Glioma Associated Antigen MAGE-D4:Preparation Of Truncated Protein And Antibody Serological Survey

Objective:In previous study,we have found that MAGE-D4 was capable of immunogenicity in varies types of cancers with MAGE-D4 autologous antibody induced.However,the antigen,used in detection of serum antibody is full-length MAGE-D4 protein which contains MHD that may affect the antigen-antibody reaction.Thus,the aim of this study is to construct the recombinant plasmid of MAGE-D4 N-terminal gene,preparing the N-terminaltruncated protein of MAGE-D4 in vitro.Serum anti-MAGE-D4 antibody in varies types of brain tumor will be detected and prognostic impact of anti-MAGE-D4 antibody will be further analyzed.Methods:(1)Antigenic epitopes of MAGE-D4 were predicted and analyzed by a well-established B cell and T cell epitope online software of IEDB-AR,then region for amplification was determined.(2)With the cDNA contemplate of MAGE-D4 prepared before,the fragment of MAGE-D4(containing 398 amino acids of N-terminal half)gene was amplified by PCR.The recombinant plasmid of pCold II/MAGE-D4 was transformed into JM109.After blue-white testing and DNA sequencing,the recombinant plasmid was identified and transformed into BL-21(DE3)pLysS.(3)The expression of His-tag/MAGE-D41398aa truncated fusion protein was induced by IPTG,and the optimal conditions including concentration and time of IPTG for expression were also explored.After identified by PAGE electrophoresis,Western-Blotting and mass spectrometry,purification of His-tag/MAGE-D41-398aa truncated fusion protein was performed by Ni-NTA arogase amylose-resin column.(4)With the purified His-tag/MAGE-D41-398aa truncated fusion protein as coating antigen,MAGE-D4 autologous antibody in sera of 175 samples of 3 types of brain tumors and 63 samples of normal were detected by indirect ELISA assay.Results from MAGE-D4 truncated and full-length protein was compared.Clinical impact of the prevalence of anti-MAGE-D4 antibody was statistically analyzed.Results:(1)Results from IEDB demonstrated that both N-terminal and MHD region of MAGE-D4 protein contain B cell and T cell epitopes;(2)DNA sequencing showed that recombinant plasmid of pCold II/MAGE-D4 was successfully constructed;(3)His-tag/MAGE-D41.398aa truncated protein was expressed,and the optimal conditions for efficient expression of truncated fusion protein were established:which is first shaking incubation in 37 temperature for 2 hours,after cooling to below 20 temperature quickly;IPTG 0.9mmol/L of IPTG was added,continuous incubation in 20 temperature for 24 hours.(4)The results of ELISA showed that,anti-MAGE-D4 antibodies were detected in 21.14%(37/175)of sera samples from 3 types of brain tumor patients.Of these,the observed percentages of autoantibody response in astrocytoma,glioblastoma,ependymoma,meningeoma and medulloblsastoma were 22.22%(24/108),22.22%(8/36),50%(5/10),8.57%(3/35)and 28.57%(4/15),respectively.There were no significantly differences between MAGE-D4 serum immunoreactivity and patients’ characteristics.The differences of median overall survival was not significantly between patients with anti-MAGE-D4 positive and with anit-MAGE-D4 negative(13 months Vs.16 months,P=0.752).Univariate analysis for overall survival identified age(>45 years),WHO grade(Ⅲ-Ⅳ),histopathologic diagnosis(glioblastoma)as significant prognostic factors.Multivariate analysis showed that histopathologic diagnosis(glioblastoma)was independently prognostic for survival.Compared to the results of indirect ELISA based on MAGE-D4 full-length protein,there were no significant difference between the positive rates detected by the two proteins,with 65.71%of coincidence rate.Conclusion:This study indicates that antigenic epitopes do not entirely locate in N-terminal half MAGE-D4 protein.Thus,performing immune-associated studies with truncated protein instead of full-length protein may be crucial The prokaryotic recombinant plasmid of N-terminal half MAGE-D4 gene was successfully constructed,and MAGE-D4 truncated protein with high purify was obtained.The indirect ELISA based on MAGE-D4139gaa truncated protein suggests that MAGE-D4 is tumor associated antigen with immunogenicity in different types of brain tumor,and is capable of inducing anti-tumor immune responses.However,clinical implications of serum MAGE-D4 levels should be validated in a large population of patients with glioma.