Anti human RANKL monoclonal antibodies developed by PAE technology and uses thereof
A technology of antigen and antibody fragments, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, and resistance to vector-borne diseases, etc. It can solve the problems that immunotherapy cannot be used, and achieve good curative effect and Safety and cost reduction effects
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Embodiment 1
[0080] Example 1: Panning for human RANKL-specific variable region fragments
[0081] (1) Whole human combination Antibody library construction
[0082] Using more than 3,000 blood samples from blood donors from different provinces and ethnicities, a super-large-scale antibody library was constructed by referring to the methods provided in the following literature. The phage lysate prepared from the constructed antibody library was diluted to 10E10 with YT medium containing 7% DMSO, divided into 1 ml small portions, and stored at -80°C for future use.
[0083] 1. Hoogenboom HR, and G Winter, 1992, By-passing immunisation: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J MolBiol, 227(2): 381-388.
[0084] 2. Griffiths AD, SC Williams, O Hartley, IM Tomlinson, P Waterhouse, WL Crosby, RE Kontermann, PT Jones, NM Low, TJ Allison, TD Prospero, HR Hoogenboom, A Nissim, JPL Cox, JL Harrison, M Zaccolo, E Gherardi, G Winter, 1994, Is...
Embodiment 2
[0117] Example 2: Preparation of recombinant monoclonal antibody protein
[0118] 1. Preparation of endotoxin-free plasmid DNA: Inoculate 100 ml of LB medium with bacteria carrying phCMV-II / 3K7F5L and phCMV-II / 3K7F5H plasmids, and prepare plasmid DNA with Ultrapure Plasmid DNA Purification Kit from Qiagen.
[0119] 2. Transfection and culture of CHO cells: The plasmid DNA prepared above and LipoFamine2000 of Invitrogen Company were used to transfect mammalian cells, and the transfection process was carried out by the method provided by the manufacturer. Alternatives such as PEI35000 can also be used.
[0120] 3. Transfection conditions: (1) Add CHO DHFR(-) cells (ATCC No. CRL-9096) to 5 ml of freshly prepared Ex302 medium (purchased from Sigma-Aldrich) to a final density of 2×10E5 cells / ml. After incubation at 37°C for 48 hours, the cells were collected by centrifugation at 450×g for 10 minutes. (2) Add 1ml of fresh DMEM medium and gently tap the bottom of the tube to resusp...
Embodiment 3
[0123] Embodiment 3: the expression of Fab form and the preparation of recombinant protein
[0124] (1) Construction of expression vector
[0125] The DNA fragment with the above-mentioned SEQ ID NO: 1 and SEQ ID NO: 3 was cloned into pCOM3H vector (Wu SC, Lin YJ, Chou JW, Lin CW.2004, Construction and characterization of a Fab recombinant protein for Japanese encephalitis virus neutralization. Vaccine. 25, 23(2): 163-71). The 5'-light chain with NheI and NotI sites was digested with NheI / NotI and ligated into pCOM3H precut with the same combination of enzymes. Then the heavy chain with XhoI and SpeI was digested with the same combination of enzymes and ligated to its XhoI / SpeI site. After transforming Escherichia coli DH5α competent cells, detect and isolate single clones on agar plates containing IPTG / X-gal and ampicillin, and confirm the selected clones by enzyme digestion. A correct clone was inoculated into 500ml LB medium containing ampicillin for 6 hours, and then in...
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