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Process for preparing hybridoma cell for secreting anti-human asparagine hydroxylase monoclonal antibodies

An asparagine hydroxylase mono- and asparagine hydroxylase technology, which is applied in the field of preparation technology of hybridoma cells, can solve the problems of long immunization cycle, poor specificity and low efficiency, and achieves short immunization cycle and non-specificity. The effect of less reaction and high efficiency

Active Publication Date: 2008-11-19
SHANXI LIFEGEN
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Problems solved by technology

[0011] The object of the present invention is to provide a hybridoma cell preparation process for secreting anti-human asparagine hydroxylase monoclonal antibody, which solves the technical problems of poor specificity, long immune cycle and low efficiency in the background technology

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  • Process for preparing hybridoma cell for secreting anti-human asparagine hydroxylase monoclonal antibodies
  • Process for preparing hybridoma cell for secreting anti-human asparagine hydroxylase monoclonal antibodies
  • Process for preparing hybridoma cell for secreting anti-human asparagine hydroxylase monoclonal antibodies

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Embodiment Construction

[0050] The present invention uses plasmid DNA immunization and recombinant protein immunization to perform combined immunization with the best effect, and the specific implementation steps are as follows:

[0051] (i) Taking recombinant eukaryotic expression plasmid DNA containing human asparagine hydroxylase HAAH coding gene. The recombinant eukaryotic expression plasmid DNA vector containing human asparagine hydroxylase HAAH coding gene can be pCDNA series, pCMV series, pCI-S, pMEP4-S or pLXSN-S and so on. The purity of recombinant eukaryotic expression plasmid DNA containing human asparagine hydroxylase HAAH encoding gene is preferably 1.6≤OD260nm / OD280nm≤1.8.

[0052] (ii) DNA immunization: inject the recombinant eukaryotic expression plasmid DNA containing the gene encoding human asparagine hydroxylase HAAH into the skeletal muscle of the mouse, preferably the quadriceps femoris of the hind limb of the mouse. The injection dose is 10-200 μg / 50-200 μl / monkey. DNA immuniz...

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Abstract

The invention relates to a preparation process for a hybridoma secreting a monoclonal antibody repelling human asparaginate hydroxylase. The method comprises the following steps: taking the recombinant enkaryon expression plasmid DNA of a human asparaginate hydroxylase HAAH coded gene, injecting the DNA into the skeletal muscle of a white rat for DNA immunity, carrying out human asparaginate hydroxylase HAAH recombinant protein immunity after 1 to 4 weeks, taking the splenic cell and myeloma cell of the immune white rat for cell fusion after 2 to 5 days, carrying out antibody detection, and cloning the positive cells in the antibody detection till obtaining the hybridoma secreting the monoclonal antibody repelling human asparaginate hydroxylase. The invention solves the technical problems of bad specificity, long immune cycle and low efficiency in the prior art. The hybridoma has clear immunogenicity, high purity, few non-specific reactions, and low use level of antigen with a total immune cycle of only 6 to 8 weeks.

Description

technical field [0001] The invention relates to a preparation process of hybridoma cells, in particular to a preparation process of hybridoma cells capable of secreting anti-human asparagine hydroxylase monoclonal antibody. Background technique [0002] Human asparagine hydroxylase (HAAH) is an α-ketoglutarate-dependent dioxygenase. Its main biological function is to participate in post-translational protein modification during protein synthesis, and it can catalyze the epidermal growth factor contained in various proteins. β-carbon atom hydroxylation of aspartate and asparagine residues within the (EGF)-like domain. [0003] Proteins containing epidermal growth factor-like domains include Notch and its ligands, blood coagulation factors and other proteins with important biological functions, among which Notch and its similar molecules are related to cell differentiation, especially its intracellular region is closely related to tumorigenicity Therefore, the relationship be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/18G01N33/577
Inventor 薛小平黄庆生张明杰
Owner SHANXI LIFEGEN
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