Generation of Mouse Hepsin Protein and Anti-Mouse Hepsin Antibody, through DNA Vaccine and Protein Immunization

Hepsin屬於第二型穿膜絲胺酸蛋白酶，人類Hepsin生理上於肝臟大量表現，而腎臟、胰臟、前列腺與甲狀腺皆可測得其微量mRNA，病理上在前列腺癌、腎細胞癌、卵巢癌與子宮內膜癌皆可測得Hepsin mRNA。許多活體外實驗指出 Hepsin可能與凝血機制、肝細胞生長與胚胎發育有關聯，但Hepsin基因剔除小鼠除了仍可正常的生長發育並繁衍下一代之外，亦具有正常的凝血功能與肝功能，且肝細胞再生能力與野生型小鼠沒有差異。近來文獻指出Hepsin於癌組織細胞中過量表現而被推測與癌細胞生長與轉移相關，雖有許多針對Hepsin蛋白質的研究，但Hepsin在正常生理功能與各種不同癌症病理中所扮演的角色仍眾說紛紜。 為了透過小鼠模式釐清Hepsin的正常生理功能及在病理中扮演的角色，我們需要可應用於ELISA、西方墨點法、免疫組織切片與甚至是可抑制Hepsin活性的抗體進行後續的研究，但市售的抗小鼠Hepsin蛋白質抗體不符需求，於是在本篇論文中期望可以得到具有活性的小鼠Hepsin基因重組蛋白質，並藉由蛋白質免疫或DNA vaccine的方式得到可做各項分析的優良抗體，並進一步應用於Hepsin蛋白質功能分析。 在本篇論文中，我嘗試由E. coli系統與哺乳類動物細胞系統大量表現小鼠Hepsin蛋白質，在成功於哺乳類動物細胞表現具活性野生型(WT)與活性區突變(S352Y)的兩種小鼠Hepsin膜外區蛋白質後，以Nickel親和力管柱進行蛋白質純化。接著對18隻野生型BALB/c小鼠與40隻本實驗室產製的Hepsin基因剔除小鼠(B6.129/Sv.Hpntm1SWL)分別進行蛋白質免疫與DNA vaccine免疫，實驗結果發現Hepsin基因剔除小鼠免疫後血清抗體效價較野生型BALB/c小鼠高100倍。 取12隻抗體效價高的小鼠與NS-1進行細胞融合，在DNA vaccine的部分得到5個細胞株，而蛋白質免疫的部分仍在進行中。取2個DNA vaccine得到的單株抗體細胞株打入野生型BALB/c與NOD-SCID後天免疫缺乏小鼠後收取腹水，發現這兩個由B6.129/Sv.Hpntm1SWL與BALB/c融合的細胞株幾乎無法在野生型BALB/c小鼠中生長，在20隻野生型BALB/c小鼠中只有2隻可取得腹水，但經純化後得到抗體效價低；而8隻NOD-SCID小鼠全部都可取得腹水且抗體效價可高18.3倍。除了單株抗體的製備，本研究同時也將純化的小鼠Hepsin膜外區蛋白委託生技公司製備多株抗體，得到血清後純化。 取得純化知單株與多株抗體後，分析抗體專一性與靈敏度。實驗結果指出單株與多株抗體可運用於ELISA、西方墨點法與可於免疫組織切片染色測得小鼠Hepsin過量表現組織中的Hepsin蛋白質。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.誌謝 ....................................................i文摘要 ...............................................iibstract ...............................................iv錄 ...................................................vi目錄 ................................................vii目錄 ...............................................viii寫表 .................................................ix言 ................................................... 1料與方法 ............................................. 7驗結果 .............................................. 23驗討論 .............................................. 28來研究方向與展望 .................................... 36amp;#63851;考文獻 .............................................. 71錄 .................................................. 7