Generation of Mouse Hepsin Protein and Anti-Mouse Hepsin Antibody, through DNA Vaccine and Protein Immunization
Date Issued
2008
Date
2008
Author(s)
Yang, Bi-Huei
Abstract
Hepsin is type II transmembrane serine protease. Physiologically, hepsin mRNA is abundantly expressed in liver and low levels in other tissues such as kidney, pancreas, prostate and thyroid. Pathologically, hepsin mRNA can be detected in prostate cancer, renal cell cancer, ovarian cancer and endometrial cancer. Several in vitro studies have shown that hepsin may play some roles in blood coagulation, hepatocyte growth, and embryonic development. However, homozygous hepsin knockout mice (hepsin-/-) were viable, fertile, and grew normally. Hepsin-/- mice maintained the normal functions in hemostasis, embryogenesis and liver functions compared to wild type mice. Recently, hepsin has been identified as one of the overexpression genes in several cancers, hepsin may be involved in the tumor cell growth and metastasis. Although there are lots of researches metion of hepsin protein, the physiological and pathological function of hepsin remains unclear. For determining the role of hepsin in physiology and pathology through mouse model, we need more sensitive and specific antibodies, which can be applied in ELISA, western blot, IHC staining and hepsin activity inhibition, compared with commercial anti-mouse hepsin antibodies for later research. This study aims at generation of functional mouse hepsin recombinant protein, and produced the best hepsin antibodies through DNA vaccine and protein immunization that can be used in several analysis described above for further studies. In this study, I tried to abundantly express mouse hepsin protein in E. coli and mammalian cell systems. In the mammalian cells, I have successfully expressed the functional (WT) and unfunctional (S352Y) mouse hepsin extracellular domain. These hepsin reconbinate protein contained His tag at N-terminal and myc tag at C terminal, and purified through nickel affinity column. 18 wild type BALB/c mices and 40 hepsin knockout mices (B6.129/Sv. Hpntm1SWL), which were generated in our laboratory were then immunized with mouse hepsin extracellular protein or DNA vaccine. Surprisingly, I found that antibody titer of post-immune serum in hepsin knockout mice is 100X higher than in wild type BALB/c mice. I selected 12 mices with higher antibody titer and took their spleen cells performed fusion with NS-1 cells. Finally, I obtained 5 hybridomas by DNA immunization, and the hybridoma by protein immunization keeps going. I choosed 2 hybridoma obtained by DNA immunization and injected them into wild type BALB/c mices and NOD-SCID mices. I found that the hybridomas which fused from B6.129/Sv.Hpntm1SWL and BALB/c strain, almostly cannot grow in wild type BALB/c mice. 2 of 20 mices grew ascites, but with very low titer of anti-hepsin antibody. On the contrary, 8 of 8 NOD-SCID mice grew ascites with high titer. In this study, except production of monoclonal antibody, I also request biotechnical commpany to produce polyclonal antibody by purified mouse hepsin extracellular protein. After analyse the specificity and the sensitivity of anti-mouse hepsin monoclonal and polyclonal antibodies, as datas shown, it can be used in ELISA, western blot and detect hepsin in mouse hepsin overexpressed tissue by immune- histochemistry.
Subjects
protein
antibody
SDGs
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