| Tubulins are heterodimers composed of two polypeptide chains, a chain and β chain. These two subunits are coded by either highly conservative and dispersed genes or mini-gene family. Morphological observations on numerous cellular and subcellular structures and the analysis on the biochemical features and assembly of tubulins have enriched the content of cytoskeleton system. Recently, with the great progress of molecular genetics, some new acquaintance has been made with the structures, functions, expression and regulation of tubulin gene.Kinesin is plentiful in various kinds of cells, different stages of cell development and multicellular organisms. Most kinesins appear in cytoplasm and some of them are connected to organells, including vesicles and endoplasmic reticulum, which are attached to cell membranes, kinesin is a tetramers, consisting of two heavy chains of 120Kda(α chain) and two light chains of 70KDa(β chain). It is an important motor-protein responsible for the transport of organells and vesicles. In addition to participating in cellular physiological activities, such as the fusion of cell nuclei, generation and dissociation of spindles, migration of chromosomes, kinesin also plays a role in regulating the depolymerizing rate of microtubules, maintenance of its stability, damage and repair of cell membranes.The brain is one of the active organs in energy metabolism. The oxygen consumption of brain is great and the ability of brain to tolerate hypoxia is poor. Severe hypoxia for 3-5 minutes can markedly damage the brain. The function of neurons can hardly be restored after exposure to complete anoxia for 5 minutes. Anoxia for 30 minutes leads to irreversible nerve damage. The cerebral cortex and hypo-cortex visual pathway are especially vulnerable to hypoxia. The poor tolerance of brain to hypoxia is related to the high level of brain ATP, which is synthesized rapidly and utilized quickly within the brain. The studies on the molecular mechanism of hypoxia are of greatsignificance for military forces in highland.The upper stream coding sequence of 3 tubulin is conservative and with satisfactory epitope. Therefore the upper stream coding sequence of tubulin of 325bp was cloned and the expression vector was constructed. The products expressed were employed as antigen to immunize adult rabbits conventionally and the antibody obtained. This makes it possible to study the expression of rat tubulin under hypoxia with immunohistochemical assay. The synthesis and prokaryotic expression of tubulin gene in ratsThe coding sequence of 3 tubulin in 156-480bp upstream was optimized according to the favorable codons of E.coli. The double chains of the sequence optimized were divided into 16 single fragments, F1HF8 and R1-R8. These sequences were chemically synthesized and there were overlapping regions of 20bp between each pair of complement adjacent fragments. F1 and R1 were 19bp long and the others 38bp. The restriction endonuclease sites of Bautt I Hind III were induced into the both ends of Fl and Rl. The expected sequences were obtained by means of artificial synthe'sis and PCR assay. The clone vector and prokaryotic expression vector of 3 tubulin were constructed and the target proteins expressed. The polyclonal antibody against rat tubulin was prepared by immunizing the rabbits with the expressed protein as satisfactory semi-antigen. The prokaryotic expression vector of rat tubulin was successfully constructed and the target protein was expressed by means of genetic engineering and protein recombination. The expressed tubulin and its polyclonal antibody were prepared. And then the expression of rat tubulin was detected in hypoxia and anti-hypoxia condition, which was helpful for the study on physiological changes and corresponding reactions of animals under hypoxia and anti-hypoxia.Effects of Compounded Chinese Medicinal Prescription on Tubulin Expression in Hypoxia RatsThe protein expressed was purified with dialysis and electrophoretic elution. The adult rabbits were immu...
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