A kind of preparation method of mouse anti-human kiaa0100 protein monoclonal antibody
A monoclonal antibody and mouse technology, applied in anti-animal/human immunoglobulin, instruments, measuring devices, etc., can solve problems such as inability to express proteins, inability to clarify subcellular localization, and hinder biological functions
Active Publication Date: 2021-08-27
THE SECOND AFFILIATED HOSPITAL OF XIAN JIAOTONG UNIV
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Problems solved by technology
Due to the lack of efficient and specific antibodies, it is impossible to study the expression of human KIAA0100 protein, and the subcellular localization of human KIAA0100 protein cannot be clarified, which also hinders the study of its biological function
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Embodiment 1
[0032] Experimental method:
[0033] 1.1 Expression of recombinant protein antigens:
[0034] 1.1.1 Problem of nuclear expression codon optimization:
[0035] Use bioinformatics software Optimum TM Codon (http: / / www.genscript.com / codon_opt.html) The prokaryotic expression codon optimization was performed on the human KIAA0100 protein fragment (1557-2234).
[0036] The amino acid sequence of recombinant protein antigens is SEQ ID NO: 1.
[0037] 1.1.2PET30A-KIAA0100 (1557-2234) Construction and Identification of recombinant plasmid:
[0038] After the nucleotide sequence of recombinant protein antigens, Nanjing Jinsi Biotechnology Co., Ltd. was subjected to sequence synthesis, and the NDEI and HindIII enzyme sinks were cloned into the PET30A vector, and the PET30A-KIAA0100 (1557-2234) recombinant plasmid was constructed. The recombinant plasmid was converted to E. coli BL21 (DE3) induced cells, and positive clones were selected using LB solid medium containing 100 μg / ml kanamycin...
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Abstract
The invention provides a method for preparing mouse anti-human KIAA0100 protein monoclonal antibody by adopting lymphocyte hybridoma technology. The antibody lays a foundation for detecting the expression of human KIAA0100 protein and studying the structure and function of human KIAA0100 protein in the future. Using the antibody prepared in the present invention, we detected the expression of human KIAA0100 protein in U937 cells by western blot analysis method; the subcellular localization of human KIAA0100 protein in U937 cells was clarified for the first time in the world by immunocytochemical analysis method. The results showed that the human KIAA0100 gene had two protein products with molecular weights of 254 kDa and <250 kDa in U937 cells; in some U937 cells, the human KIAA0100 protein was mainly localized on the cell membrane; in the cell membrane and cytoplasm. In summary, the mouse anti-human KIAA0100 protein monoclonal antibody prepared by the present invention can efficiently and specifically recognize human KIAA0100 protein.
Description
Technical field [0001] The present invention relates to a method of preparing a mouse anti-human kiAa0100 protein monoclonal antibody. Background technique [0002] The use of lymphocyte hybridoma techniques to prepare monoclonal antibody techniques were first created by MILSTEIN and Kohler in 1975, and monoclonal antibodies prepared by this technique have been widely used in the field of diagnosis and treatment of biology and diseases. The monoclonal antibody can be used to detect the expression of the antigenic protein, analyzing the structure of the antigenic protein, detecting the structural relationship between the antigenic antibody, and the like. [0003] The basic principle of lymphocyte hybridoma technology is to use cell fusion, screening, and other cell fusion agents (or electrical fusion, laser fusion) such as polyethylene glycol cells and antigen-induced B cells (or electrical fusion, laser fusion), such as polyethylene glycol. And established hybridoma cell lines th...
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IPC IPC(8): C07K16/18G01N33/68G01N33/577
Inventor 张王刚崔鹤蒙昕古流芳高洁刘宇宏
Owner THE SECOND AFFILIATED HOSPITAL OF XIAN JIAOTONG UNIV