| AIM: Monoclonal antibody HAblS is a high affinity and specific anti-hepatoma antibody. It has a great advantage and potential in human hepatoma carcinoma diagnosis and therapy. At present, it is the only one mAb that being approved into phase II clinical test in China. But mAb HAblS itself has no therapeutic function and the radioisotope linked HAblS is not broadly accepted by patients and physicians. So developing high specific therapeutic antibodies having the same target as HAblS is a new research direction. Construct HAbISO/CD 147 prokaryotic expression vector and try to get enough HAbl8G/CD147 protein so that can be used into developing antibodies is the primary aim of this project. Whether HAbl8G/CD147 protein expressed by prokaryotic expression system has immunogenicity or not is the key question, because only the expressed protein with immunogenicity that can be used screening new antibodies. So detecting the immunogenicity of HAbl8G/CD147 expressed by prokaryotic expression system is the second aim of the project. Finally, the natural HAbl8G/CD147 protein play an important role in infiltration and metastasisof hepatoma cells, as it can induce fibroblast cells around the hepatoma tissue secreting MMPs (Matrix metalloproteinases). MMPs have the ability of destroying local histological construct and basement membrane barrier. However, the mechanism of HAbl8G/CD147 inducing MMPs and other functions are not clear. So doing the functional experiments about the purified HAbl8G/CD147 expressed by prokaryotic expression system will help us understand the role of it in metastasis and lay the foundation of further research of this protein. Methods: The experiments includes four parts1. Construction of prokaryotic expression vector of HAbl 8G/CD147gene. The full length cDNA of HAbl8G/CD147 was obtained by PCR method, recombinant vector pBluescript/HAblSG was the template. The enzyme site BamH I and Xho I were designed in primers. The fragment of the cDNA cut by BamH I and Xho I was linked with the prokaryotic expression vector pRSET-C cut with the same enzymes. The constructed vector was identified by restriction endonucleases digesting and sequencing.2. High efficient expression of HAbl8G/CD147 gene in E.coli BL21(DE3), purification and identification of fusion protein.The E.coli BL21(DE3) was transformed by the recombinant prokaryotic expression vector pRSET-C ?HAblSG. The protein was induced by IPTG (0.5m mol/1), the control were the expression induced without IPTG and the E.coliBL21(DE3) transformed by the empty vectorpRSET-C. The expression products were identified by SDS-PAGE and Laser Thin Layer Scan. 3. Purification and identification of HAb 18G/CD147 fusion protein.The expression products were purified by Ni-NTA affmity-chromatography. The purified protein was further identified by SDS-PAGE.4 . Functional experiments of HAbl8G/CD147 prokaryotic expression protein in vitro.4.1 ELISA assay of purified fusion proteinDilute the protein from 1:250 concentration in 96 well plate as the antigen. Add HAb 18 as the first antibody and enzyme linked goat-anti-mouse antibody as the second antibody. Colorize with OPD, read data by ELISA assay machine.4.2 Gelatin Zymography of the purified fusion proteinFive groups of cells were designed :(l)fb;(2) HHCC; (3) HHCC+fb (4)purified fusion pratein + fb; (5)positive control group (purified eukaryotic expression protein +fb). Collect the culture supernatant, abandon cells debris by gradient centrifugation, collect sedimentation after deposition and desalt. 10 u 1 samples individually was add to SDS-PAGE electrophoresis (the glue contained 0.1% gelatin). After electrophoresis, elute, incubate, stain discolor in turn.Results:1. Construction of prokaryotic expression vector of HAbl8G/CD147.1.1 Electrophoresis showed a 1.7kb band by cutting PCR products and a 3.0kb band by cutting vector pRSET-C.1.2 Electrophoresis showed a 1.7kb band and a 3.0kb band by cutting vector pRSET-C ?HAblSG wit...
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