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目的通过对人精囊蛋白1(semenogelin I,SEMGI)第165~281位氨基酸序列片段(△SEMGI)的原核表达、纯化与鉴定,分析不同质量浓度下的该片段对精子活率影响。方法通过PCR扩增△SEMGI片段目的基因,与p MAL-c2x-tev载体重组;重组质粒转化ROSETTAⅡ后IPTG诱导表达,目的蛋白经过Amylose层析柱、Ni柱、分子筛纯化、免疫印迹法和蛋白质谱分析鉴定。最终,在4个不同蛋白质量浓度下孵育并分析其对人精子活率的影响。结果蛋白质谱分析证实△SEMGI蛋白片段重组成功,其在不同质量浓度下对精子活率均有抑制作用(P<0.05)。结论重组精囊蛋白1的C端片段具有抑制精子活率功能,为进一步研究蛋白结构提供基础。
OBJECTIVE: To purify and identify the amino acid sequence of amino acid sequence from the 165th to the 281st amino acids of semenogelin I (SEMGI), and to analyze the effect of different concentration of this fragment on sperm motility. Methods The target gene of △ SEMGI fragment was amplified by PCR and recombined with p MAL-c2x-tev vector. The recombinant plasmid was transformed into ROSETTAⅡ and induced by IPTG. The recombinant protein was purified by Amylose, Ni column, molecular sieve, immunoblotting and protein profiling Analysis and identification. Finally, four different protein concentrations were incubated and analyzed for their effect on human sperm viability. Results Protein spectrum analysis confirmed that the △ SEMGI protein fragment was successfully recombined, which inhibited the sperm motility at different concentrations (P <0.05). Conclusion The C-terminal fragment of recombinant seminal vesicle protein 1 has the function of inhibiting sperm motility and provides the basis for further study of protein structure.