Gene immunity preparation method for human I-type deoxythymidine kinase monoclonal antibody

Abstract

The invention relates to a novel process for preparing monoclonal antibody of human I-type thymidine kinase by immuno gene. The concrete method is that RNA is extracted in the cell of Hela which is conducted by aminopterin first, TK1 cDNA is amplified through reverse transcription of PCR, TA is conducted to be DNA sequencing to confirm right gene sequence of TK1 after being cloned and is constructed by subcloning pSecTag2 / Hygro B into the point (pSecTag / TK1) between HindIII and XhlI. Protein which is inserted by expression of segments can be secreted to extracellular receptor from eukaryotic cells. Immunized mice with plasmid DNA is directly recombined by pSecTag / TK1, hybridoma is prepared by fusion of myeloma cells and spleen cells, and the hybridoma of monoclonal antibody with anti-TK1 is screened. Pluralities of TK1 monoclonal antibodies are prepared via mouse ascites. At last, the collected mouse ascites is frozen and centrifugated in high speed, the clear liquid on the mouse ascites is extracted by the method of noctanoic acid-ammonium sulphate and purified by the method of protein G column affinity chromatography, and TK1 monoclonal antibody which can be used to detect TK1 markers in clinical sample.

Description

technical field [0001] The invention relates to a method for preparing human type I thymidine kinase monoclonal antibody by gene immunization, which adopts genetic engineering and molecular immunology technology, through the cloning of human type I thymidine kinase gene and the recombination construction of eukaryotic secretory vector A new method of gene immunization to prepare human type I thymidine kinase monoclonal antibody was implemented. The monoclonal antibody prepared by this method should belong to the product of genetic engineering. Background technique [0002] Technical background about human thymidine kinase type I (hTK1): hTK1, its Chinese name is human thymidine kinase type I, and its Chinese abbreviation is human thymidine kinase type I; its English name is Human thymidinekinase, and the English abbreviation is hTK1; its enzymatic Chinese name is adenosine triphosphate or adenosine triphosphate: thymidine 5-phosphotransferase, EC.2.7.1.21, kinase, and its e...

AI Technical Summary

Problems solved by technology

The tissue TK1 antigen extracted by biochemical methods is not only cumbersome, long-term, difficult, and extremely complicated to purify, it requires a lot of preparation reagents, manpower, material and financial resources, and even if a large amount of tissue can be extracted to obtain TK1, the yield rate is very small. , it is difficult to meet the needs of monoclonal antibody preparation and practical application; although using prokaryotic cells to express TK1 protein to prepare its monoclonal antibody has obvious progress and advantages compared with TK1 extracted from a large number of tissues, it also has the difficulty of purifying TK1 antigen, and the operation cumbersome, technically complex and other issues; especially since TK1 commercial reagents or standard antigens have not been seen in the world at present, the preparation methods and standards of TK1 monoclonal antibodies reported by each cannot be unified, and the specific epitope of TK1 is in Which peptide segment is still unknown

Therefore, no matter what method the purified TK1 (eukaryotic or prokaryotic origin) more or less loses some valuable antigenic determinants, especially TK1 is a multimer under physiological conditions, so the current TK1 monoclonal antibody The research of the hTK1 antibody still needs to be further in-depth. These are some deficiencies, defects and existing problems of the current preparation method of hTK1 monoclonal antibody. Only by overcoming these can it be possible to unify the standard of TK1 antibody and prepare a very suitable TK1 monoclonal antibody