重组SCIRR39多克隆抗体的制备及检测

Preparation and Characterization of Polyclonal Antibody Against Rat Spinal Cord Injury and Regeneration Related Protein 39

目的：在大肠杆菌中表达大鼠脊髓损伤与修复蛋白39（SCIRR39）的C端抗原表位，并制备其多克隆抗体。方法：从大鼠脊髓全横断损伤脊髓cDNA中扩增1386bp的Scirr39基因编码框，亚克隆该基因编码蛋白C端359～461位氨基酸残基的DNA片段，插入表达载体，转化大肠杆菌BL21（DE3），IPTG诱导表达，SDS-PAGE分析表达情况，切胶纯化目的蛋白；利用多克隆抗体制备技术，制备重组SCIRR39蛋白的多克隆抗体；用ELISA方法检测抗体效价，Western印迹检测抗体的特异性。结果：SCIRR39蛋白C端抗原表位与GST的融合蛋白在大肠杆菌中以可溶形式高表达，相对分子质量为37．9×103；获得抗SCIRR39蛋白C端抗原表位的兔抗血清，其效价达到1：104；Western印迹显示多克隆抗体能特异识别重组SCIRR39蛋白的C端抗原表位。结论：在原核系统中表达纯化了重组SCIRR39抗原表位蛋白，制备的重组蛋白多克隆抗体将用于检测SCIRR39在脊髓损伤过程中的表达变化。

Objective： To express the recombinant rat spinal cord injury and regeneration related protein 39 （SCIRR39） C-terminal epitope in E.coli BL21 （DE3）, and prepare polyelonal antibody against it. Methods： The gene coding SCIRR39 with the length of 1386 bp was amplified from rat spinal cord eDNA by PCR, then site 1077 to 1383 bp was amplified from it and was inserted into pGEX-4T-2 to construct the GST-SCIRR39-C ex- pression plasmid. Then it was transformed into E.coli BL21（DE3） and induced by IPTG. The recombinant GST- SCIRR39-C was visualized by SDS-PAGE and was purified from the gel, and the GST tag was removed. Poly- clonal antibody against Scirr39-C antigen was prepared by polyelonal antibody preparation techniques and detected by ELISA and Western blot. Results： The recombinant GST-SCIRR39-C was expressed at high level and partly soluble, and its molecular was 37.9 kD. The rabbit serum could specifically recognize the recombinant SCIRR39-C, and the titer reached 1：104. Conclusion： The recombinant SCIRR39-C has been expressed in E.coli BL21（DE3） and was purified to prepare the polyclonal antibody against it. The polyclonal antibody will be used to explore the molecular mechanism of CNS injury and repair.