Prokaryotic Expression, Antibody Preparation, And Identification Of Human NY-ESO-1

Objectives:Prokaryotic expression vectors were constructed for NY-ESO-1 cancer-testis antigen and GST fusion genes. Their monoclonal& polyclonal antibodies were prepared and preliminarily identified. Besides, their specificity was tested by Western-blot and their application in cancer treatment was studied through immunohistochemistry.Methods:1 Acquisition of Target and Construction of Recombinant Expression VectorsSpecific primers were designed for NY-ESO-1, and NY-ESO-1 segments were amplified at cDNA library of testis, which were cloned into the downstream of GST tag inside the prokaryotic expression vector pGEX-4Tl after double digestion by EcoR I and Xho I that were introduced from upstream and downstream primers for the purpose of constructing the recombinant expression vector pGEX-4T1-NY-ESO-1.2 Culturing BacteriaThe vector pGEX-4T1-NY-ESO-1 was transferred to E. coli BL21 (DE3) pLysS to culture bacteria.3. Induced Expression, Purification and Identification of Target ProteinIPTG was used to induce the expression of NY-ESO-1/GST. After being eluted and purified by urea of different concentration, expression products were identified by SDS-PAGE and Western blot to acquire NY-ESO-1/GST infusion protein.4 Preparation of Monoclonal AntibodyBALB/c mice were immunized with NY-ESO-1/GST fusion protein and monoclonal anti-NY-ESO-1 antibody was prepared by conventional methods. Meanwhile, hybridoma cell lines of the monoclonal anti-NY-ESO-1 antibody were obtained through cell fusion, HAT selection, cloning and amplification via limiting dilution.5 Preparation of Polyclonal AntibodyRabbits were immunized by NY-ESO-1/GST fusion protein to prepare polyclonal NY-ESO-1 antiserum, and polyclonal NY-ESO-1 antibody was acquired.6 Antibody Identification and ImmunohistochemistryAntibody titer was measured by indirect ELISA and its specificity was tested by Western-blot. Additionally, its application in detecting normal testicular tissues and cancer was examined by immunohistochemical techniques.Results:Recombinant plasmid was identified by double digestion via restriction endonucleases EcoR I and Xho I, and IPTG induced the expression of NY-ESO-1/GST. The results of SDS-PAGE analysis suggested that the relative molecular mass (Mt) of the expression products was 44,000, which was in agreement with the theoretical value, and the products primarily existed in the form of inclusion bodies. The results of Western blot demonstrated that NY-ESO-1/GST could have specific reaction with monoclonal anti-GST antibody (mAb), and it was found to be infusion protein. After preparing monoclonal antibody, a monoclonal NY-ESO-1 cell line was obtained through immunization, the titer of which was measured to be 10-7 by indirect ELISA. Identified by Western blot and immunochemistry, the antibody was determined to be highly specific and have no cross reactions with the protein of the vector (GST). High-titer NY-ESO-1 antiserum was obtained after immunizing rabbits and it was verified to be highly specific by Western blot. The immunohistochemical test showed that the expression of its target genes was visible.Conclusion:The prokaryotic expression vector pGEX-4Tl-NY-ESO-1 was successfully constructed for NY-ESO-1 gene. Highly pure NY-ESO-1/GST fusion protein existing in the form of inclusion bodies was obtained. Furthermore, a cell line was successfully established for the monoclonal anti-NY-ESO-1 antibody. Owing to good identification of Western blot, rabbit anti-human NY-ESO-1 polyclonal antibody was smoothly prepared and identified. Promising results were obtained and laid a foundation for exploring biological functions of NY-ESO-1.