Transient Transfection Of Human Gastric Cancinoma Cell Line SGC7901 With Sense And Antisense Expression Vector Of β3GalT7 Gene And Comparative Proteome Analysis Of The Models

Object: UDP-gal :beta GlcNAc beta 1,3-galactosyltransferase polypeptide 7(β3GalT7) which is a new member of glycosyltransferases belongs to theβ-3 glycosyltransferase family which had been first electro-cloned by our lab. This thesis aims to analysis the proteome of human gastric cancinoma cell line SGC7901 in whichβ3GalT7 is moderate-expressed ,and by constructing 2-DE profiles of up-regulation and down-regulation subcellular models and setting up the platform of the Comparative proteome research, to look for the proteins of which the expression will change with the variation ofβ3GalT7, all in all, made the first step in the research of the function ofβ3GalT7。Methods:1β3GalT7 positive-sense expression vector pEGFP-C1-T7s and antisense expression vector pEGFP-C1-T7 as which are preserved by our lab run double digestion by restriction enzymes, PCR and sequencing identification2 Plasmid which packed by cationic liposome is used to design a gradient transfection system, to follow the principle of selecting the highest transfection efficiency and lowest cell death rate in order to optimize the transfection condition.3 Construct positive-sense and antisense expression vector. Use the optimal transfection system to transfect SGC7901. 24h after transfection we drew the RNA, use RT-PCR to identify the change ofβ3GalT7 on transcriptional level. 48h after transfection we drew the total protein, use Westernblot to identify the change ofβ3GalT7 on protein level.4 Use two-dimentional gel electrophoresis, image scanning and analysis system of Amersham corperation. Choose rational PH and length range of the IPG strips. Optimize the lyse method, the quantity of loading sample, electrophoresis condition and staining method. Construct 2-DE profiles ofβ3GalT7 up-regulation and down-regulation subcellular models and human gastric cancinoma cell line SGC7901.5 Apply ImageMater 2D Platinum5.0 to analysis 2-DE profiles of those protein samples. To analysis suited protein spot groups by Groups report , fix quantity of protein spots use Vol% normalization and screen those protein spots whose expression quantity had prominent changed.Results:1. The sense-expression vector pEGFP-C1-T7s and the antisense-expression vector pEGFP-C1-T7as which conserved in our laboratory are all constructed rightly except a few spots mutation.2. Compared the transfection efficiency of 6 transfection system, accoding to the optimizing principle of the highest effiency and the lowest cell death rate, we chose the transfection proportion:plasmid : 0.75μg/1*10^5cells ,liposome: 1.5μl per 1*105cells.3. The results of RT-PCR and WesternBlot all indicated thatβ3-GalT7 were up-expressed in the subcellular models which had been trsfected with sense-expressed vector pEGFP-C1-T7s, and on the contrary ,β3-GalT7 were down-expressed in the subcytic model which had been transfected with antisense-expressed vector pEGFP-C1-T7as .The two models were successfully constructed.4. First of all , we Used 11cm IPG strip of pH =3-10 IPG gel in Isoelectric Focusing Electrophoresis(IEF), the quantity of loading sample was 200μg, Coomassie brilliant blue stained profiles indicated that the distribution of protein spots focus on the range of PH=3.8-7.3, the effect of isolation showed not so good. Then selected 24 cm dry IPG strip of pH 4-7,draw the Coomassie brilliant blue stained profiles and the silver stained profiles respectively, the result showed that the protein spots focused totally ,there was no obvious cross strips or upright strips, it can be used for downstream.comparative proteome analysis5. The gray level analysis of 2-DE profiles suited protein spots group of transfection group and control group indicate that there were 22 protein spots had prominent change, 7 of which up-regulating and the other 15 down-regulating. We had got the isoelectric points and molecular weight of these protein spots.Conclution: Under optimal transfection condition, we successfully constructed human gastric cancinoma cell line SGC7901 up-regulation and down-regulation subcellular models, and identification results of PT-PCR and WesternBlot are same. At the same time we constructed the technique platform of two-dimentional gel electrophoresis and got the 2-DE profiles with good repeatability and dissociative effect. We got the information of protein spots by Proteomics analysis of subcellular models. Further study should be carried out by mass spectrum identification.