Sp2/0 cell immortality improvement based hybridoma technique

An immortalization, hybridoma technology, applied in the field of cell hybridization and whole genome amplification and storage, can solve the problems of death, not reaching the ideal confluence, floating death while growing, etc.

Inactive Publication Date: 2019-06-21
翁炳焕
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Problems solved by technology

However, if it is transferred to the preparation of hybridoma cells hybridized between human gene mutant lymphocytes and mouse sp2/0 tumor cells, we found that although they can hybridize, there are the following three problems: (1) Human gene mutant hybridoma cells are attached Floating death while growing on the wall: There are always about 1/2 to 1/3 of the cells floating in the culture

Method used

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  • Sp2/0 cell immortality improvement based hybridoma technique
  • Sp2/0 cell immortality improvement based hybridoma technique
  • Sp2/0 cell immortality improvement based hybridoma technique

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Embodiment Construction

[0022] Combine below figure 1 , figure 2 , image 3 and Figure 4 , the embodiments of the present invention are described in detail.

[0023] 1. Construction of recombinant retroviral vector (pcDNA3.1-hTERT)

[0024]1. Reagents: pcDNA3.1 retroviral vector was purchased from Clontech, USA, hTERT (strain 776) was purchased from Invitrogen, USA, PCR amplification kit and calcium phosphate precipitation transfection kit were purchased from Invitrogen, USA, E.coliDH5-alpha large intestine Escherichia coli were preserved in this unit, Endotoxin-free plasmid purification kit was purchased from QIGEN, Germany, EcoR I and BamH I endonucleases were purchased from Fermentas, Lithuania, T4 DNA ligase was purchased from Roche, Germany, neomycin derivatives G418 was purchased from Sigma Company of the United States, and fetal bovine serum and DMEM medium were purchased from Gibcol Company of the United States. The vectors involved in the present invention also include pCDNA3.1, pCMV,...

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Abstract

An sp2/0 cell immortality improvement based hybridoma technique for the field of biology is characterized in that hTERT with an immortality characteristic is adopted for transfection of rat sp2/0 tumor cells through a pcDNA3.1-hTERT recombinant retroviral vector to obtain immortality characteristic improved recombinant vector packaged rat sp2/0 tumor cells, then hTERT is guided into hybridoma cells through cell screening and fusion, and accordingly the immortality characteristic of the hybridoma cells is enhanced, the cell fusion success rate and fusion rate are increased, the cell survival rate in a cloning screening process is increased, the cell mortality rate in positive clone cell subculturing is decreased, confluence in cell adherence growth is increased, and a foundation is laid forindustrial amplification of fusion cells and further amplification of pathogenic genes in the fusion cells.

Description

technical field [0001] The invention relates to a hybridoma technology based on sp2 / 0 cell immortalization improvement in the field of biology, which is mainly used for cell hybridization and whole genome amplification and storage in the fields of scientific research, pharmacy and diagnosis. Background technique [0002] Hybridoma technology is lymphocyte hybridoma technology, also known as monoclonal antibody technology. [0003] Kohler and Milstein (1975) proved that myeloma cells were hybridized with spleen cells of immunized animals to form monoclonal antibodies that can secrete homogeneous and highly specific antibodies against the antigen. The technology is commonly known as hybridoma technology. Myeloma cells can be continuously passaged in vitro, while spleen cells are terminal cells that cannot be propagated in vitro. For example, if the myeloma cells of mice are hybridized with lymphocytes secreting certain antibodies or factors, the hybrid cells not only have th...

Claims

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Application Information

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IPC IPC(8): C12N15/867
Inventor 翁炳焕杨昊堃
Owner 翁炳焕
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