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目的 :原核表达神经生长相关蛋白 4 3(GAP 4 3) ,并制备抗GAP 4 3的单克隆抗体 (mAb)。方法 :从含有GAP 4 3cDNA的质粒中 ,用PCR扩增GAP 4 3cDNA的全长编码序列 ,克隆入表达载体pGEX 4T 1中 ,构建GAP 4 3的高表达工程菌 ,并以IPTG诱导表达目的蛋白。通过亲和层析法纯化表达的GST GAP 4 3融合蛋白 ,并以此为抗原制备mAb。结果 :酶切鉴定证明 ,获得含有目的基因片段的重组质粒。表达的GST GAP 4 3融合蛋白以可溶性的形式存在。ELISA检测表明 ,获得的抗GAP 4 3mAb效价为 1∶10 8,其IgG的亚类为IgG2a,亚型为κ型。用Westernblot检测大鼠脑匀浆蛋白 ,在相对分子质量(Mr)为 5 0 0 0 0处有一条特异性带。用此mAb进行荧光免疫法检测表明 ,在致敏豚鼠的肺切片中 ,有GAP 4 3阳性的神经纤维。结论 :所获抗GAP 4 3mAb的特异性强、效价高 ,对进一步研究GAP 4 3在神经系统中的作用提供了有用的试剂。
OBJECTIVE: Prokaryotic expression of nerve growth-associated protein 4 3 (GAP 4 3) and preparation of anti-GAP 4 3 monoclonal antibody (mAb). METHODS: The full-length coding sequence of GAP 4 3 cDNA was amplified by PCR from the plasmid containing GAP 4 3 cDNA and cloned into the expression vector pGEX 4T 1 to construct a highly expressed GAP 4 3 -enhanced engineering bacterium. The target protein was induced by IPTG . The expressed GST GAP43 fusion protein was purified by affinity chromatography and mAb was prepared as antigen. Results: Digestion identification proved that the recombinant plasmid containing the target gene fragment was obtained. The expressed GST GAP43 fusion protein is in soluble form. The results of ELISA showed that the titer of anti-GAP 4 3 mAb was 1:10 8, the IgG subclass was IgG2a, and the subtype was κ. Western blot was used to detect protein in rat brain homogenate, with a specific band at molecular weight (Mr) of 50000. Fluorescence immunoassay using this mAb showed that GAP 43-positive nerve fibers were found in lung sections of sensitized guinea pigs. Conclusion: The obtained anti-GAP 4 3 mAb is highly specific and has high titer, which provides a useful reagent for further study on the role of GAP 4 3 in the nervous system.