Kim Dae Won, Eum Won Sik, Choi Hee Soon, Kim So Young, An Jae Jin, Lee Sun Hwa, Sohn Eun Joung, Hwang Seok-Il, Kwon Oh-Shin, Kang Tae-Cheon, Won Moo Ho, Cho Sung-Woo, Lee Kil Soo, Park Jinseu, Choi Soo Young
Department of Biomedical Science and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea.
J Biochem Mol Biol. 2005 Nov 30;38(6):703-8. doi: 10.5483/bmbrep.2005.38.6.703.
We cloned and expressed human pyridoxal-5'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin B6, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin B6 abnormalities.
我们使用pET15b载体在大肠杆菌中克隆并表达了人磷酸吡哆醛（PLP）磷酸酶，它是维生素B6的辅酶活性形式。在小鼠体内针对纯化的人脑PLP磷酸酶产生了单克隆抗体（mAb），并获得了四种识别不同表位的抗体，其中一种可抑制PLP磷酸酶。使用生物传感器技术测定这四种mAb与PLP磷酸酶的结合亲和力，结果表明它们具有相似的结合亲和力。以抗PLP磷酸酶抗体作为探针，我们研究了它们在各种哺乳动物和人类组织及细胞系中的交叉反应性。在蛋白质免疫印迹上获得的免疫反应条带的分子量约为33 kDa。几种哺乳动物细胞系经类似分级分离的提取物均产生了一条分子量为33 kDa的单带。我们认为，这些PLP磷酸酶mAb可作为有价值的免疫诊断试剂，用于检测、鉴定和表征与维生素B6异常相关的各种神经疾病。
