Human Calcium Binding Protein S100A9 Gene Cloning Expression With Purified And Study The Role Of SH-SY5Y Cell

Objective: To construct the human S100A9(rh-S100A9)recombinant prokaryotic expression plasmid pET28a-S100A9.To induce the expression of the protein in different conditions,isolate and purify the two sources of rh-S100A9 proteins both from suspernatant and inclusion body.To compare whether both sources of rh-S100A9 proteins have different biologic effects on the neuroblastoma SH-SY5 Y cell lines,and preliminarily explore the probable mechanism.Method: Gene synthesis was used to achieve the S100A9 prokaryotic expression recombinant plasmid pET28a-S100A9 and then single enzyme digestion method and Polymerase Chain Reaction(PCR)were used to dentify the construction.Isopropylthio-?-D-galactose glucoside(IPTG)was employed in inducing rh-S100A9 protein expression in different conditions,olyacrylamide gel electrophoresis(SDS-PAGE)and Western blot were employed to show whether the rh-S100A9 was expressed in different conditions.Nickel affinity chromatography method was applied respectively to isolate and purify these two sources of rh-S100A9 protein,and then lyophilized after dialysis.CCK-8 cell viability test was used to detect the effect on SH-SY5 Y neuroblastoma cell viability of the rh-S100A9 derived both from supernatant and inclusion body.AO/EB double staining,flow cytometry,cell cycle analysis and ROS assay were used to detect the proliferation inhibition mechanism.Result: Olyacrylamide gel electrophoresis(SDS-PAGE)and Western blot showed the rh-S100A9 was abundantly expressed in the supernatant in 18?,and in the inclusion body in 37?.The CCK-8 proliferation inhibition test results showed that the rh-S100A9 protein inhibited the growth of SH-SY5 Y cells dramaticly(P<0.05)in low concentration(0.05 mg/mL),but no significant difference between the two sources of rh-S100A9.AO/EB double staining and flow cytometry showed that both proteins promote the apoptosis of SH-SY5 Y cells.Comparison about apoptosis between the two sources of proteins also showed there was no significant difference.The cell cycle detection showed SH-SY5 Y can be blocked in G2 / M phase by S100A9.The ROS test results showed that S100A9 may activate the reactive oxygen species in SH-SY5 Y cells.Conclusion:In our experiment,we successfully obtained adequate amount of the recombined S100A9 protein from supernantant and inclusion body,further more,these two sources of protein show consistent biological effect on SH-SY5 Y cells proliferation inhibition and it has an relationship with cell apoptosisas for its working mechanism.S100A9 can inhibite proliferation of SH-SY5 Y through apoptosis and obviously blocked the cell division in G2 / M phase,and can lead to increased intracellular ROS.