Hybridoma technology and rare disease whole genome immortal gene pool construction method

Abstract

The invention relates to the field of biomedicine and discloses a hybridoma technology and a rare disease whole genome immortal gene pool construction method. The hybridoma technology comprises the steps: transferring an SV40LT gene and a neomycin resistant gene into a mouse sp2 / 0 oncocyte to prepare a packaged sp2 / 0 cell; fusing the packaged sp2 / 0 cell with a human cell and obtaining a hybridomacell line through G418 and HAT dual screening, wherein the hybridoma cell line has the parental cell hereditary character, is insensitive to G418 and HAT combined screening, can be cryopreserved permanently and can infinitely amplify in vitro, and rare disease gene mutation cell whole genome is stored in the hybridoma cell line; storing a mutant gene in the hybridoma cell line, storing in hybridoma cell line in a hybridoma cell bank and utilizing infinite reproduction of the hybridoma cell to copy a rare disease gene. Therefore, a rare disease whole genome immortal gene pool can be established, the gene pool can be applied to storing and industrially preparing inherited disease whole genome, and recyclable genome is provided for clinic, teaching, scientific researches, pharmacy and diagnosis.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a hybridoma technology and a method for constructing a rare disease whole-genome immortal gene bank, which is used for cell hybridization, monoclonal antibody preparation, 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 s...

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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 medium due to death, and the confluence of living cells attached to the wall is generally 30-50%, and usually does not reach 80-90% The above ideal confluence; (2) Hybridoma cells adhere to the wall while growing and dying: usually about 1 / 2 to 1 / 3 of the cells adhere to the wall and die, and usually do not reach more than 80 to 90% of the living cells Ideal confluence for adherent growth; (3) Mononuclear cells isolated from human peripheral blood mainly contain lymphocytes and monocytes. The life span of the former is about 7 days, and the life span of the latter is several months or even longer. As a result, there will be the following types of cells, namely effective fusion cells (fusion cells formed by the fusion of mouse sp2 / 0 tumor cells and human gene mutant cells), invalid fusion cells (between mouse sp2 / 0 tumor cells, and between human lymphocytes) , human monocytes, fusion cells formed by fusion between human lymphocytes and human monocytes), unfused cells (rat sp2 / 0 tumor cells, human lymphocytes, human monocytes), in fusion experiments In this method, only effective fusion cells are expected to grow, and the earlier the death of invalid fusion cells and unfused cells is, the more conducive to the growth of effective fusion cells. The prior art is to use HAT to screen mouse sp2 / 0 tumor cells and their mutual fusion cells in 7 to 14 days to die, while lymphocytes, monocytes and their fusion cells all die naturally, there is no screening method, no artificial killing method, that is, unfused lymphocytes and fusion cells formed between lymphocytes need 7 days began to die, unfused monocytes, fusion cells formed between monocytes and fusion cells formed by monocytes and lymphocytes can live for several months or even longer, which is not conducive to the effective growth of fusion cells

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