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目的 从人的子宫颈黏液分离和鉴定新抗菌多肽,探讨子宫颈黏液抗菌机制。方法 用5%乙酸提取人子宫颈黏液可溶物,应用电泳凝胶抗菌蛋白分析法分析其抗菌蛋白,制备酸性尿素聚丙烯酰胺凝胶电泳及反相高效液相色谱分离纯化抗菌多肽。凝胶琼脂糖弥散法抗菌检测抗菌活性。纯化的抗菌多肽进行氮端氨基酸序列测定,根据其序列设计简并引物,利用简并聚合酶链反应(PCR)技术扩增其全长cDNA。通过BLAST硷基比对程序分析比较与已知蛋白的同源性。进一步应用常规基因重组技术构建其原核表达质和制备重组多肽。采用最小抑菌浓度和最小杀菌浓度检测重组多肽的抗菌效能。结果 从子宫颈黏液中分离纯化到一个具抗菌活性的多肽,通过蛋白测序、简并PCR及BLAST等方法鉴定该蛋白为HMGN2高游动性蛋白。制备获得重组HMGN2多肽,重组HMGN2抗大肠杆菌和铜绿假单胞菌的最小抑菌浓度分别为10 .42μg/ml±3. 13μg/ml和27. 78μg/ml±8 .33μg/ml,与人中性粒细胞防御素相当;最小杀菌浓度分别为20 .83μg/ml±6 .25μg/ml和55 .56μg/ml±16 .67μg/ml。结论 除已知的抗菌肽外,HMGN2亦可能是宫颈黏液含有的抗感染效应分子之一。
Objective To isolate and identify new antimicrobial peptides from human cervical mucus and explore the antimicrobial mechanism of cervical mucus. Methods Human cervix mucilaginous solubles were extracted with 5% acetic acid. The antibacterial proteins were analyzed by electrophoresis gel antimicrobial protein analysis. Acidic urea polyacrylamide gel electrophoresis and reverse phase high performance liquid chromatography were used to separate and purify antimicrobial peptides. Gel agarose dispersion method antibacterial activity detection Purified antimicrobial peptides were used for the determination of the amino acid sequence of the N-terminus. Degenerate primers were designed according to their sequences and their full-length cDNAs were amplified by degenerate polymerase chain reaction (PCR). Homology with known proteins was compared by BLAST base alignment program analysis. Further, conventional prokaryotic recombination technology was used to construct the prokaryotic expression vector and prepare the recombinant polypeptide. The antibacterial efficacy of the recombinant polypeptide was tested by the minimum inhibitory concentration and the minimum bactericidal concentration. Results A polypeptide with antibacterial activity was isolated and purified from cervical mucus. The protein was identified as HMGN2 highly motile protein by protein sequencing, degenerate PCR and BLAST. Preparation of recombinant HMGN2 polypeptide, recombinant HMGN2 anti-Escherichia coli and Pseudomonas aeruginosa minimum inhibitory concentration of 10 .42μg / ml ± 3. 13μg / ml and 27. 78μg / ml ± 8. 33μg / ml, and human Neutrophil defensins; the minimum bactericidal concentrations were 20.83μg / ml ± 6.25μg / ml and 55.56μg / ml ± 16.67μg / ml, respectively. Conclusion In addition to the known antimicrobial peptides, HMGN2 may also be one of the anti-infection effector molecules in cervical mucus.