| The optic nerve, like most mature central nervous system(CNS) pathways, does not regenerate after injury. Like most CNS neurons, mature retinal ganglion cells (RGCs) fail to regenerate injured axons beyond the injury site. Instead, more than 90% of all RGCs undergo apoptosis after an intraorbital optic nerve crush (ONC).Thus, how to delay the degeneration of RGCs , activate the regeneration procedure of injured axons, make them survive and regeneration long axons has been the focus of ophthalmology and CNS research field. Recent studies have found that activated macrophages in eyes have significant effect to promote axonal regeneration on RGC. Oncomodulin, which is expressed and secreted by macrophage, is considered the most important mediator to this effect. Further, oncomodulin promotes axon regeneration through the inhibitory environment of the mature optic nerve in vivo and also acts upon peripheral sensory neurons. However, the exact mechanism of regenerative effect of oncomodulin is still unclear. At present, both at home and abroad have not been reported. Inland, there are still no reports about the research of structure and nerve regeneration function of oncomodulin. Therefore, it can provide some experimental evidence for the study on the new ways of nerve regeneration to doing related studies on oncomodulin.In this study, we obtained OM gene from activated rat macrophages by RT-PCR, constructed pET22b(+)-OM prokaryotic expression vector and successfully expressed the protein. This study plays foundation for further purification , activity study and mechanism study of promoting axonal regeneration on OM.Objective:1. To clone the OM gene, construct prokaryotic expression plasmid and identify it.2. TO induce OM expression and identify.Methods:Experiment 1 : Construction and identification of prokaryotic expression plasmids with rat oncomodulin gene1. Select SD Rat, than extract rat peritoneal macrophages to culture and activation. The cells were identified by macrophages specific antibody CD68.2. Total RNA was extracted from activated macrophages, and design primers to obtain whole length of oncomodulin gene by RT-PCR. The result was identified by 1% agarose gel electrophoresis.3. The oncomodulin gene was cloned into pCU57 vector .Then the gene was inserted into pET-22b(+) expression vector and identified.Experiment 2: Expression and identification of rat oncomodulin gene prokaryotic expression vector in E.coliPET-22b(+)-OM recombinant plasmid was transformed into E.coli BL21,induced by IPTG, and observed expression product by SDS-PAGE, then identified by western-blot.Result:1. OM gene was been seen by 1% agarose gel electrophoresis. It is about 346bp length, just consistent with the expected size of the target gene.2. Recycled the destination gene OM and connected it with the intermediate vector PUC-57, then transformed it into E. coli DH5a. The positive clones about 500bp length were obtained by PCR examination of bacterial colony, which were consistent with the expected size. Extracted the PUC-57-OM plasmid, then it was confirmed being consistent with the expected sequence by DNA sequencing.3. The PUC-57-OM plasmid was cut by endonuclease, then connected with PET- 22b(+) vector. The connection product was transformed into E. coli DH5a. Extracted the PET-22b(+)-OM plasmid, then it was confirmed being consistent with the expected sequence by identification of endonuclease and DNA sequencing.4. PET- 22b (+)-OM recombinant plasmid was transformed into E.coli BL21. The protein expression was induced by IPTG. The analytic result of SDS-PAGE showed that the protein exists mainly in soluble form. Then the protein was identified as OM by western blot.Conclusion:It provides a practical and feasible way to obtain OM that rat oncomodulin was successfully expression in Prokaryotic cells. This study plays foundation for follow-up experiments including further purification, activity study and mechanism study of promoting axonal regeneration on OM. |
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