The Study On Function Of Red Protein S Expressing In Eukaryotic Cell With Biophysis Method

Nowadays, with the ending of human genome project, people pay more attention on investigation of the function various genes, which has become major mission in the field of life science , and establish a new subject named functional genomics. However, it is difficulty to study the function of genes only depending on information from construction genomics, if we want to learn about the function of certain gene, firstly, we must investigate the phonetype of the organism which is absence of this gene or in which this gene has been mutated in certain functional structural domain. This type organism is usually called gene modify organism that is constructed by gene targeting technology, therefore, gene targeting technology is a essential method and technology plat during studying the function of gene.Gene-targeting technology is a kind of genetic manipulation altering genetic information, production and development of which bases on homological recombination technologies and stem cell technologies. Studies using gene-targeting have proven invaluable in research of gene function, but which is limited in manipulation. As far knock out technology that is in the forefront of this approach, which engineers a targeted gene deletion or mutation in modeanimals resulting in inactivation of gene expression and change of phenotype. However, in many case, particular genes take some important role in embryonic development, inactivation of which will result in embryonic abortion. Therefore, it is necessary to establish a system that can conveniently and quickly knock out target gene in vitro in cell level, by which we can study the gene function in cell level, on the other hand, Up to now, Cre/LoxP still is unique system in constructing genetic modify animal, which knock out or knock in target gene depending on natural recombination. However, homological recombination rate in eukaryotic cell is so far lower than it in prokaryotic cell. Therefore, it is a hot problem needed resolving urgently to acquire a rapid homological recombination system, which can be manipulated easily.recent researches show that Red homological recombination system from A, phage has been used successfully to accelerate the process of gene recombination in prokaryotic organism. If this system could be used in eukaryotic organism, it would accelerate significantly the process of gene targeting and decrease the cost of constructing gene modify animal. At the same time, gene targeting technology could be used extensively in all field of life science. However, it is until today that is still in the stage of theory research, related research has not been reported yet.To study the function of Red gene in the eukaryotic cell, it is necessary to identify the expressive and distributive characteristic of Red protein in eukaryotic cell. This study is to work out this problem, which we elucidate from two parts: First, to prepare anti-Red Antiserum, Red recombinant protein encoding gene (gam, bet and exo) were amplified from A. phage genome by PCR and cloned into expression vector pDH2. high expression engineering bacteria of Red recombinant protein was constructed; three proteins were induced by raising the temperature. Use these proteins to immune rabbit to prepare corresponding antiserum, which was further purified by binding with the whole proteins of E.coli cell, The titer and specificity of polyclonal antibody were detected by Western blotting; Then we detect the characterisric of Red proteins expressed in eukaryotic cells by immunofluorescence technology.