中文摘要：

目的构建新糖基转移酶基因Glt8d2的原核表达载体，诱导融合蛋白的表达，并对其进行纯化；制备兔抗GltSd2蛋白多克隆抗体并进行鉴定。方法应用RT—PCR技术，以HepG2细胞mRNA为模板，扩增Glt8d2目的基因片段，构建原核表达载体pET-32a（＋）-Glt8d2。转化大肠埃希菌B121，异丙基-D-半乳糖苷诱导并通过SDS—PAGE凝胶电泳分析、Western印迹分析、生物质谱分析证实Gh8d2重组蛋白表达正确。大量表达后利用Ni’亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰大白兔，获得抗Gh8d2蛋白的多克隆抗体。以纯化的Glt8d2重组蛋白为抗原，分别以免疫前后的新西兰兔血清作为第一抗体，利用Western印迹和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测。结果扩增获得Glt8d2基因片段，成功表达了Gh8d2重组蛋白，经SDS—PAGE凝胶电泳和Western印迹分析得到证实。成功获得重组蛋白及兔抗Gh8d2多克隆抗体。ELISA检测证实多克隆抗体效价〉1：320000。免疫组织化学分析显示，该基因编码的糖基转移酶在肝实质细胞内呈胞质模式表达分布。结论利用大肠埃希菌B121能够成功表达Gh8d2重组蛋白，获得高特异性、高效价兔抗Glt8d2重组蛋白的多克隆抗体，为今后研究Glt8d2基因的生物学功能研究奠定了基础。

英文摘要：

Objective To express and purify giycogene Glt8d2 recombinant protein, and to prepare rabbit polyclonal antibody specific for GltSd2 recombinant protein. Methods GltSd2 cDNA was ligated into the prokaryotic expressive vector pET-32a （ ＋ ） . The resultant plasmid was transformed into E. coil BI21 （DE3） Glt8d2 recombinant protein expression was induced under IPTG condition and analyzed with western blotting, followed by Ni ＋ affinity column purification. The GltSd2 recombinant protein product was further confirmed by mass spectrometry. New Zealand rab-bits were immunized with the purified pET-32a （ ＋ ） -Glt8d2 fusion protein to gain polyclonal antibody. Antibody speeificity and potency were evaluated by Western blot and ELISA. Results The Glt8d2 fusion protein was highly expressed. The protein was produced mainly in the inclusion body. ELISA showed the titer of the polyclonal antibody higher than 1：320 000. Western blot con- firmed the high specificity of Gh8d2 antibodies. Immunohistochemistry staining of liver tissue showed that this glycosyhransferase are mainly seattered in the eytoplasm of hepatocytes. Conclusion The recombinant Gh8d2 fusion protein and the Glt8d2 specific potyclonal antibody will be useful for investigation of Gh8d2 biologieal function.