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Preparation and Identification Truncated Protein of Testis Cancer Antigen OY-TES-1 Carboxy-terminal
Author: XuLiXiang
Tutor: XieXiaoZuo
School: Guangxi Medical University
Course: Learning organizations learn and Embryology
Keywords: OY-TES-1 Fusion protein Clone Purification
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 12
Quote: 0
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Abstract
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Objective: To construct OY-TES-1 carboxy -terminal truncated protein (OY-TES-1-C) the recombinant plasmid , in vitro expression of OY-TES-1-C protein , purification and identification of the expressed product , OY-TES-1 lay the foundation for future research . Methods: (1) extraction of human testis tissue RNA , reversal of the cDNA was synthesized ; ( 2 ) Amplification of the OY-TES-1 carboxy terminal 253 (S291-G543) amino acids of the cDNA sequence ; (3) The PCR amplified product was inserted into the prokaryotic expression vector pMAL-C2, and to construct the recombinant plasmid pMAL-C2-OY-TES-1-C , and transformed into DH5α bacteria to amplification of the recombinant plasmid pMAL-C2-OY-TES-1-C ; (4) by blue-white screening . DNA sequencing to sieve out the correct recombinant plasmid pMAL-C2-OY-TES-1-C , the recombinant plasmid was transformed Rosetta bacteria ; (5) The recombinant bacteria were induced with IPTG , and optimize the use of IPTG concentration and add timing , and induction temperature and induction time ; (6) expression of the fusion protein MBP - OY- TES- ??1- C , purified by affinity column of Amylose-resin ; under optimized conditions (6) the purified MBP-OY-TES-1 protein -C Western Blot identification . Results: The recombinant plasmid pMAL- C2 - OY -TES - 1 - C sequencing with the same known sequence ; successfully induced to express the fusion protein MBP - OY- TES- ??1- C . PMAL-C2-OY-TES-1-C expressed in vitro optimal conditions : 37 ° C and cultured with shaking for 3 hours, adding IPTG (final concentration of 0.7 mmol / L ) and cultured for 4 hours and then shaken at 32 ℃ ; expressed expected MW found a fusion protein . Conclusion: successfully constructed a recombinant plasmid pMAL-C2-OY-TES-1-C , MBP-OY-TES-1-C fusion protein expressed by E.coli .
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