The Extraction And Charactastic Reseach Of Glycoprotein From Secretory Cells Of Changbaishanensis Oviduct

Rana c. Changbaishanensis is a subspecies of Rana temporaria chensinensis David,it is the only valuable and economic rana that used in medicine, nutrition and food in the world. The dried femal rana oviduct in preovulatory of Rana c. Changbaishanensis is called rana oil (oviductus rannae). Rana oil is another nutrition product after ginseng and cartialgenous,it has effect on invigorating the kidney, profit sperm,moistening lung to mnourish yin. It also has an effect on resisting tired and regulating body endocrine secretion.So it is called "soft gold" in medical science. physiological activity.Oviduct glycoprotein is a kind of special protein that secreted by oviductal epithelium, it has an high content in oviduct during ovulation period,fertilization and early embryonic development.Many evident has indicated that oviduct glycoprotein has an important function on fertilization, early embryonic development, sperm-egg fusion, and diagnosing and curing genital system disease of mammal or amphibian. but the report about rana chensinensis oviduct glycoprotein hasn't seen.In this paper, using saturated ammonium sulfate salt fractionation on Rana c. Changbaishanensis oviduct glycoprotein, it can determine the composition of Rana c. Changbaishanensis oviduct glycoprotein crude extract by adopting SDS-PAGE electrophoretic. The separation, purification of Rana c. Changbaishanensis oviduct rinse adopted DEAE Cellulose-52 ion-exchange chromatography and Sephadex G-100 sieve chromatography. After that the sterling Rana c. Changbaishanensis oviduct glycoprotein can be get.Then undertaking the qualitative analysis of it. It can provide support to glycoprotein comparision of Rana c. Changbaishanensis oviduct secretory cell in vitro culture. So the study has an important significance on protecting wild rana resources, promoting ecological balance and wild rana resources persistence utilization. The content and results of this study were acquired as follows:1. Cultivating oviduct secretory cell of Rana c. Changbaishanensis.By shear, tear treatment oviduct of Rana c. Changbaishanensis. Centrifugation to remove fallopian tubes to collect the supernatant into the package is a good culture flasks at 37℃incubator for culture. When the cells were covered flask, the further expansion of cultivation, collected cells after culture medium to expand.2. The separation and purification of Rana c. Changbaishanensis oviduct glycoproteinThe experiment determined, under oviduct secretory cell of Rana c. Changbaishanensis in the optimal glycoprotein salt of 70% ammonium sulfate saturation, getting the most accumulation is 0.3mg/100mg,and salting collection. After the precipitate centrifuged supernatant by SDS-PAGE electrophoresis to determine the existence of protein and molecular weight of 116kDa, and the same to Rana c. Changbaishanensis oviduct protein content. Using isoelectric focusing electrophoresis, the resulting is:p1 is pH=5.2. Due to high level of hydrophily nature of glycoprotein chains, it is recommended to using DEAE Cellulose-52 ion exchange column chromatography, as well as further purification of glycoprotein isolated by using Sephadex G-100 molecular sieve chromatography, finally using SDS-PAGE electrophoresis to determine glycoprotein purity, and the resulting is molecular weight of 116kDa.3. The result of qualitative analysis of glycoprotein, which is produced by cultivating oviduct secretory cell of Changbaishan Rana.(1) Ultraviolet with the wavelength:Qualitative analysis of glycoprotein, which is produced by cultivating oviduct secretory cell of Changbaishan Rana,Sample purified would be scanned completely by ultraviolet with the wavelength coverage from 200nm to 900nm, the sugar features more than 200 run Feng,280 run in absorbing the protein;(2)Infrared spectrum: we would also process the sample by using infrared spectroscopic analysis within the wavelength coverage of 4000-400 cm-1,the same is getting glycoprotein.