人LAK细胞抗菌活性多肽HLP-3P21的分离纯化和鉴定

目的　分离纯化人LAK细胞小分子抗菌多肽 ,鉴定其分子特性 ,检测其抗菌活性。方法　采用密度梯度离心法分离人外周血单个核细胞 ,并用IL 2和PHA刺激培养。 5 %乙酸匀浆细胞获得其酸溶性提取物。应用制备酸性尿素聚丙烯酰胺凝胶电泳技术和反相高效液相色谱技术分离纯化多肽 ,Tricine SDS PAGE鉴定其相对分子质量 (Mr) ,运用琼脂糖弥散法鉴定其抗菌活性 ,采用Edman降解法测定其氮端序列 ,并进行肽质谱分析。结果　从人LAK细胞酸溶性提取物中纯化出一多肽 ,Tricine SDS PAGE表观Mr 为 17× 10 3,命名为HLP 3P2 1(humanlymphocytepeptide) ,具有抗致病性大肠杆菌ML 35P及铜绿假单胞菌ATCC6 5 92 2的活性。其氮端序列为PKRKAEGDAK ,肽质谱的检索分析未发现匹配度大于 5 0 %的蛋白质分子。结论　HLP 3P2 1可能为LAK细胞一新的抗菌效应分子。

Isolation and characterization of an antibacterial polypeptide from human LAK cells

Objective To isolate and purify new peptides with antibacterial activity from human LAK cells. Methods Preparative acid-urea polyacrylamide electrophoresis and reverse phase HPLC were performed to isolate and purify polypeptides from acid extract of human LAK cells. The molecule mass was analyzed by Tricine-SDS-PAGE. Radial agar diffusion assay was used to analyze the antibacterial activities of isolated polypeptides. Edman degradation method and peptide-mapping were used to analyze amino-sequence and define the molecular. Results A 16-17kD bioactive polypeptide that had antibacterial against activities E.coli ML-35P and Pseudomonas aeruginosa ATCC65922 was purified from human LAK cells, which was named as HLP-3P21. N-terminal sequence of HLP-3P21 was PKRKAEGDAK. The result of peptide-mapping indicated that there was no 50% intensity matched protein in data base. Conclusion HLP-3P21 may be a new effector molecule of human LAK cells. [