The main challenge in the development of antibody is to select the appropriate antigen particularly when a truncated protein is used for immunization or as vaccine antigen. In previous studies, fragment selection was mainly based on epitopes and less often on the structure. Fewer studies have paid attention to the prediction of the truncated protein 3D structure and retained its similarity in the native and truncated proteins. Here we used in silico analysis to select two fragments of Pyruvate Kinase M2 (PKM2), as a tumor marker. One fragment, M-tPKM2, had a shorter sequence with one epitope although the predicted 3D structure was similar to the native PKM2. The other fragment, R-tPKM2, had a longer sequence and thus more epitopes, but had a different structure from the native PKM2. Recombinant truncated proteins were expressed in E. coli and purified via affinity chromatography. Secondary structure elements in purified proteins were determined by Circular Dichroism, then they were utilized to develop antibodies in mice. Both antigens could elicit high immune response against themselves (OD450 = 3.326 ± 0.562 for M-tPKM2; OD450 = 3.562 ± 0.110 for R-tPKM2). However, significantly higher response against PKM2 was observed among the mice immunized with M-tPKM2 (p < 0.0001 by One way ANOVA followed by Tukey's post hoc comparison). Also, the monoclonal antibody produced against the M-tPKM2 could recognize the native PKM2 in the MCF7 cells. Our finding suggested that for the purpose of designing an antigen with the ability to produce a potent antibody against the target protein, it is better to select sequences which have a similar structure in truncated and native proteins, even at the cost of having shorter sequences and fewer epitopes.