in step 2 after being amplified by 2 nd antibody and developed by the following development procedure proteins showing positive were collected those proteins were recognized by the anti mycoplasm spp antibody and therefore would be suitable as candidate antigens for active ingredient of subunit vaccines the hybridization was conducted by western blotting briefly the 2d gel after electrophoresis was transferred to a pvdf membrane then the membrane was incubated and hybridized sequentially with 1 st antibody the serum containing anti mycoplasm spp antibody and 2 nd antibody ap conjugated anti pig igg afterward a color reaction was conducted by using nbt bcip solution the result of the color reaction of western blotting was shown in fig2 wherein 10 proteins positive to the immuno hybridization with anti mycoplasm spp antibody were marked as candidate antigens for being used as active ingredients of subunit vaccines according to the color reaction of the western blotting the gel corresponding to the positive location on the membrane was cut by micropeptide and analyzed by mass spectrometry the obtained data of the mass spectrometry was then matched with amino acid sequence and protein database to identify those proteins please see the following table 1 said 10 proteins positive to the immune hybridization with anti mycoplasm spp antibody were listed escherichia coli jm109 was used.