Clone,Expression And Purification Of Cancer Testis Antigen Psp32

Objective:In order to exploit and utilize the Psp32 antigen and to evaluate its role in diagnosis and therapy of malignant tumor,we amplified and then recombined and expressed Psp32,then purify and identify the expression product.Methods:(1)Extract the total RNA of human testis,synthesis to cDNA by reverse transcriptase;(2)The cancer testis antigen Psp32 gene was amplified by reverse transcriptase polymerase chain reaction(RT-PCR)techniques;(3)then it was inserted into the expression vector pMAL-C2.The recombinant vector was transformed into E.coli DH5 a.The right recombinant clone was selected by a serial of assay,such as blue-white select,PCR,incision enzyme and DNA sequencing.(4)The precise clone was transformed into TB₁ and induced by IPTG in the optimal condition.(5)The expression products was analyzed through the SDS-PAGE method to search the best induction conditions.(6)After the induced bacteria broken by the ultrasound,purify the supernatant through the Amylose-resin column can obtain the purified MBP-Psp32.Then identify the expression product by protein mass chromatographic analysis.Result:The.recombinant clone pMAL-C2/Psp32 was identical with the knowing sequence by DNA sequencing,and succeeded induce the expression of the interest protein MBP-Psp32.Determined the optimal induced condition that was firstly shaking 3 hours in 37℃,,then in 30℃for 5 hours with the final concentration 0.3mmol/L of IPTG.The fusion protein was identified by SELDI-TOF-MS.Conclusion:The expression vector pMAL-C2 is suitable for recombining and expressing the gene Psp32,It is important for us to do the foundation for the postorder research of Psp32.