Construction And Identification Of Glial Fibillary Acidic Protein Prokaryotic Expression Vector

[objective] To perform gene clone to the whole gene of GFAP, and to construct and identify the prokaryotic expression plasmid. [Methods] Isolated mRNA from human brain glioma tissue was subjected to RT-PCR to obtain GFAP fragment, then express, link it with prokaryotic expression plasmid pGEX-4T-2 to express and purify GFAP on the basis of E.Coli BL21.Finally identify the expected protein by method of Western-blot. [Result] After construction of expectant recombinant plasmid, the pGEX-4T-2-GFAP was digested with restriction enzyme, and identified with DNA sequencing and Western-blot, so as to confirm its construction. [Conclusion] Successful construction of GFAP in prokaryotic expression vector could provide basis for further study of genetic engineering vaccine effectively.BACKGR0UND:Glial Fibillary Acidic Protein (GFAP) plays an important role in the nerve injury.0BJECTIVE:To perform gene clone to the whole gene of GFAP, and to construct and identify the prokaryotic expression plasmid.DESIGN, TIME AND SETTING:The single sample experiment was performed at Lab in Chang Zheng Hospital from September to November 2008.MATIERIALS:The pGEM-T Easy vector was provided by Promega Corporation, the original expression plasmid pGEX-4T-2 was from Lab in Chang Zheng Hospital.METH0DS:Isolated mRNA from human brain glioma tissue was subjected to RT-PCR to obtain GFAP fragment, then express, link it with prokaryotic expression plasmid pGEX-4T-2 to express and purify GFAP on the basis of E.Coli BL21.Finally identify the expected protein by method of Western-blot.MAIN OUTCOME MEASURES:Identification of total RNA; PCR amplification product; enzyme digestion of recombinant expression vector; Western-blot.RESULTS:After construction of expectant recombinant plasmid, the pGEX-4T-2-GFAP was digested with restriction enzyme, and identified with DNA sequencing and Western-blot, so as to confirm its construction.C0NCLUSION : Successful construction of GFAP in prokaryotic expression vector could provide basis for further study of genetic engineering vaccine effectively.