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目的构建穿膜肽TAT与EGFP融合蛋白的原核表达系统,研究该融合蛋白对人成熟精子的穿膜作用和对精子运动参数的影响。方法以p EGFP-N1载体为模板,设计N端和C端含有TAT序列的引物,用PCR技术扩增TAT-EGFP和EGFP-TAT基因,扩增产物插入载体p ET28a,构建成重组质粒p ET28a-TAT-EGFP和p ET28a-EGFPTAT。将重组质粒转入大肠杆菌DE3中,IPTG诱导TAT-EGFP和EGFP-TAT融合蛋白表达。表达产物经Ni-NTA亲和层析柱纯化后SDS-PAGE电泳鉴定。将融合蛋白TAT-EGFP和EGFP-TAT分别加入正常人精子中孵育,荧光显微镜观察融合蛋白TAT-EGFP和EGFP-TAT穿膜情况。结果成功构建了高表达重组质粒p ET28a-TAT-EGFP和p ET28aEGFP-TAT,纯化了分子质量约为32 k D的融合蛋白TAT-EGFP和EGFP-TAT。融合蛋白TAT-EGFP和EGFP-TAT在正常人成熟精子中有穿膜作用。不同浓度的融合蛋白TAT-EGFP和EGFP-TAT对正常人成熟精子存活率及运动参数无明显影响。结论通过对融合蛋白TAT-EGFP和EGFP-TAT表达纯化及活性分析,证实穿膜肽TAT在精子中的跨膜转运作用,为将来精子研究提供了实验基础。
Objective To construct a prokaryotic expression system of the transmembrane peptide TAT and EGFP fusion protein to study the transmembrane function of the fusion protein on human mature sperm and its influence on sperm motility parameters. Methods The primers of TAT sequence were designed at the N-terminal and C-terminal using p EGFP-N1 vector as a template. The TAT-EGFP and EGFP-TAT genes were amplified by PCR. The amplified product was inserted into vector p ET28a to construct recombinant plasmid p ET28a -TAT-EGFP and p ET28a-EGFPTAT. The recombinant plasmid was transformed into E. coli DE3, IPTG induced TAT-EGFP and EGFP-TAT fusion protein expression. The expressed product was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE electrophoresis. The fusion proteins TAT-EGFP and EGFP-TAT were added to normal human spermatozoa and incubated. The transfection of TAT-EGFP and EGFP-TAT was observed by fluorescence microscopy. Results High expression recombinant plasmids p ET28a-TAT-EGFP and p ET28aEGFP-TAT were successfully constructed, and the fusion proteins TAT-EGFP and EGFP-TAT with molecular mass of about 32 kD were purified. The fusion proteins TAT-EGFP and EGFP-TAT have a transmembrane action in normal human mature spermatozoa. Different concentrations of fusion proteins TAT-EGFP and EGFP-TAT had no significant effect on the survival rate of mature human sperm and the exercise parameters. Conclusion The purification and activity analysis of TAT-EGFP and EGFP-TAT fusion proteins confirmed the transmembrane transport of transmembrane peptide TAT in sperm and provided the experimental basis for future sperm research.