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目的在大肠杆菌中表达GST-MPS-1融合蛋白并制备兔抗GST-MPS-1抗体。方法将MPS-1全长cDNA克隆至pGEX-5X载体内,转化大肠杆菌BL21,用IPTG诱导GST-MPS-1融合蛋白的表达。以分离纯化表达的蛋白免疫新西兰兔,制备抗GST-MPS-1抗体。用Western blot和细胞免疫荧光染色法,检测兔抗GST-MPS-1抗体的特异性。结果经测序和酶切鉴定确认,MPS-1基因以正确的方式插入到载体中。此重组表达载体经IPTG诱导后,可高效表达相对分子质量(Mr)为36000的GST-MPS-1融合蛋白。以融合蛋白免疫兔获得的抗血清,经ELISA检测效价达到1×105。Western blot和细胞免疫荧光染色证实,该多克隆抗体能与MPS-1蛋白特异性结合。结论兔抗MPS-1特异性抗体的获得,为进一步检测MPS-1蛋白的表达和功能分析奠定了基础。
Objective To express GST-MPS-1 fusion protein in E. coli and prepare rabbit anti-GST-MPS-1 antibody. Methods The full-length MPS-1 cDNA was cloned into pGEX-5X vector and transformed into E. coli BL21. The expression of GST-MPS-1 fusion protein was induced by IPTG. Anti-GST-MPS-1 antibody was prepared by immunizing New Zealand rabbits with isolated and purified protein. The specificity of rabbit anti-GST-MPS-1 antibody was detected by Western blot and immunofluorescence staining. Results Sequencing and restriction enzyme digestion confirmed that the MPS-1 gene was inserted into the vector in the correct manner. After induced by IPTG, the recombinant expression vector could express GST-MPS-1 fusion protein with a relative molecular mass (Mr) of 36000. The antiserum obtained from the rabbit immunized with the fusion protein reached a titer of 1 × 10 5 by ELISA. Western blot and immunofluorescence staining confirmed that the polyclonal antibody could specifically bind to MPS-1 protein. Conclusion The obtaining of rabbit anti-MPS-1 specific antibody lays the foundation for the further detection of MPS-1 protein expression and function analysis.