Guo Ai-lin, Cao Yun-xin, Wang Wen, Ding Bo, Huang Yu-qiang, Wen Jian-ming
Department of Pathology, Zhongshan Medical College, SUN Yat-sen University, Guangzhou 510080, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2004 Sep;20(5):578-81.
To express phosphatase 2 of regenerating liver (PRL-2) fusion protein in E.coli and to prepare specific hen egg yolk immunoglobulin(IgY) against PRL-2.
A full-length human PRL-2 gene coding sequence was cloned into expression vectors pGEX-4T-2 and pET21a, then transformed into E.coli. Fusion protein GST-PRL-2 was expressed in E.coli via IPTG induction. The expressed proteins were purified from lysates through Glutathione Sepharose 4B and the Ni-NTA agarose columns, respectively. Egg-laying hens were immunized using the purified GST-PRL-2 with Freund's complete or incomplete adjuvant. The specificity of the resulting antibody was identified by Western blot.
A high level of expression of target protein was detected by Western blot after IPTG induction and purified protein was obtained through Glutathione Sepharose 4B and the Ni-NTA affinity chromatography agarose columns, respectively. Western blot analysis showed that the anti-PRL-2 polyclonal antibody can recognize 6 x His-PRL-2 fusion protein.
The hen egg yolk immunoglobulin(IgY) against PRL-2 expressed in E.coli has good specificity which provides an useful reagent for the detection of PRL-2.
在大肠杆菌中表达再生肝磷酸酶2（PRL-2）融合蛋白，并制备针对PRL-2的特异性鸡卵黄免疫球蛋白（IgY）。
将人PRL-2基因全长编码序列克隆到表达载体pGEX-4T-2和pET21a中，然后转化到大肠杆菌中。通过IPTG诱导在大肠杆菌中表达融合蛋白GST-PRL-2。分别通过谷胱甘肽琼脂糖4B柱和镍-亚氨基二乙酸琼脂糖柱从裂解物中纯化表达的蛋白。用纯化的GST-PRL-2与弗氏完全或不完全佐剂免疫产蛋母鸡。通过蛋白质免疫印迹法鉴定所得抗体的特异性。
IPTG诱导后通过蛋白质免疫印迹法检测到目标蛋白的高表达水平，并分别通过谷胱甘肽琼脂糖4B柱和镍-亚氨基二乙酸亲和层析琼脂糖柱获得纯化的蛋白。蛋白质免疫印迹分析表明，抗PRL-2多克隆抗体可识别6×His-PRL-2融合蛋白。
在大肠杆菌中表达的针对PRL-2的鸡卵黄免疫球蛋白（IgY）具有良好的特异性，为PRL-2的检测提供了有用的试剂。
