The Isolation, Purification And Screening Of The Tumor-prevention In Vitro Of Glycoprotein From Chlorella Pyrenoidosa

Cklorella pyrenoidosa is man-cultured health supplement contained kinds of compounds.The hot water extracts(CVE) and power of Chlorella pyrenoidosa have been reported to possess various pharmacological effects including antitumor activities and potent biological response modifiers that modulate immune responses against bacteria and viruses,and it mainly is the glycoprotein extracts that play an important role.The study on Chlorella pyrenoidosa focused on the extracts and its functions in vivo,but the report on the and tumor-prevention is rare.Traditional methods of screening the drug for antienoplastic in the light of dieases and compounds suited only to random screening.In order to use this resource,the isolation,purifition of glycoprotein from Chlorella pyrenoidosa were carried and a rapid,sensitive,high throughout and cheap screening method in vitro was constructed to study the tumor-prevention of glycoprotein from Chlorella pyrenoidosa.It will be of scientific significance in researching and developing the new synthesized and natural tumor-prevention.The glycoprotein from Chlorella pyrenoidosa were isolated and purifed firstly. The technology and condition of glycoprotein extraction from Chlorella pyrenoidosa were studied.Water extraction was used to extract glycoprotein,the optimum extraction conditions of glycoprotein were chosen by considering the effect of each factor and subsequently doing response surface experiment.The better condition of glycoprotein extraction is:the ratio of raw material to water is 1:21.9,extracting temperature is 86.0℃and extracting time is 5.0 h;Sevag method was used to remove protein.The optimum conditions was chosen by considering the effect of each factor and subsequently doing orthogonal experiment,and the better condition of removing protein is:the ratio of sample liquid to Sevag liquid is 2,the ratio of chloroform to 1-butanol is 4,removing protein times is 3,the time of removing protein is 15 min.The crude glycoprotein was further purified by Sephadex G75 gel,dialysis, concentration.After vacuum freeze-drying,two white flocculent purified glycolproteins were obtained.Elution patterns on sephadexG-200 column chromatography proven CGP I was homogeneous and CGPⅡwas not homogeneous.The results of HPLC indicated the purity of CGPⅠand CGPⅡwere 98.35%and 78.84% respectively.Chemical composition of CGPⅠwas studied.The results of SDS-PAGE of CGPⅠshowed a single spot and the molelular weight is 63,700;CGPⅠcontains 17 amino acids and two acidic amino acids(Asp and Glu) is higher using amino acid analyzer;Lack of absorption at 260.00nm by UV scaning indicated that CGPⅠcontained no nucleic acid.It was proved that the main monosacchrides were glucose, galactose and xylose by gas chromatography.The type of the glucosidic bonds was pyranose by IR and O-glycosidic linkage was found byβ-elimination reaction.The antioxidant activities in vitro of glycoprotein from Chlorella pyrenoidosa were researched through determinations of reducing power and scavenging hydroxyl free radical,superoxide free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical,the results showed that the different concentrations CGPⅠhad stronger reducing power and scavenging effect on superoxide free radical,hydroxyl free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical in a concentration of dose-dependant manner.The scavenging capacity of CGPⅠin the hydroxyl free radical was stronger than Vc,but the scavenging capacity to superoxide free radical, hydroxyl free radical,1,1-diphenyl-2-picrylhudrazyl radical and alklyl radical was lower than Vc.A transgenic cell model was established to screen preliminarily in vitro the tumor prevention of glycoprotein from Chlorella pyrenoidosa.The TK promoter was amplified from plasmid pRL-TK of recombinant PCR and the SacⅠenzyme site was added.Then the 500 bp and 150 bp segments obtained from the first round of the recombinant PCR and the 635 bp segment from the second round PCR were both identified then cloned into pMD18-T vector.The TK promoter was amplified from it and cloned into pEGFP to construct pTK-GFP,the ARE enhancer was inserted into the upstream of TK to construct the vector pARE-TK-GFP.At last the TK and ARE-TK segments were amplified and cloned into pEGFP-N1 so the eukaryotic expression vectors pTK-GFP/Neo and pARE-TK-GFP/Neo were constructed.The two vectors were transfected into HepG2 cells and clones resistant to 800μg/ml G418 were isolated and named as HepG2-TK-GFP and HepG2-ARE-TK- GFP of which the green fluorescence was obvious under the fluorescent microscope.The different concentration(0.32,1.6,8,40,200μg/ml)of glycoprotein was added into the transgenic cells and the PDTC and lentinan were as controls.The result indicated that the best concentration is 200μg/ml and the fluorescence intensity has dosedependency with the different concentrations of glycoprotein in a certain range while the controls have not.In the study,a glycoprotein from Chlorella pyrenoidosa was obtained,and it anti-oxidation activities was proved;According to the the antioxidant mechanism of tumor-prevention,A green fluorescent protein(GFP) transgenic cell model under the transcriptional control of TK promoter and ARE was constructed to evaluate the tumor-preventive activity of glycoprotein from Chlorella pyrenoidosa.The glycoprotein could induce the cis-expression of ARE and promote the fluorescence intensity of GFP,and the induced level of GFP have dose-dependency with the concentrations of glycoprotein.So,the tumor-prevention of the glycoprotein from Chlorella pyrenoidosa was made sure preliminarily.