Monoclonal antibody FnAb12 and application thereof
An antibody and carrier technology, applied in the field of biomedicine, can solve problems such as difficulty in obtaining protein molecules
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Embodiment 1
[0162] Example 1 Screening and preparation of monoclonal antibodies
[0163] In this example, the constructed single-chain antibody phage library is screened, and the specific screening method is as follows figure 1 b2M and hFcRn&b2M were first labeled with biotin, and then the biotin-labeled b2M was mixed with the phage antibody library for subtraction screening, and the phage bound to b2M was removed with magnetic beads coupled with avidin protein. Then, the supernatant was mixed with biotin-labeled hFcRn&b2M protein, and the phages bound to hFcRn were collected by magnetic beads. After amplification, the next round of panning was performed. After three consecutive rounds of panning, a single phage was picked for amplification. The positive phages were identified by phage enzyme-linked immunosorbent assay, then sequenced, the genes were cloned into a new expression vector, the antibodies were expressed and purified, and subsequent binding and affinity identifications were pe...
Embodiment 2
[0169] Example 2 Preparation of fusion protein
[0170] The nucleotide sequence expressing GLP1 (7-36 amino acids, containing three mutation sites of A8G, G22E, R36G) was connected to the 5' end of the nucleotide sequence of FnAb8 or FnAb12 with a (G4S)3 linker, and the gene After the sequence was synthesized, it was inserted into the expression vector pcDNA3.1 (GenScript Biotechnology Co., Ltd.), and then the plasmid was transfected into mammalian cells to prepare the protein.
[0171] The amino acid sequence of the GLP1 (AA7-36) protein used in this example is as follows:
[0172] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGG (SEQ ID NO.: 2)
[0173] In this example, GLP1 (AA7-36) was successfully fused and expressed with two single-chain antibodies respectively, and a fusion protein was prepared. Named G12.
[0174] The amino acid sequence of fusion protein G8 is as follows:
[0175] HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGS QAVLTQPPSASGTPGQRVTI SCSGSSSNIGSNSVNWYQQLPGTAPKLLIY...
Embodiment 3
[0178] Example 3 In vitro activity detection of fusion protein
[0179] Surface plasmon resonance (SPR) was used to identify the binding of GLP-1 fusion protein to its corresponding receptor protein. Biacore T100 was used to immobilize GLP-1R-Fc protein and hIgG on a CM5 chip by chemical coupling. 200nM of FnAb8, G8, FnAb12 and G12 respectively flowed over the chip surface at high speed to detect their binding signals.
[0180] Biacore was used to identify the binding of GLP-1 fusion protein to hFcRn. hFcRn was coupled to a CM5 chip, and 200 nM of FnAb8, G8, FnAb12 and G12 were flowed over the chip surface at high speed to detect their binding signals.
[0181] The experimental results in this example prove that G8 and G12 can specifically bind to GLP-1R. And both G8 and G12 can bind to FcRn protein and maintain the pH-dependent binding. image 3 Binding of G12 to GLP-1R-Fc protein is shown. GLP-1R-Fc or hIgG was coupled to a CM5 chip, and 200 nM of G8, FnAb8, G12, and FnAb...
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