Human tumor-related protein hyrdC: prokaryotic expression,antibody preparation and its preliminary detection in gastric cancer

Objective:To prepare pure prokaryotically expressed protein of hyrdC and the monoclonal antibody against hyrdC,and to detect the hyrdC protein in gastric cancer with the prepared monoclonal antibody.Methods: The target DNA sequence of hyrdC was obtained by RT-PCR and subsequently inserted into prokaryotic expression vector pET32a.The TRX-hyrdC fusion protein was purified and was used to immunize 6-week-old BALB/c female mice to prepare monoclonal antibodies.The supernants of hybridoma cells were screened by enzyme-linked immunoabsorbent assay(ELISA) and identified by Western blot.The prepared monoclonal antibody was used to detect the expression of hyrdC protein in gastric cancer and adjacent normal tissues by immunohistochemical method(SP).Results: The sequence of hyrdC ORF was conformed to the reported in GenBank.Two lines of hybrids secreting monoclonal antibodies against hyrdC were established,both belonging to IgG2a subtype.Western blot showed that the prepared monoclonal antibodies specifically combined with human hyrdC protein.Immunohistochemistry(SP) results showed that the expression of hyrdC protein in gastric cancer was significantly higher than that in normal gastric mucous membrane(P0.001).Conclusion: The monoclonal antibody against hyrdC has been successfully obtained.There are significant differences in the expression of hyrdC between the gastric tumor tissues and normal tissues.