Sun Qi-fei, Gao Yuan, Liu Xi-bao, Sun Qi-hong, Wang Song-ling
Molecular Laboratory for Gene Therapy, Faculty of Stomatology, Capital University of Medical Sciences, Beijing 100050, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2006 Jul;22(4):521-3, 538.
To prepare the rabbit antibody against human sialin and identify its properties.
Recombinant expression vector pGEX-5X-1-sialin was constructed, in which the sialin cDNA encoding the 1-38 aa was fused to the C-terminal of the gene encoding the GST protein. The GST-sialin (N1-38) fusion protein was expressed in E. coli JM109 at 37 degrees C in the presence of IPTG at 0.1 mmol/L for induction for 3 hours, purified by GSTrap FF, and then used as the immunogen to prepare the rabbit polyclonal antibody. The properties of antiserum against human sialin were identified by ELISA, Western blot and immunocytochemistry.
The recombinant expression plasmid pGEX-5X-1-sialin was constructed. The GST-sialin (N1-38) fusion protein was highly expressed with a molecular weight of 30 kDa, and the yield of the fusion protein was about 20% to 30% in total E. coli protein. The titre of antiserum against human sialin was 1:32,000. Western blot analysis proved the rabbit polyclonal antibody could identify both GST-sialin (N1-38) fusion protein and GST. Besides, it specially recognized a 55 kDa band expressed in the human submandibular gland (HSG) cell line. The antigen recognized by the antibody was located in the cytoplasm and nucleus of HSG cell.
The successful preparation of the polyclonal antibody against human sialin will provide efficient affinity reagent for further functional study of sialin expressed in human salivary glands.
制备抗人唾液酸转运蛋白的兔抗体并鉴定其特性。
构建重组表达载体pGEX-5X-1-唾液酸转运蛋白，其中编码1-38个氨基酸的唾液酸转运蛋白cDNA与编码谷胱甘肽S-转移酶（GST）蛋白的基因C末端融合。GST-唾液酸转运蛋白（N1-38）融合蛋白在大肠杆菌JM109中于37℃、0.1 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）存在下诱导表达3小时，通过GSTrap FF纯化，然后用作免疫原制备兔多克隆抗体。采用酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法（Western blot）和免疫细胞化学法鉴定抗人唾液酸转运蛋白抗血清的特性。
构建了重组表达质粒pGEX-5X-1-唾液酸转运蛋白。GST-唾液酸转运蛋白（N1-38）融合蛋白高表达，分子量为30 kDa，融合蛋白产量约占大肠杆菌总蛋白的20%至30%。抗人唾液酸转运蛋白抗血清的效价为1:32,000。蛋白质免疫印迹分析证明兔多克隆抗体可识别GST-唾液酸转运蛋白（N1-38）融合蛋白和GST。此外，它能特异性识别在人下颌下腺（HSG）细胞系中表达的一条55 kDa条带。该抗体识别的抗原位于HSG细胞的细胞质和细胞核中。
成功制备抗人唾液酸转运蛋白的多克隆抗体，将为进一步研究人唾液腺中表达的唾液酸转运蛋白的功能提供高效的亲和试剂。
