OX40 antibody as well as preparation method and application thereof
An antibody and affinity technology, applied in the field of OX40 antibody and its preparation, can solve the problems of small toxic side effects and high antibody titer
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[0304] The preparation method of the nucleic acid is a conventional preparation method in the art. Preferably, it includes the following steps: obtaining the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining the nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis .
[0305] Those skilled in the art know that substitutions, deletions, alterations, insertions or additions can be appropriately introduced into the base sequence encoding the amino acid sequence of the above protein to provide a polynucleotide homologue. The homologue of the polynucleotide in the present invention can be prepared by replacing, deleting or adding one or more bases in the gene encoding the protein sequence within the scope of maintaining antibody activity.
[0306] carrier
[0307] The invention also provides a recombinant expression vector comprising the nucleic acid.
[0308] The recombinant expression v...
Embodiment 1
[0378] The preparation of embodiment 1OX40 antibody
[0379] 1.1 Preparation of Immunogen A
[0380] The nucleotide sequence containing the amino acid sequence Leu29-Ala216 encoding the extracellular domain of the human OX40 protein (the human OX40 protein sequence is derived from the Uniprot database, and the protein sequence number is P43489) was cloned into the pTT5 vector carrying the human IgG Fc fragment (hFc) And prepare the plasmid according to the established standard molecular biology method, the specific method is referring to Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). The prepared plasmid was transiently transfected into HEK293 cells (polyetherimide (PEI), purchased from Polysciences) and expanded for culture. After 4 days, the cell culture fluid was collected, and the cell components were removed by centrifugation to obtain the culture su...
Embodiment 2
[0397] Example 2 Production and Purification of Lead Antibody
[0398] The concentration of antibodies produced by hybridoma cells is low, only about 0.1-10 μg / mL, and the concentration varies greatly. Moreover, various proteins produced by cell culture in the culture medium and fetal bovine serum components contained in the culture medium have varying degrees of interference with many biological activity analysis methods, so small-scale (1-5 mg) antibody production and purification are required.
[0399] The hybridoma cells obtained in Example 1 were inoculated into T-75 cell culture flasks, and expanded with DMEM medium containing 10% FBS. When the growth state is good, the cell culture spinner bottle is inoculated, and the production medium (Hybridoma serum free medium, purchased from Invitrogen Company) containing 2.5% FBS (Low IgG) is used. Add 500mL of production medium to each 2L culture spinner bottle, and inoculate the cell density at 1.0×10 5 / mL. Close the bottle...
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