Cloning And Prokaryotic Expression Of Endothelial Monocyte Activating Polypeptide Ⅱ Gene From Silkworm，bombyx Mori

Edothelial monocyte activating polypeptideⅡ(EMAP II) was decomposed as anendothelial and monocyte-activating polypeptide from proEMAP II, based on its ability to inducetissue factor in endothelial cells and monocytes and to evoke chemotactic migration ofblood leukocytes and monocytes. EMAP II has been widely used in traeting tumorsexperiment because it was identified as an anti-angiogenic tumor vasculature in vivo andcould promote apopotosis.In order to produce more activity EMAP II,we cloned andexpressed EMAP II form B. Mori,using prokaryotic expression system and discuss thedifferences in transcription level in this research.We firstly designed specific primers according to the sequence information frompublic B. mori genomic and EST databases, then cloned the silkworm BmEMAP II cDNAand DNA sequence. A sequence of1186bp including3extrons and2introns with an870bp open frame, a ORF of which encodes a289-aa polypeptide with a predicted molecularmass of about32kDa.BmEMAP II was probably a hydrophilic protein because there wasno obvious transmembrane domains and signal peptide. The protein might be subcellularlylocalized somewhere different from the mitochondria or extracellular.TheNeighbor-Joining(NJ) evolutionary tree suggested that the distance between the silkwormB. mori and the Chilo suppressalis was the nearest, while BmEMAP II was far away fromthose of Homo spaiens and Mus musculus.Meanwhile, the total RNA of female silkworm Bombyx mori was extracted to analyzethe expression of edothelial monocyte activating polypeptideⅡin different developmentalstages and tissues, then reversely transcribed and synthesized the first strand cDNA byM-MLV. Using the18s RNA of silkworm Bombyx mori as a reference gene, we carried thesemi-quantitative PCR. The BmEMAP II expressed difference in different stages andtissues because it was existed in all tissues except the moth of body wall and wings in thepupal.The ORF was ligated to pET-28a(+) vector for prokaryotic expression by IPTGinduction. Western blot analysis proved that we succeeded in obtaining the recombinantBmEMAP II protein.induced by IPTG under different concentrations. Through SDS-PAGEelectrophoresis and Western blot analysis, the induced fusion protein was successfullyexpressed. This will provide the foundation for further study on eukaryotic expression andfunction of edothelial monocyte activating polypeptideⅡ gene.