Preparation And Identification Truncated Protein Of Testis Cancer Antigen OY-TES-1 Carboxy-terminal

Objective: To construct the recombinant plasmid of C-terminal half OY-TES-1 gene(OY-TES-1-C), express this truncated protein in vivo, and provide basis for further study of OY-TES-1.Methods: (1) Total RNA was extracted from normal human testis, and cDNA was synthesized by the reverse transcriptase. (2) The fragments of OY-TES-1-C (containing 253 amino acids of C- terminal half) gene was amplified by Polymerase Chain Reaction (PCR). (3) PCR product of OY-TES-1-C was inserted into the expression vector pMAL-C2. The recombinant plasmid was transformed into E.coli DH5α. (4) The recombinant clone was selected by blue-white selected test, and DNA sequencing. (5) The clone with correct sequence was transformed into Rosetta. (6) The expression of the OY-TES-1-C was induced by IPTG. The expressive optimization of recombinant plasmid including concentration, time, temperature and duration of IPTG was tested; (7) Purification of fusion protein of MBP-OY-TES-1-C was performed by the amylose-resin column, then the purified MBP-OY-TES-1-C was tested by the Western blot .Result: The sequence of recombinant plasmid of C-terminal half OY-TES-1 was identical with its known sequence. The fusion protein of MBP-OY-TES-1-C was successfully expressed. The optimal condition for the expression of MBP-OY-TES-1-C was established, which is first shaking incubation of recombinant clone in 37℃for 3 hours, then adding IPTG (0.7mmol/L), continuous incubation in 32℃for 4 hours. Expressed in line with the expected molecular weight of the fusion proteinConclusion: The recombinant plasmid of C-terminal half OY-TES-1 gene (OY-TES-1-C) was successfully constructed. The fusion protein MBP-OY-TES-1-C was high efficiently expressed. This study provides the basis for further study of OY-TES-1 gene.