Construction Of Transgenic Cell Model For Screening The Cancer Chemoprevention Of Glycoprotein Extract From Chlorella Pyrenoidosa

It is the first time to report that the glycoprotein extract from Chlorella pyrenoidosa have chemopreventive activity in china. The crude glycoprotein was extracted with hot water and precipitated by ethanol after deproteinization, the best extraction process and conditions were chosen by optimize the effect of four factors as follows: the ratio of raw material to water, temperature, extracting time and extracting times. Because the phaseⅡenzyme play an important role in preventing cancer , the promoter of the enzyme gene has DNA motifs called anti-oxygen response elements (ARE) which are very conservative and can regulate the expression of the down stream genes. Based on this principle, we constructed the eukaryotic GFP reporter vector pARE-TK-GFP/neo under the transcriptional control of TK promoter adjacent to which ARE enhancer was inserted and the control vector pTK-GFP/neo. The two vectors were transfected into HepG2 cells and clones resistant G418 were isolated. After that the different concentrations of glycoprotein were added into the 96-well plate in which transgenic cells were seeded, 48h later the different induced levels of GFP were measured to indicate the effect of the glycoprotein. The results were as follows:1. The optimum extraction process and conditions were chosen by considering the effect of each factor and subsequently doing orthogonal experiment: the ratio of raw material to water is 1:20, extract two times under the temperature of 70℃and each times with 6h. This is the best condition to extract glycoprotein because the obtained sugar components were the highest. The homogeneity was examined with Sephadex-200 column chromatography and SDS-PAGE, and the molecular weight was established to be about 63.7kDa on SDS-PAGE.2. The TK promoter was amplified from plasmid pRL-TK of recombinant PCR and the Sac I enzyme site was added. The 500bp and 150bp segments obtained from the first round of the recombinant PCR, the 635bp segment from the second round PCR were 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, they were both identified by PCR. 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, which were identified by enzyme cutting and sequencing.3. 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. The green fluorescence was obvious under the fluorescent microscope. The different concentration of glycoprotein was added into the transgenic cells and the known chemopreventive agents PDTC and lentinan were as controls. The result indicated that the best concentration is 200μg/ml and the fluorescence intensity has dose-dependency with the different concentrations of glycoprotein in a certain range while the controls have not.This study primary constructed transgenic cell model which can induce the phaseⅡenzyme. It was not only provides a basis for the screening of natural or synthetic cancer chemopreventive agents, but also provides experimental evidence for the Chlorella pyrenoidosa developed as chemopreventive agents.