Construction and expression of prokaryotic expression plasmids with rat oncomodulin gene

Abstract

Objective To construct and express the prokaryotic recombinant vectors for rat oncomodulin gene, and to identify their expression. Methods Peritoneal macrophages were extracted from six selected SD rats and activated. Total RNA was extracted from activated rat macrophages,and design primers to obtain whole length of oncomodulin gene by RT-PCR. The oncomodulin gene was cloned into pUC57 vector. Then the gene was inserted into pET-22b(+) expression vector and and the inserting plasmid was transformed into Escherichia coli. host DH5α. The positive clone was characterized by PCR examination of bacterium liquid pET-22b(+)/OM and DNA sequence analysis.Then the recombinant plasmids were transformed into E. coli BL21 and the expressions of recombinant plasmids were induced by adding isopropylthiogalactoside (IPTG). The expression product was identified by Western blot assay. Results The target DNA sequence of oncomodulin was obtained by RT-PCR which was about 350 bp length, exactly in accordance with prospective. The positive clones about 500 bp length were obtained by PCR examination of bacterial colony pET-22b(+)/OM plasmid, which were consistent with the expected size. The result of DNA sequence analysis for recombinant plasmids extraction revealed that the gene encoding oncomodulin was coloned into vector pET-22b(+)accurately. The protein bands with the molecular weight of about 11.7 kD were induced by IPTG in the recombinant plasmids. Western blot assay revealed that the protein was OM. Conclusion The prokaryotic recombinant vectors for rat oncomodulin gene is successfully constructed and expressed. It might provide a foundation for further study on the functional test of oncomodulin that can promote regeneration of damaged optic nerve. Key words: Oncomodulin; Cloning; Gene expression; Rats