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The Extraction and Charactastic Reseach of Glycoprotein from Secretory Cells of Changbaishanensis Oviduct

Author: XuZuo
Tutor: LiuJingSheng
School: Jilin Agricultural University
Course: Aquatic Products Processing and Storage Engineering
Keywords: Changbai Mountain Rana Fallopian tube secretory cells The extraction and purification of glycoprotein Qualitative Analysis
CLC: S966.35
Type: Master's thesis
Year: 2011
Downloads: 61
Quote: 0
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Abstract


Changbai Mountain forest frog (Rana chensinensis Changbaishanensis) is mainly distributed in China's Changbai Mountain in Northeast and Xiaoxing'anling region, belonging to a subspecies of Chinese forest frog has important medicinal value and health tonic. The dry goods preovulatory female animals tubal commonly known the frog oil (toad oil), is following ginseng, deer horn after another nourishing the development and utilization of health care products, has Bushenyijing, the effect of the lungs Yin. Tubal glycoproteins by oviduct epithelial cells synthesize and secrete specific proteins involved in constitute tubal microenvironment important role in the reproductive physiology of mature gametes before fertilization, fertilization, and early embryonic development, and to varying degrees by the body hormone regulation. However, the extraction and separation of the Changbai Mountain forest frog the tubal secretion of cell culture product glycoprotein has not been reported. The study of the the Changbai Mountain Rana fallopian tube secretory cells cultured in vitro. With different saturation ammonium sulfate salting oviduct secretory cell culture supernatant, the culture solution of the protein is isolated after electrophoresis method determined for the glycoprotein, and finally with DEAE Cellulose-52 anion exchange column chromatography and Sephadex G-100 gel filtration purified glycoproteins isolated, pure glycoprotein, study its biological characteristics, which can protect wild Rana resources, promote ecological balance, achieve wild Rana sustainable use of resources, etc. are of great significance, as well as to provide a reliable test for the artificial production of oil Rana and biopharmaceutical basis. In this thesis, the content and the results are as follows: 1. The Changbai Mountain Rana tubal secreting cells cultured using cut, shredded treatment of the Changbai Mountain Rana tubal organization. Oviduct tissue was removed by centrifugation, collecting the supernatant into the package is a good flasks were cultured in a 37 ° C incubator. The confluence culture bottles, further expand the culture, the cell sap collected after expanding culture. 2 Changbaishan Rana oviduct secretory cell culture product of the separation and purification of glycoproteins experimentally determined The Changbaishan Rana fallopian secreting cell culture product glycoprotein salting optimum ammonium sulfate saturation of 70%, the resulting protein precipitate maximum for 0.3mg/100mg collected after salting precipitate was centrifuged, the supernatant by SDS-PAGE electrophoresis to determine the presence of protein and a molecular weight of 116KDa flushing with Changbai chensinensis oviduct lumen same liquid for sugary protein. Using isoelectric focusing electrophoresis method of determination of the isoelectric point (pI) of the glycoprotein results: the p workers for pH = 5.2. Then the isolated glycoprotein was purified using DEAE Cellulose-52 anion exchange column chromatography and Sephadex G-100 molecular sieve chromatography, and finally, the purity of the protein by SDS-PAGE and identifying the sugar, and a molecular weight of 116kD. Changbai Rana oviduct secretory cell culture product glycoprotein Qualitative Analysis of Results (1) UV Full scan: the purified sample in the 200nm-900nm wavelength range ultraviolet full scan derived 200nrm at polysaccharide characteristic peaks at 280nm protein absorption peak, and thus can be qualitatively sugar protein in oviduct secretory cell culture fluid; (2) Infrared Full scan: qualitative sugar protein in oviduct secretory cell culture medium in the range of 4000-400 cm-1 for infrared full scan.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > Other aquaculture > Frog breeding > Rana
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