中文摘要：

目的制备人类功能未知基因C16orf54重组蛋白的多克隆抗体,为该基因功能研究奠定基础。方法分离纯化LX2细胞的总RNA,逆转录为cDNA,PCR扩增C16orf54基因的目的片段,构建重组蛋白表达质粒,转化大肠杆菌BL21,制备C16orf54重组蛋白。鉴定表达正确后,利用纯化的C16orf54重组蛋白免疫新西兰大白兔,获得抗C16orf54重组蛋白的多克隆抗体。利用制备的多克隆抗体,采用免疫组织化学技术观察该蛋白在肝细胞内的表达特征。结果 Western blot及生物质谱技术分析证实,重组蛋白表达正确。所制备的多克隆抗体效价〉1：320 000,Western blot分析显示,多克隆抗体的特异性良好。免疫组织化学分析显示,该蛋白主要表达于肝实质细胞周围,呈膜型和浆型分布;胃黏膜下组织细胞弱表达。结论 C16orf54多克隆抗体被成功制备。C16orf54主要表达于肝实质细胞周围及胃黏膜下基底层细胞。

英文摘要：

Objective To prepare the polyclonal antibody of C16orf54,and identify the specificity and potency of the antibody.Methods Total RNA was obtained from cultured LX2 cells and reverse transcripted into cDNA.PCR was used to amplify the cDNA of C16orf54.The plasmid of C16orf54 recombinant protein was constructed and transformed into E.coil BL21.Purified recombinant protein was used to prepare the polyclonal antibody of C16orf54.The immunohistochemistry technique was used to observe the expression of C16orf54 in liver tissue.Results Western blot and Mass spectrometry confirmed that the recombinant protein was accurately expressed.After immunized with recombinant protein to rabbit,ELISA analysis indicated the titer of polyclonal antibody 1∶ 320 000.The high specificity was confirmed by Western blot.Immunohistochemistry staining of C16orf54 demonstrated that C16orf54 highly expressed around liver cells,and lowly scattered in few cells in gastric tissue.Conclusion The recombinant C16orf68 specific antibody was obtained.The C16orf54 mainly expressed in the around of hepatocytes and few gastric cells.