子宫内膜黏液抗菌肽的分离纯化及鉴定

目的从人子宫内膜黏液酸溶性提取物纯化及鉴定抗菌多肽,探讨子宫内膜的抗菌机制。方法采用酸性尿素聚丙烯酰胺凝胶电泳琼脂糖弥散法分析人子宫内膜黏液酸溶性提取物的抗菌活性,切下相应的抗菌条带进行洗脱电泳,并用反相高效液相色谱进一步分离纯化。用琼脂糖弥散法检测纯化分子抗菌活性;Trieine-SDS-PAGE电泳检测其相对分子质量。根据N端氨基酸序列和质谱分析的测序结果推导纯化分子的氨基酸序列,并进行生物信息学分析。结果从人子宫内膜酸溶性提取物中纯化出一种相对分子质量为6777的抗菌肽HUP-39,经鉴定为人血红蛋白α链N端第33-95的氨基酸残基片段。该片段富含碱性氨基酸,包含3个完整的α螺旋跨膜结构域,对大肠埃希菌氨苄西林耐药株ML-35p、标准株ATCC25922和临床分离株54080有较强的抑菌活性。结论本研究从人子宫内膜黏液酸溶性提取物中分离纯化出的抗革兰阴性菌多肽,为人血红蛋白α链片段。子宫内膜黏液中的抗菌分子不仅来源于上皮细胞和白细胞,也可来源于红细胞。

Antimicrobial peptide may originate from hemoglobin and play a role in the antibacterial mechanisms of endometrium

OBJECTIVE: To isolate and purify antimicrobial peptides from human uterus mucus. METHODS: Acid-soluble extract was obtained from the specimens of endometrium mucus from 3 hysteromyomas patients during total hysterectomy. Acidic urea-polyacrylamide gel electrophoresis was used to analyze the acidic extract. The corresponding antimicrobial band HUP was further isolated and purified by electrophoretic elution and reversed-phase high-performance liquid chromatography (RP-HPLC). The antimicrobial activity of the fractions was analyzed by agarose radial diffusion assay. The molecular weight was determined by Tricine-SDS-PAGE. According to the results of the N-terminal sequencing and Mass Spectrometry analysis, the amino acid sequences of the purified molecules were deduced. The deduced amino acid sequence of the antibacterial fragment was further analyzed by ExPASy and OMIGA softwares. RESULTS: An antibacterial peptide named HUP-39 was purified from the human uterine mucus with a molecular weight of 6.777 Ku. N-terminal sequencing and Mass Spectrometry analysis suggested that this antimicrobial peptide should be hHEM-alpha 33-95 amino acid fragment. ExPASy and OMIGA analysis showed that it contained 3alpha-helical transmembrane domains and its pI was 8.38. The fragment was mainly against Escherichia coli ML-35p, E. coli ATCC 25 922, and the clinically isolated strain E. coli 54 080. CONCLUSION: An antimicrobial peptide has been isolated and purified from human uterine mucus, a hHEM-alpha 33-95 amino acid fragment. The antimicrobial molecule in the uterine mucus originates not only from epithelial cells and leucocytes, but also from erythrocytes.