Bioinformatics Analysis Of Human GACE And Its Antibody Preparation

PURPOSE: Analysing antigen epitope of human gACE by bioinformatics and preparing polyclonal antibody of human gACE. These results will make foundation for further research of human gACE functions.METHODS: Amino acids sequences of human gACE were analyzed using bioinformatics resourses such as ExPASy( http://www.expasy.org/tools ),TMHMM(http: //www.cbs.dtu.dk./services/TMHMM),protparam ( http://www.expasy.o rg/cgi-bin/proparam ),protscale ( http://www.expasy.org/cgi-bin/protscale ),sppider( http://www.sppider.cchmc.org/sppider_doc.html),SOPMA and DNAstar( protean ) . On the base of these analyses, we predicted the possible physicochemical property, hydrophilicity, antigenicity, surface accessibility, flexibility, secondary structures and transmembrane structures of the protein of human gACE. According to the homology analysis and the principal of antibody designing, a antigen epitope containing 11-amino acid residues which located at sequence 281-291(YVRRALHRHYG)of gACE was chosen as the candidate for our study. After synthesizing this peptide, it was conjugated with KLH. The connector was used to immunize rabbit to obtain anti-rabbit gACE polyclonal antibodies. The specificity and titer of anti-rabbit gACE polyclonal antibodies were evaluated by ELISA, Western blot and immunohistochemistry.RESULTS: 1.The antigenicity was predicted by bioinformatics analysis. 2.The polyclonal antibody of gACE was successfully obtained. 3. The titer of the polyclonal antibody against human gACE was up to 1:16000. 4. Western blot analysis revealed that the polyclonal antibody could specifically react with gACE which was extracted from human spematozoon and with the standard sample of recombinated human gACE from Hela cells. 5. The result of immunohistoch- emistry also confirmed that the localization of gACE on human spermatozoon, which was in consistent with reports from relative literatures.CONCLUSION: The antigen epitope of gACE was predicted by bioinformatics analysis. On the base of the prediction, polyclonal antibody with high specificity was successfully obtained.