modification)

[0046] 10) Retrovirus infection of target cells

[0047] 11) Screening of stably infected cell lines (puro selection of stably infected HEp-2 cells)

[0048] 12) Amplification of stable strains

[0049] The retroviral vector system consists of two parts: the packaging cell line (such as 293FT cells) and the defective virus itself (retroviral vector).

[0050] In the ret...

Embodiment 2

[0053] Concrete steps for constructing HEp-2 cell line expressing NS1 gene:

[0054] 1. Carrier Construction

[0055] 1 Gene sequence analysis

[0056] Analysis of the NS1 gene sequence submitted on NCBI (NCBI accession number is AF013254), the result: the NS1 gene is available Bam H I and EcoR I was digested and placed behind the promoter. The pBaBb-puro expression vector is pBABE-Puro Retroviral Vector, CELL BIOLABS, INC.

[0057] The coding sequences of NS1 genes are respectively: 420bp, and primers NS1-F and NS1-R were designed according to the above information.

[0058] PCR amplification of NS1 gene

[0059] According to the characteristics of the NS1 gene fragment and primers, design the PCR program:

[0060] Reaction system: 20μl

[0061] 5×PrimeSTAR Buffer (Mg 2+ plus): 4μl

[0062] 2.5mM dNTPs: 1μl

[0063] 10p upper primer: 1μl

[0064] 10p lower primer: 1μl

[0065] PrimeSTAR HS (2.5U / μl): 0.3μl

[0066] Sterilized water: 11.7μl

[0067] Templa...

Embodiment 3

[0154] Example 3 Comparison of the sensitivity of cells to RSV virus before and after improvement.

[0155] Material:

[0156] Virus: RSV B strain (purchased from ATCC, lot: 5271356), serially diluted to 10 -1 —10 -8

[0157] Cells: cells before improvement: HEp-2 cells, cells after improvement: HEp-2-NS1 cells.

[0158] method:

[0159] 1. Prepare cells before and after improvement up to 85%;

[0160] 2. Wash the cell surface 1-2 times with PBS;

[0161] 3. After adding 50uL virus diluent to each well, centrifuge at room temperature for 60 minutes at 3000 rpm;

[0162] 4. Add 50uL of corresponding 4% DMEM to each well and culture at 34°C. Observe for 2-4 days.

[0163] For cytopathic (CPE) results see figure 1 , 2 , 3, 4. The arrows in the figure indicate cytopathic changes.

[0164] The results in the figure show that after the modified HEp-2-NS1 cells were inoculated with RSV virus, the cell lesion appeared earlier than that of the unmodified HEp-2 cells, and th...

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PUM

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Abstract

The invention provides a human laryngeal epithelium cancer cell HEp-2-NS1 with a preservation number (China Center for Type Culture Collection) CCTCC NO: C201092. The human laryngeal epithelium cancer cell can inhibit interferon and can be rapidly propagated. The invention also provides a preparation method and application of the human laryngeal epithelium cancer cell.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to human laryngeal epithelial cancer cells, a preparation method and application. Background technique [0002] Viral infectious diseases are the main diseases among human infectious diseases today. According to recent data, among the newly discovered infectious diseases in the past 30 years, about 60% of the diseases with clear pathogens are caused by viruses, and about 50% of respiratory diseases are caused by viruses, the typical example of which is SARS coronavirus and highly pathogenic avian influenza virus. The AIDS and SARS epidemics that have emerged in my country in recent years have brought new challenges to clinical medicine and virology research. The highly pathogenic avian influenza epidemic in Southeast Asia since 2003 has attracted worldwide attention, and a large number of poultry and a small number of people have been infected in my country. So far, there ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N7/08
Inventor 杨子峰秦笙王玉涛周荣王丹芬关文达占扬清莫自耀钟南山
Owner STATE KEY LAB OF RESPIRATORY DISEASE
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