Study On The Separation And Purification Of Gecko Anti-tumor Protei

ObjectiveGecko is a traditional Chinese medicine,which has clear curative antineoplastic effect in clinic applications.Our previous results showed that aqueous extracts of fresh gecko,the macromolecular fractions(Peak 1)separated from fresh gecko by Sephadex G-25,0.1 M NaCl component obtained from Sephadex G-25 peak 1 through DEAE-cellulose DE-52 weak anion exchange chromatography,0.5 M(NH4)2SO4 component obtained through penyl sepharose fast flow all had strong anti-tumor effect in vivo.The purpose of this subject is to isolate and purify the anti-tumor activity protein from gecko.Methods1.Separation and purification methods of anti-tumor component from geckos(1)Gel filtration chromatography(Sephadex G-25):Sephadex G-25 was used for the purification of gecko coarse extraction liquid with high purity water.(2)Ion exchange chromatography:DEAE cellulose DE-52 weak anion exchange medium was used for the purification of Sephadex G-25 peak 1 with 0.1 M NaCl solution,0.2 M NaCl solution,0.3 M NaCl solution and 0.5 M NaOH solution.(3)Hydrophobic interaction chromatography:Phenyl agarose FF hydrophobic medium was used for the purification of 0.1 M NaCl component with 1.0 M(NH4)2SO4 mother solution,0.5 M(NH4)2SO4 mother solution,0 M NaCl mother solution,high purity water and 0.5 M NaOH solution.(4)Native polyacrylamide gel electrophoresis(Native-PAGE):0.5 M(NH4)2SO4 component was further purified by Native-PAGE at the condition concentrated glue(5%)voltage 100 V,separation glue(6%)voltage 200 V.(5)75℃ heat treatment:0.5 M(NH4)2SO4 component was further purified by 75℃heat treatment for 10 min,and then the supernatant was collected after centrifugation with 10000 r·min-1 for 15 min.(6)Ultrafiltration:75℃ heat treatment component was further purified by Amicon Ultra-50 mL with molecular mass cut-off were 50 kDa and 100 kDa.And the sample was separated after centrifugation with 3000 g·min-1 for 15 min.(7)Ion exchange chromatography:Source 15Q strong anion exchange medium was used for the purification of 75℃ heat treatment component with 0.05 M NaCl solution,0.1 M NaCl solution,0.2 M NaCl solution,0.5 M NaCl solution and 0.5 M NaOH solution.And the molecular weight of vary components were cut from 50 kDa to 100 kDa with Amicon Ultra-50 mL.2.Methods for screening the activities(1)In vivo:Select H22 tumor-bearing ICR mice for screening the vivo anti-tumor activities.(2)In vitro:The proliferation inhibition of Bel-7402 cell was determined by MTT experiments.And the morphological changes of Bel-7402 cells were observed directly using an inverted microscope.3.Methods for the determination of protein concentrationThe Bradford method was used for determining the concentration of protein.4.Methods for the component analysis of the gecko sampleChoose the SDS-PAGE electrophoresis and Native-PAGE electrophoresis to analysis the protein of sample.ResultsBase on the above methods,purification and activity screening test results are as follow:1.Native-PAGE electrophoresisFour parts of the gel were cut down for the MTT test.They were referred to as upper,upper-middle,lower-middle and lower parts.The MTT experiments showed that the tumor inhibition rate of the four parts were 5%,49.27%,78.42%and 13.16%respectively.But the further purification gave up this method because of its poor repeatability.2.75℃ heat treatmentThe inhibitive rate of tumor of 75℃ heat treatment component in vivo was 43.99%,and had significant difference(P=0.013<0.05)compared with control group.But it had no significant difference(P=0.854>0.05)compared with 0.5 M(NH4)2SO4 component.The results of SDS-PAGE electrophoresis and Native-PAGE electrophoresis showed that 75℃heat treatment component had less strips compared with 0.5 M(NH4)2SO4 component.Above all,the results showed that the anti-tumor component may be a heat resistant substance.This method was used in the further study.3.UltrafiltrationThe three parts of ultrafiltration were collected for the vivo test.The inhibitive rate of tumor of<50 kDa component,50 kDa-100 kDa component and>100 kDa component in vivo were-6.68%,36.87%and 20.98%,and 50 kDa-100 kDa component had significant difference(P=0.018<0.05)compared with control group.The MTT test also testifid 50 kDa-100 kDa component had an effective anti-tumor effects in vitro,and the inhibition rate of proliferation was increased with the concentration of protein from 10 μg·mL-1 to 100 μg·mL-1.4.Source 15Q strong anion exchange chromatographyThe method of linear gradient elution of Source 15Q strong anion exchange chromatography had no significant effect,and the method of phase elution was used in the further study.The inhibitive rate of tumor of 0.05 M NaCl component,0.1 M NaCl component,0.2 M NaCl component and 0.5 M NaCl component in vivo were 35.67%,24.32%,35.04%and 5.01%respectively.SDS-PAGE electrophoresis and Native-PAGE electrophoresis had less strips.The results showed that the anti-tumor substance was in 0.1 M NaCl component.ConclusionThe gecko crude extract was effectively purified by gel filtration chromatography,DEAE cellulose DE-52 weak anion exchange chromatography,hydrophobic interaction chromatography,Native PAGE gel electrophoresis,75℃ heat treatment,ultrafiltration and Source 15 Q strong anion exchange chromatography.And the anti-tumor component may be a heat resistant substance.SDS-PAGE electrophoresis has less strips and a comparatively single active protein was isolated.Speculate the range of the anti-tumor substance molecular weight is 50 kDa-100 kDa.