| The urokinase-type plasminogen activator(uPA)could hydrolyze plasminogen to plasmin,as its main function,leading to degrade the extracellular matrix (ECM) and basement membrane. uPA also could activate latent collagenase and promote degradation of ECM and vascular basement membrane with plasminogen. In the primary lesion and metastatic lesion of ovarian cancer patients,uPA has a high expression. Melittin is the major component of the Apis mellifera venom. Melittin with a strong hemolysis in the destruction of the cell surface pHospHolipid bilayer and the formation of abnormal ion channels, leading to cell death.Recombinant rhuPAa-melittin has a dual role in the feature, one the hand , melittin release of anti-tumor cells, once rhuPAa-melittin combined with the uPAR in ovarian cancer cells; on the other hand, rhuPAa-melittin could combined with double-stranded uPAR binding sites, as compete with uPA, thus weaken the activity of uPA degrades the extracellular matrix, inhibition of tumor cell adhesion, migration and invasion. Therefore, in this experiment, recombinant rhuPAa-melittin ,provided by laboratory, is taken to establish an efficient pichia pastoris expression system for expression and purification.high purity rhuPAa-melittin protein has been obtained ,then detected activity in normal cells and ovarian cancer. Preliminary investigate rhuPAa-melittin on the treatment of ovarian cancer and its mechanism.In this study, pichia pastoris as a bioreactor of recombinant rhuPAa-melittin is carried out the following tasks:1. rhuPAa-melittin in pichia yeast expression secretion Utilize the lab in rhuPAa-melittin to linear ,and transform rhuPAa-melittin into Pichia pastoris X-33.Identified the yeast genome by PCR,and screened highly effective and stable secretion expression rhuPAa-melittin pichia pastoris strain.Establish expression of rhuPAa-melittin secreted pichia pastoris expression system.2. rhuPAa-melittin in the large-scale fermentation and purification Optimize conditions of large-scale production rhupAa-melittin by using pichia pastoris expression system on fermentation. Fermentation is divided into three pHases: the first pHase training pHase yeast added glycerol, pichia pastoris grew up in the basal medium added PTM1 and biological factors,and increased cell biomass. The second stage row of additional glycerol, glycerol of the medium is consumed, the dissolved oxygen increased.when wet weight of bacteria less than 180g / L, then began to flow plus 50% glycerol, biomass continues to increase. The third stage the induced expression, also known as methanol feeding stage, methanol has been added,leading to induce the expression of recombinant protein. At the beginning,methanol fed slowly, because the cell needs some time to adapt to methanol as a carbon source. At various stages of pHysiological state and the difference of bacterial cell volume cause the difference of demanding for methanol. As the expression of host cell proteins is different, consumption rate of pichia pastoris methanol is also different. purification of rhuPAa-melittin protein divided two steps: purified by the SepHarose SP XL c ation exchange column chromatograpHy and Source TM30RPC sparse inverse chromatograpHy, concentrated rhupAa-melittin in 15% SDS-PAGE to detect purity.3. rhuPAa-melittin bioassayAdding the preliminary purified rhuPAa-melittin in medium which normal cells and SKOV3 human ovarian cancer cells were cultured for 48h, 72h, and then measured at 490nm wavelength absorbance, to reflect the number of living cells,indirectly, reflect on the culture rhuPAa-melittin cell line.In summary: The conditions of rhuPAa-melittin in the fermentation and purification has been optimized successfully.Obtain high-purity rhuPAa-melittin protein and high-yield strain.Detection of normal cells and ovarian carcinoma cells, demonstrated rhuPAa-melittin have strong anti-tumor effect, no killing effect on normal cells. Recombinant rhuPAa-melittin as a targeting treament provides a theoretical and experimental basis.
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