ABSTRACT
An optimized two-dimensional polyacrylamide gel electrophoresis (2-DE) system for analyzing plant proteins was developed by evaluating different reagents and concentrations used in the sample extraction solutions and lysis buffers. Two main sample preparation methods, referred to as trichloroacetic acid (TCA)-acetone method and phenol extraction-ammonium acetate/methanol (phenol-NH4Ac/methanol) precipitation method, were compared. Four ecotypes of reed plants (Phragmites communis Trin.) from the desert region of north-western China were used as experimental materials: (1) swamp reed (SR) which grows in water about 1 m deep; (2) dune reed (DR) which grows on 5～10 m high sand dunes; (3) heavy salt meadow reed (HSMR) which grows on low-lying salt flats; and (4) light salt meadow reed (LSMR) which grows in the transition area between DR and HSMR growing areas. The optimized phenol-NH4Ac/methanol precipitation method consisted of extracting leaf proteins of different ecotypes of reed with water-saturated phenol and then precipitating with a 5-fold volume of 0.1 mol/L NH4Ac in methanol, followed by dissolving in the lysis buffer. The optimized protein lysis buffer consisted of 7 mol/L urea, 2 mol/L thiourea, 4% CHAPS, 2% Ampholine(pH 3.5～10∶pH 5～8 = 1∶4) and 65 mmol/L DTT. The prepared protein sample (80 ?g) was then separated by 2-DE gel and detected by silver staining method. This improved 2-DE system resulted in a 2-D protein profile of higher resolution and higher protein yields as analyzed by PDQuest software. Good results were also obtained when this 2-DE system was used in 2-D analysis of proteins from other plant materials, such as rice leaves, indicating that it is a suitable 2-DE system for analyzing leaf proteins of different plant species.
ABSTRACT
·AIM: To investigate the preparation of endostatin protein and its biologic activity on vascular endothelial cell.· METHODS: pBlast-hEndostatin and pBlast-Mcs were identified by digesting with Nhe Ⅰ and Sal Ⅰ, by PCR reaction, by sequencing, and by Alignments of PCR products with gene bank using NCBIBLAST software. The identified pBlast-hEndostatin as well as pBlast-Mcs were then purified with QIAGEN Endofree plasmid maxi kit.The purified plasmids transfected human fibroblasts. The expression of endostatin was detected by RT-PCR, Westem-Blot and immunohistochemistry. The endostatin prorein produced by transfected fibroblasts was purified by ultrafiltration and affinity chromatography. The inhibitory action of endostatin on human umbilical vein endothelium was measured by MTT assay.· RESULTS: pBlast-hEndostatin was found to contain human endostatin gene. Endostatin protein was produced by transfected fibroblasts. The inhibitory ratio of 2.5,5,10,20,40,80mg/L endostatin on human umbilical vein endothelium for 48h were 8.5%,13.1%,27.7%,38.1%,56.7%,63.8% respectively. IC50 value was 34.5mg/L.No inhibition action was found on fibroblasts.·CONCLUSIONS: Endostatin protein can be produced by the transfected fibroblasts. The produced endostatin has inhibitory action on human umbilical vein endothelium and has no inhibition action on fibroblasts.