Study On Expression Of ProPO Gene For Penaeus Monodon In Prokaryotic And Eukaryotic Cells

ProPO gene, playing important role, is the one of the specific immune factors inhumoral immunity for prawn. To establish the proPO vitro expression system willsignificantly improve the exploration of the proPO's function in immune procession.The proPO gene from Penaeus monodon was cloned into pET-28a(+) and expressed inE.coli. Both result of SDS-PAGE and Western blot displayed a special band about 78.5 kDa.The PO activity could be directly detected after the expression was induced at lowertemperature, however the PO activity only could be detected after the process ofdenaturation and renaturation when expression was induced at normal temperature.The proPO gene was cloned into pIZT/V5-His, transformed into Sf9 cell. The greenfluorescence can be observed by fluorescence microscope. After the Zeocin^TM antibioticscreening, the positive rate of the transgenic cells was 70%. That GFP and proPO sequencecan be amplified from the genome of the transgenic cells by PCR, and SDS-PAGE shows aband whose molecular weight is about 67 kDa with PO activity proves proPO has beenexpressed in sf9 successfully.To establish pIZT/ V5-His/Bm-N expression system, pIZT/ V5-His-proPO wastransfected into Bm-N cells. Finally those cells with green fluorescence were obtained. Theresults of PCR detection and enzyme activity assay indicated a novel pIZT/V5-His/Bm-Nexpression system had been set up.