Wu Yin-qiao, Wang Meng-wei, Wu Ben-yan, Wang Gang-shi, You Wei-di, Wang Wei-hua
Department of Gastroenterology, South Building, General Hospital of PLA, Beijing 100853, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2009 Aug;25(8):681-3.
To prepare the polyclonal antibody against gastric cancer-related protein GCRG224.
The thioredoxin/GCRG224 fusion protein was expressed in E.coli. The polyclonal antibody against GCRG224 was obtained by immunizing a rabbit with the purified GCRG224 protein. The titer and specificity of the antibody were determined by ELISA and Western blot, respectively. The expression of GCRG224 in paraffin-embedded tissue sections from normal gastric mucosal tissues and advanced gastric cancer was determined using immunohistochemistry technique.
The thioredoxin/GCRG224 fusion protein with relative molecular mass of 16.8 kDa was over-expressed in E.coli. The purity of the expressed product directly purified from a denaturing polyacrylamide gel was about 100%. The polyclonal antibody against GCRG224 was prepared. ELISA detection proved the titer of antiserum against GCRG224 was about 1:256,000. Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically. GCRG224 was found to have higher expression in gastric cancer tissues than in normal ones by immunohistochemistry.
The successful preparation of the polyclonal antibody against GCRG224 lays a foundation for further study of the biological function and the possible role of GCRG224 in the development of gastric carcinoma.
制备抗胃癌相关蛋白GCRG224的多克隆抗体。
硫氧还蛋白/GCRG224融合蛋白在大肠杆菌中表达。用纯化的GCRG224蛋白免疫家兔，获得抗GCRG224的多克隆抗体。分别用ELISA和Western印迹法检测抗体的效价和特异性。采用免疫组织化学技术检测GCRG224在正常胃黏膜组织和进展期胃癌石蜡包埋组织切片中的表达。
相对分子质量为16.8 kDa的硫氧还蛋白/GCRG224融合蛋白在大肠杆菌中过量表达。从变性聚丙烯酰胺凝胶直接纯化的表达产物纯度约为100%。制备了抗GCRG224的多克隆抗体。ELISA检测证明抗GCRG224抗血清的效价约为1∶256 000。Western印迹分析表明，该抗血清能特异性结合表达的融合蛋白。免疫组织化学发现，GCRG224在胃癌组织中的表达高于正常组织。
抗GCRG224多克隆抗体的成功制备为进一步研究GCRG224的生物学功能及其在胃癌发生发展中的可能作用奠定了基础。
