【作者】 刘璐；

【导师】 刘健；

【作者基本信息】 合肥工业大学 ， 食品科学， 2013， 硕士

【摘要】 在肥胖、糖尿病、粥样动脉硬化和腹主动脉瘤等代谢疾病发病机制中，肥大细胞起着重要的作用。小鼠肥大细胞蛋白酶4、6（mMCP4、mMCP6）是小鼠肥大细胞特异蛋白酶，在肥大细胞相关疾病生物学中具有关键的调控作用。但是，市场上并没有较好的商品化抗体，在一定程度上限制了这些肥大细胞蛋白酶细节作用机制的研究。因此，原核表达并制备这些蛋白酶的抗体，将有利于深入探讨肥大细胞的相关作用机制。本研究，构建了小鼠肥大细胞蛋白酶4(mMCP4)和6(mMCP6)的原核表达体系，诱导表达并纯化了包涵体蛋白，并通过多次皮下注射制备了兔抗mMCP4和兔抗mMCP6多克隆抗体。其具体方法包括：(1)提取小鼠脾细胞总RNA，运用RT-PCR技术获得目的基因mMCP4、 mMCP6；(2)构建重组原核表达载体pET28a-mMCP4、pET28a-mMCP6，将其转化到大肠杆菌BL21中，以异丙基硫代半乳糖苷(IPTG)诱导融合蛋白表达；(3)利用Ni亲和层析柱纯化mMCP4、mMCP6融合蛋白；(4)纯化后重组蛋白作为抗原免疫兔子获得mMCP4多克隆抗体、mMCP6多克隆抗体；(5)采用ELISA法测抗体效价，western blot测抗体特异性。其具体结果表明：(1)经测序验证，所扩增目的基因mMCP4、mMCP6序列准确，所构建重组表达质粒pET28a-mMCP4、 pET28a-mMCP6成功；(2)经SDS-PAGE电泳验证，所诱导并纯化的mMCP4、mMCP6融合包涵体蛋白分子量符合预期，条带单一；(3)经ELISA测定，所得兔抗mMCP4血清和兔抗mMCP6血清效价分别达到1:128000和1:32000；(4)经western blot和免疫组化分析，表明mMCP4、mMCP6多克隆抗体具有较好的特异性。总之，通过克隆、原核表达和多次免疫，我们成功地获得两种具有良好的效价和特异性的抗体，可用于免疫学检测法，能满足利用之进一步研究肥大细胞的生物学功能的要求。更多还原

【Abstract】 Mast cells play an important role in the pathogenesis of metabolic diseases,such as obesity, diabetes, atherosclerosis and abdominal aortic aneurysm. Mousemast cell protease4and6(mMCP4and mMCP6) are specific mouse mast cellproteases which play a key role in regulation of mast cells related diseases.However, there are no useful commercial antibodies in market which limits theresearch of mast cells. Prokaryotic expression and preparation of polyclonalantibodies for these proteases will be used to the fureher study of mast cells.We constructed the recombinant expression plasmids of mouse mast cellprotease4and6which were transfected into E. coli BL21and induced to expressthe fused proteins. The fusion proteins were purified and used as antigens to injectinto subcutaneous tissue of the rabbit. Then we obtained rabbit anti-mMCP4polyclonal antibody and rabbit anti-mMCP6polyclonal antibody.Methods:(1) The target genes were amplified from mouse spleen total RNA byRT-PCR.(2) The recombinant plasmids pET28a-mMCP4and pET28a-mMCP6weretransfected into E. coli BL21and induced by IPTG to express the fused proteins.(3)Mouse MCP4and MCP6fusion proteins were purified by Ni resin affinitychromatograph.(4) The purified recombinant proteins were used as antigens toimmune rabbit and prepare mMCP4polyclonal antibody and mMCP6polyclonalantibody.(5) The titer and specificity of polyclonal antibodies were confirmed byELISA and western blot, respectively.Results:(1) The target genes were cloned by RT-PCR and the recombinantplasmids were constructed successfully.(2) The analysis of SDS-PAGE showed thatwe obtained a high expression of mMCP4and mMCP6fusion protein.(3) The titerof rabbit anti-mMCP4serum and rabbit anti-mMCP6serum were respectively about1:128000and1:32000.(4) Western blot and immunohistochemistry showed that thespecificity of mMCP4polyclonal antibody and mMCP6polyclonal antibody werehigh. In conclusion, we successfully obtained two kinds of antibodies with high titerand specificity through cloning gene, prokaryotic expression and immunization.The antibodies can be used in immunological method and satisfied with therequirements of further study of mast cells.更多还原