Recombination,Expression And Purification Of Cancer/Testis Antigens GAGE7 Protein And A Exploratory In Lung Cancer Patients Serodiagnosis

ObjectiveTo study a method to recombinate and express the gene segment of Cancer/testis -associated antigen-GAGE7 in Yeast.Find out the suitable conditions under which the fusion protein can be expressed most efficiently.Then provide a basis for the production and application of this antigen.And we detect the human specific autoantibodies against GAGE family proteins by ELISA to provid an evidence which the GAGE are considered as promising targets for immunotherapy and serodiagnosis for lung cancer.Methods1.Expression,purification and renaturation of GAGE7 protein.The gene segment of GAGE7 gene was amplified by PCR(Polymerase Chain Reaction),and then inserted into the vector PYES2/NTA at the downstream of the gene encoding the HIS-protein.The recombinant vector was transfected into E.coli DH5α.The anti-ampicillin selection assay,agarose electrophoresis,incision enzyme assay and DNA sequencing were performed to find out the right recombinant clone.The right clone was transfected into the expression host(DBY746),and the engineering bacterias induced by 2%IPTG for 0,6,12,16,24,36 hours were collected and total protein was extracted.The protein products was analyzed throught western methods to find out the best induction time under which the engineering bacterias can obtain the highest expression efficiency.After the induction bacterias broken by CelLytica^TMY Yeast Cell Lysis/Extraction Reagent and purifing the supernatant throught the HIS-select^TM-resin to obtain the purity HIS-GAGE7 protein.2.The establish of ELISA method to diagnose lung cancer after coating wells with renaturated GAGE7 protein,the serum from the patients with lung cancer serum and the healthy people were detected by indirected-ELISA and the results were analyzed by statistics.ResultsThe gene segment of GAGE7 gene have been successfully inserted into the vector PYES2/NTA at the multiple clone site,and the interest protein was expressed when the engineering bacterias induced by IPTG.When the engineering bacterias induced in 30℃,the production of the fusion protein is highter.As theTime is added,the production of the fusion protein increase in the first 18 hours,but drop after it.After the induction bacterias broken by CelLytica^TMY Yeast Cell Lysis/Extraction Reagent and purifing the supernatant throught the HIS-select^TM-resin to obtain the purity HIS-GAGE7 protein, the ELISA results analyzed by statistics indicate the AD₄₅₀ from different tectotype lung cancer groups compared to the healthy controls is higher and significant in the statistics(P＜0.01);the AD₄₅₀ fromâ…¢-â…£stage lung cancer groups compared to theâ… -â…¡stage lung cancer groups is higher and significant in the statistics(P＜0.01).Conclusion1.The expression vector PYES2/NTA can be used to recombinate and express the protein segment of GAGE7 gene in Yeast.Added the glucose(the final concentration is 0.1%) and the sucrose(the final concentration is 0.1%) to the culture medium and the first OD of yeast is 0.1.added the inductor-IPTG(the final concentration is 2%)after the OD of Yeast step up 0.3,then under the temperature to 30℃and induced 18 hours. Crushing the induced Yeast with CelLyticAa^TM Y Yeast Cell Lysis/Extraction Reagent in ice bath,with the protease inhibitor-PMSF and glass bears added in it,then purified the supernatant throught the HIS-select^TMresin column in 4℃.All these can help to improve the production and the purity of the dissoluble interest protein.2.The GAGE7 protein showed excellent immunoreactivity and it was recognized by human specific autoantibodies against GAGE family proteins;These results indicate these GAGE antigens can be used as promising molecular mkarers for early diagnosis and possible tgarets for immunotherapy of lung cancer.