| Glutathione-S-Transferase (GSTs) families, including a group of multiple physiological functional proteins, are an important detoxication enzyme of human biotransformation and mainly locate in the cytosol. With a function of catalysing the reduced Glutathione thiocytidine to combine with lyophobic compounds and change their electrophilic character to hydrophilic ones that can easily excrete from bile and urine. As such kinds as electrophilic compounds, potential toxications and lipophilic compounds were degenerated and excreted by this way, GSTs, therefore, play an important role in the process of toxication metabolism, cell protection and inhibition of carcinomatous change of cell.GSTPi, belonging to n class of GSTs families, is particular abundant in the lung. Having the high action on degenerating the poly aromatic hydrocarbon compounds, it qulifies itself one of the roles of detoxicating carcinogen and preventing cancer generation. Cloning it and developing its functions have been becoming one of the focuses on the cancer defending.In the present study, the cloning, expression and purification of maltose -Tagged Human Glutathione S-Transferase Pi in vitro had been performed. The experiment was composed of several linked processes as the following. Objective:To construct a prokaryotic expressive vector carrying human glutathione-S-transferase Pi gene and make its efficiency expression in E.coli JM109 and purification for research on toxicology and chemoprophylaxis. Methods:1. Specimens collection:The placenta tissues were obtained from patients at surgery in Zhengzhou University first affiliated hospital. The specimens were frozen in liquid nitrogen less than 20min after surgical resection.2. RNA extraction and conservation:The total RNA, extracted from specimens of placenta tissues using RNeasy?Mini Kit produced by Promega Corp, were dissolved by ddH2O (treated with DEPC), respectively, and then preserved at -70 癈 or directly to use.3. cDNA synthesis and human GSTPi gene Amplification:cDNA were synthesed with random primer by using total RNA as template. GSTPi, with template of cDNA, was amplified by polymerase chain reaction (PCR), using the pair of special primers designed according to the cDNA sequence providing by NCBI. The PCR product was identified by agaroseelectrophoresis.4. Constraction of recombination prokaryotic cloning vector pMD 18-T-GSTPi:GSTPi was amplified by PCR using LA Taq DNA polymerase, which added an A on 3 end of PCR product, and made it possible to be directly ligated with the prokaryotic cloning vector pMD 18-T vector by the T4 DNA polymerase at proper ratio. The recombination plasmids were transformated into E. coli. JM109, and the positive clones were selected using ampicillin resistance and blue-white experiment, the white ones were redetected by restriction enzyme digestion and PCR.5. Sequence analysis of GSTPiThe cDNA fragment on the plasmid was sequenced, and then compared with the sequence in GenBank. The DNA sis 2.5 was carried out to analysis the DNA sequence.6. Construction of recombination prokaryotic expression vector pMAL-C2x- GSTPi and expression:Both the GSTPi amplified by PCR and the prokaryotic expression vector pMAL-C2x were digested by EcoRJ and Sail respectively, and the purified genes fragments, collected by Gel Extract Kit after agarose electrophoresis, were ligated by the T4 DNA polymerase at proper ratio. The recombination plasmids were transformated into E. coli. JM 109, and the positive clones were selected using ampicillin resistance and blue-white experiment; the white oneswere redetected by enzyme digestion and PCR.7. The expression, identification, and purification of GSTPi Protein:The expression of GSTPi on pMAL-C2x-GSTPi was induced by IPTG at 37 C and collected after the culture for 1-5 hours. Denaturing the protein and loading protein on SDS-PAGE to locate the target protein, which was glancing identified by comparing to the protein marker, and confirmed by being incubate... |
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