小鼠MIP-1α-DT390重组免疫毒素原核表达载体的构建及鉴定

Construction and prokaryotic expression of a recombinant immunotoxin fused with mouse macrophage inflammatory protein-1α and truncated diphtheria toxin

目的构建小鼠MIP-1α基因和白喉杆菌外毒素DT390基因的原核融合表达载体,诱导并鉴定该蛋白的表达。方法通过RT-PCR获得mMIP-1α基因,通过PCR扩增质粒SRα-DT390获得DT390基因,酶切、连接,构建原核表达载体pET-32a(+)-mMIP-1α-DT390,重组质粒证实构建成功后,转化感受态大肠杆菌BL21(DE3),通过IPTG诱导融合蛋白表达,并用SDS-PAGE和蛋白免疫印迹法鉴定。结果成功构建了mMIP-1α与DT390基因的原核融合表达载体pET-32a(+)-mMIP-1α-DT390,重组载体在大肠杆菌中获得了稳定的表达,表达蛋白的相对分子质量与预期值一致,并可被抗mMIP-1α和抗白喉毒素的特异性抗体所识别。结论获得了mMIP-1α与DT390融合基因在原核系统中的稳定表达,为研究其临床应用奠定了基础。

Objieetive To construct a new recombinant immunotoxin expression vector by fusing mouse macrophage inflammatory protein-1α gene and a tnmcated diphtheria toxin （DT390） gene, and examine the expression of mMIP-1α-DT390 fusion protein in Escherichia coli. Methods mMIP-1α cDNA was cloned from mouse hver tissue through reverse transcription-polymerase chain reaction （RT-PCR）, and the DT390 gene was cloned from a eukaryotic expression vector Sra-DT390 by PCR. The obtained genes were digested by restriction endonucleases, and then inserted to the expression plasmid pET-32a（ ＋ ） to produce a recombinant vector pET-32a（ ＋ ）-mMIP-1α-DT390. The confirmed vector was transformed to E. coli BI21 （DE3） and induced by IPTG, and then the expressed protein was obtained and detected by SDS-PAGE and Western-blotting. Results The new recombinant immunotoxin expression vector PET-32a（ ＋ ）-mMIP-1α-DT390 was constructed successfully. The mMIP-1α-DT390 fusion protein was expressed in E. coli BL21 （DE3）, and the molecular weight of the fusion protein was identical to the expected value. Furthermore, the protein could reacted with the specific antibody against mMIP-1α and DT390, respectively. Conclusion pET-32a（ ＋ ）-mMIP-1α-DT390 recombinant immtmotoxin expression vector may have some potential value in clinical application.