| Objective:In order to research the biological activities of human osteoblast-stimulating factor, the study was aimed at inducing P. pastoris GS115/pPIC9k-osf-1to express recombin ant human osteoblast-stimulating factor.Identifying and purifying the protein,analysing its biological activity.Methods:1.The expression of human osteoblast-stimulating factor①Selecting individual colony of P. pastoris GS115/pPIC9k-osf-1from YPD medium into Buffered Glycerol-compl ex Medium,The culture of bacteria was in30℃until OD600of Buffered Glycerol-complex Medium was4.②The culture was centrifuged at room temperature, The bacteria was suspended in Buffered Methanol-complex Medium to ensure the OD600of Buffered Methanol-complex Medium was1.③The bacteria was induced in25℃,Adding methanol into the Medium every24h to keep the concentration of the Medium is1.0%,Collecting the fermentation broth in0h、12h、24h、36h、48h、72h、96h、120h.Collecting the supernatant after centrifugation,Analyzing the expression of the protein by SDS-PAGE.④The protein was transfered into PVDF membrane by wetting transfer after SDS-PAGE,The transfer condition was100V for1h, PVDF membrane was in block buffer for1h in room temperature,Adding first antibody of Goatpolyconal to OSF-1into the buffer,The PVDF membrane was incubated in room temperaturefor1h in the first antibody solution,Then PVDF membrane was washed3times.Adding second antibody of Peroxidase-Conjugated AffiniPu re Rabbit Anti-Goat IgG(H+L)into the buffer,The PVDF membrane was incubated in room temperature for1.5h in the second antibody solution,Then PVDF membrane was washed3times,Adding DAB/Nicl2in the buffer with development in dark environment.⑤Analysing the concentration of protein in supernatant by Bradford.2.The purification of human osteoblast-stimulating factor expressed by recombin-ation yeast①Collecting the fermentation broth in optimal time,The fermentation broth was first purified by ultrafiltration.②Then the supernatant was dialysed against refolding buffer (20mM Na-Phosphate, pH7.6,) at the temperature of4degree for20hr with changing the buffer every4hr.③Washing SP-Sephadex C-504times with double distilled water and refolding buffer.④Degassing of resin and filling the column,The supernatant was applied to the SP-Sephadex C-50column, equilibrated with refolding buffer at the rate of1.Oml/min, The column was washed with refolding buffer and bound protein was eluted with phosphate-buffered salt solution (20mmol/Lphosphate buffer,2.0M NaCI,pH7.6),Collectingthe protein by Ep pipe.⑤Taking the washed out protein40ul, analysing the protein by discontinuous SDS-PAGE,Collecting the goal protein according the SDS-PAGE result.⑥Ultra-filtered the goal protein at the speed of10000rpm/h for20min.The elution was analyzed by SDS-PAGE and Western-blot after ultrafiltration.The protein was analyzed by Bradford.3.The biological activity assay of purified human osteoblast-stimulating factor Measuring the cell proliferation by CCK-8①The medium contained10%fetal bovine serum, The negative group added medium with nothing,The concentration of hrOSF-1in experimental group was added at0.1μg/mL、1.0μg/mL、10.0μg/mL,The concentration of PTH positive group was0.041178μg/mL②Making MC3T3-E1cell suspension (5x103cells/ml),The cell suspension was seeded into96-well plates,The volume was about100ul,The cells were cultured for24hours without any drug additions.③Then the cells was in different concentrations of hrOSF-1、PTH、negative control in plates,The plates were incubated at37℃for3days, during which the plates were exchanged every24hr,Watching the cell growth everyday.④In the third day adding10uL CCK-8into the medium,The plates was incubated for3hours,To observe the cell proliferation of the protein to MC3T3-E1,OD value was measured by ELISA in450nm after incubation.Measuring the cell differentiation by PNPP①Makeing MC3T3-E1cell suspension (2x104cells/ml),The cell suspension was seeded into24-well plates,The plates were incubated at37℃for24huors without any additions②Then adding different concentrations of OSF-1、PTH、negative control into plates,The negative group added medium with nothing,The concentration of hrOSF-1in experimental group was added at0.1μg/mL、1.0μg/mL、0.0μg/mL,The concentration of PTH positive group was0.041178μg/mL,the volume was400ul,the plates were incubated at37℃for3days,during which the plates were exchanged every24hr,Watching the cell growth everyday③In the third day soaking up the mediun in the plate with PBS washing2times,Adding200ul TritoX-100in the plate, cells was splitted for 50minutes.Sucking the cell lysis、phenol standard application liquid、ouble distilled water into96-well plates,The volume was30uL,adding50ul matrix and loading buffer into plates with water bath for15min.④To observe the cell differentiation of the protein to MC3T3-E1OD value were measured by ELISA in492nm after adding chromogenic reagent to test ALP activity,The volume of chromogenic reagent was150ul.Results:1.A special stripe was appear near the molecular weight of18kDa in SDS-PAGE, The stripe was corresponded to the prediction, the optimal time of the most expression was96hr. The protein was proved to be OSF-1by Westem-blot,The concentration of primary supernatant was86.27μg/mL by Bradford.2.Collecting the fermentation broth in optimal time,the fermentation broth was purified by SP-Sephadex C-50with ion-exchange chromatography. A obvious stripe was appear near the molecular weight of18kDa in SDS-PAGE,The protein was proved to be OSF-1by Westem-blot,The purity of the protein can reach above98%through using the purifying method.3.Cell proliferation of MC3T3-E1tested by CCK8show OD value of hrOSF-1group was greater than negative control(P<0.05),OD value in hrOSF-lwas time dependence,In cell proliferationOD value in the concentration of1.0μg/mL was highest in the various concentrations of OSF-1,It was higher than the PTH group.Cell differentiation of MC3T3-E1in the aspect of ALP activity tested by PNPP showed hrOSF-1controls in various concentrations of OSF-1were higher than negative control,but lower than PTH control. OD value in hrOSF-1controls was time dependence.ALP activity was increased with the concentration of hrOSF-l,but increment in the concentration of1.0μg/mL was more obvious than the other two concentrations,Although OD value in the concentration10.0μg/mLcontinued increasing,but slower.Conclusions:In this study pichia pastoris yeast expression system was successfully induced to expressOSF-1,OSF-1had wanted bio-activity.It layed the foundation for further research of anti-osteoporotic activity and industrialized exploitation... |
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