| BackgroundOncomodulin (OM) is a kind of micromolecule protein, It was found by Macmanus in the end of 1970s, and was extract from rat,s hepatoma tissue. It was certified that OM could promote the generation of nerve, especially to optic nerve.ObjectiveTo assembe the fusion gene OM/tP (oncomodulin/truncated protamine) in vitro and construct and express this fusion gene in an expression vector.MethodsTotal RNA was refined from Macrophage which is extracted from rats edabdominal cavity, Three oligonucleotide primers were designed and used to amplify the OM gene, Coding sequence of tp was added in the 3'primer termination, The OM/ tp gene was amplified by PCR and cloned into middle expression vector PUC57, we digested the positive cloned plasmid with restriction endonuclease and connected it with prokaryotic expression vector PET22b, transformed it to E coli DH5a, sieved positive clone, extracted plasmid with plasmid mini kit, After that it was sent to be identified with sequence analysis .Finally , the OM/ t P was expressed in E coliBL21 The OM/ t P protein was detected in SDS-PAGE and western-blot.ResultsThe correct target band was obtained by agarose gel electrophoresis. After the cloned gene was connected with PET22b, transferred to E coli DH5a, the amplified target bands at 500 bp were exhibited with the consistent size with expected one. The sequence is complete coincidence with the designed sequence.compare to the bacterium without inducing, different concentration IPTG induced bacterium, the target bands at 18.4KDa appeared in the supernatant of lysate. the band exhibited with the consistent size with expected one. The two different induced concentration have little influence to the expression of the protein. SDS-PAGE and Western blot analysis displayed the detected protein was the protein encoded by the OM/tp gene.ConclusionThe OM/ t P gene can be successfully constructed and expressed, which could be used for the optic nerve generation study...
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