maltosyl-agarose亲和层析法提取人肺灌洗液中肺表面活性物质结合蛋白A及其鉴定

Purification of the surfactant associate proteins A from human bronchoalveolar lavage by affinity chromatography on maltosyl-agarose and its identification

目的:提取及纯化人肺灌洗液中肺表面活性物质结合蛋白A(SP-A)。方法:由支气管肺泡灌洗液经10000g离心40min获得的沉淀中提取SP-A。沉淀溶解于6M的尿素,SP-A蛋白重新复性后上清过maltosyl-agarose柱进行亲和层析,用梯度EDTA洗脱SP-A,并过surpose6柱纯化。聚丙烯酰胺电泳和Westernblotting鉴定。结果:经亲和层析法可以从人肺灌洗液中提取出高纯度的SP-A,聚丙烯酰胺电泳在36ku和70ku处可见清晰条带,在分子量118ku以上亦可看到3条清晰条带,经Westernblotting鉴定均为特异性SP-A蛋白。结论:采用maltosyl-agarose亲和层析法可以从肺灌洗液中获得高纯度的SP-A。为进一步研究SP-A的功能和制备SP-A单克隆抗体提供了充足的SP-A来源。

Objective To isolate and purify human surfactant associate protein A(SP-A)from human bronchoalveolar lavage.Methods The SP-A by fractionation of the pellet obtained after spinning the lung lavage at 10 000×g for 40 min were isolated.The pellet was solubilised in 6 M urea and,following renaturation,the solubilised proteins were applied to maltosyl-agarose and SP-A eluted using a gradient of EDTA.The SP-A was further purified by gel-filtration on Superose-6.The final product was identified by SDS-PAGE electrophoresis and Western blotting.Results The high purity SP-A from human lung lavage were obtained by affinity chromatography on maltosyl-agarose.There were five clear band on the SDS-PAGE gel with apparent molecular weights (Mr) of 36,70 and ≥118 kDa.We consider the higher-Mr protein was the SP-A aggregates.All the visible bands were identified to be special SP-A by Western blotting analysis.Conclusion High purity SP-A can be obtained from human lung lavage by affinity chromatography on maltosyl-agarose.This method can provide enough SP-A for further research on SP-A structure and function.In addition it can provide enough antigen for the preparation of SP-A monoclonial antibody.