Gene Cloning, Expression And Purification Of Cancer Testis Antigen OY-TES-1 Truncated Protein In N-Terminal Half

Objective:To construct the recombinant plasmid of N-terminal half OY-TES-1 gene(OY-TES-1-N),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-N (containing 268 amino acids of N- terminal half)gene was amplified by Reverse transcriptase Polymerase Reaction(RT-PCR).(3)PCR product of OY-TES-1-N was inserted into the expression vector pMAL-C2.The recombinant plasmid was transformed into E.coli DH5a.(4)The recombinant clone was selected by blue-white selected test and DNA sequencing.(5)The clone with correct sequence was transformed into E.coli TB₁.(6)The expression of the OY-TES-1-N was induced by IPTG.The expressive optimization of recombinant plasmid including concentration,time,temperature and duration of IPTG was tested;(7)The fusion protein of MBP-OY-TES-1-N was tested by the electrophoresis of SDS-PAGE.(8)Purification of fusion protein of MBP-OY-TES-1-N was performed by the amylose-resin column,then the purified MBP-OY-TES-1-N was tested by the Western blot and identified by the mass chromatographic analysis.Result:The sequence of recombinant plasmid of N-terminal half OY-TES-1 was identical with its known sequence.The fusion protein of MBP-OY-TES-1-N was successfully expressed.The optimal condition for the expression of MBP-OY-TES-1-N was established,which is first shaking incubation of recombinant clone in 37℃for 3 hours,then adding IPTG(0.3mmol/L), continuous incubation in 32℃for 4 hours.The result of the mass chromatographic analysis was showed that the fusion protein was identical with target protein.Conclusion:The recombinant plasmid of N-terminal half OY-TES-1 gene (OY-TES-1-N)was successfully constructed.The fusion protein MBP-OY-TES-1-N was high efficiently expressed.This study provides the basis for further study of OY-TES-1 gene.