Antibody targeting interleukin 17a, preparation method and application thereof
A technology of antibodies and antibody drugs, applied in the field of medicine, can solve problems such as unclear downstream signals
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[0138] Antibody preparation
[0139] Any method suitable for producing monoclonal antibodies can be used to produce the anti-IL-17A antibodies of the present invention. For example, animals can be immunized with linked or naturally occurring IL-17A homodimers or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more approaches can be used.
[0140] Any suitable form of IL-17 can be used as an immunogen (antigen) to produce non-human antibodies specific to IL-17A and screen the biological activity of the antibodies. The eliciting immunogen can be full-length mature human IL-17A, including natural homodimers, or peptides containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen...
Embodiment 1
[0172] Example 1 Anti-human IL-17A mouse monoclonal antibody-preparation method of original antibody
[0173] 1.1 Preparation of hybridoma cells that produce murine monoclonal antibodies
[0174] First, human IL-17A protein was used as the antigen, and BALB / c mice were emulsified with adjuvant for multi-point subcutaneous immunization. The serum titer of the immunized mice was monitored. After reaching the requirements, the spleen cells and myeloma (Sp2 / 0) The cells are fused, and the hybridoma polyclonal cells are obtained after HAT screening.
[0175] 1.2 Indirect ELISA-screening method for hybridoma cells
[0176] Use ELISA detection method to screen out highly specific binding polyclones, carry out monoclonal culture, and then use ELISA method to screen highly specific binding monoclonal cell lines; through HT1080 cells to IL-6 release experiment, screen for cell function The effective monoclonal cell line is then analyzed by Biacore method for affinity and half-life, and finall...
Embodiment 2
[0185] Example 2 Cloning of V-gene sequence of anti-IL-17A antibody
[0186] Based on the 5'RACE technology, the DNA sequence encoding the variable region of the mouse antibody expressed by hybridoma 7D6 / 5H8 was determined. In short, the SMART 5'RACE synthesis kit (TAKARA, No. 634859) was used to prepare gene-specific cDNA for the heavy and light chains according to the manufacturer's instructions. The PCR products were analyzed by agarose gel electrophoresis. The variable regions of both the heavy chain and the light chain are approximately 500 base pairs in size. The amplified PCR product with the appropriate band size obtained from the reaction was cloned into the vector pEASY-Blunt Simple plasmid (Beijing Quanshijin, No. CB111-02), and transformed into Stellar E. coli competent cells (TAKARA, No. 636763) )in. Clones were screened by colony PCR with universal M13 forward or reverse primers, and 2-3 clones from each reaction were selected for DNA sequencing analysis. Use Ex...
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