P-糖蛋白的融合表达研究

Expression of fused P-glycoprotein in E. coli DH5α

目的 对P 糖蛋白胞外段基因进行表达研究。方法 根据P 糖蛋白抗原性分布曲线及mdr1基因结构 ,设计了一对引物 ,PCR扩增产物插入到pGEM T中 ,经测序正确后 ,再克隆入表达载体pGEX 2T ,构建高效表达载体pGEX Pgp ,转化大肠杆菌DH5α ,进行表达、鉴定及纯化。结果 经IPTG诱导 4h ,蛋白表达即达高峰 ,SDS PAGE分析 ,表达出约 3 0kD大小的蛋白。结论 该基因片段的高效表达为进一步制备其抗体或筛选其特异性结合肽 ,进行靶向治疗等工作奠定了基础。

Objective To study the expression of the extracellular fragment of P glycoprotein in the bacteria. Methods According to the P glycoprotein antigen analyses and mdr1 gene structure, a couple of primers were designed to amplify the extracellular DNA fragment of the protein with PCR. The product was inserted into pGEM T vector followed by sequencing. The sequencing verified vectors were cloned into pGEX 2T to get a highly expression recombinant vector pGEX Pgp. The recombinant was transferred into E. coli DH5α, and its expression, identification and purification were studied. Results The highest level of recombinant expression was achieved at 4 h after IPTG induction. SDS PAGE analysis indicated that the expressed protein was about 30×10 3. Conclusion The target gene fragment expression at high level is established for the preparation of its antibody and biospanning of its specific peptide by using random phage library, founding a basis for the targeting treatment of multidrug resistance.