GAP-43原核表达载体的构建、表达及抗GAP-43单克隆抗体的制备

Construction and expression of prokaryotic expression vector and preparation of monoclonal antibody against GAP-43

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。

AIM: To express the growth associated protein 43 (GAP 43) in prokaryotic cells and prepare monoclonal antibody(mAb) against GAP 43. METHODS: Full length sequence of GAP 43 gene was amplified from the plasmid containing pGAP 43 cDNA and was cloned into the expression vector pGEX 4T 1. GST GAP 43 fusion protein was expressed in E.coli under IPTG induction. Expressed fusion protein was purified by glutathine agarose chromagraphy, Using purified protein as an immunogen, mAb against GAP 43 was prepared. RESULTS: The recombinant plasmid containing the target gene was constructed successfully. The fusion protein was expressed in E.coli in soluble form. The titer of anti GAP 43 mAb in ascites was 1∶10 8. The Ig subclass and subtype of the mAb was IgG2a and κ type, respectively. Specificity of the mAb was confirmed by ELISA, Western blot and immunofluorescence technique. CONCLUSION: The anti GAP 43 mAb obtained has strong specificity and high tilter, which provides an useful reagent for further studying the function of GAP 43 in nervous system.