葡萄球菌蛋白A的纯化新工艺

目的建立一种高效、简便、实用的分离纯化葡萄球菌蛋白A(SPA)的工艺。方法采用超声波破菌和IgG-Sepharose4B亲和层析分离纯化SPA;以Lowry法检测蛋白含量;双向琼脂免疫扩散法测定效价和特异性;用还原和非还原SDS-PAGE法检测纯度和相对分子质量。结果此法制得的3批SPA的蛋白含量分别为4·7、4·4和5·4mg/ml,收率分别为2·70、2·64和3·78g/100g湿菌。对人IgG的免疫双扩散效价均为1:64;与正常人、豚鼠、家兔和小鼠的血清反应均出现一条沉淀线,而与鸡和羊不出现沉淀线。经非还原SDS-PAGE检测只呈现一条蛋白带,相对分子质量约为160000～180000;经还原SDS-PAGE检测呈现两条蛋白带,相对分子质量分别约为67000和34000。结论已成功建立了分离纯化SPA的新工艺。

A Novel Procedure for Purification of Staphylococcal Protein A

Objective To develop a highly effective,simple and practical procedure for separation and purification of staphylococcal protein A(SPA).Methods Separate and purify SPA by ultrasonication and IgG-Sepharose 4B affinity chromatography.Determine the purified SPA for protein content by Lowry method,for titer and specificity by double immunodiffusion test,and for purity and relative molecular mass by reduced and non-reduced SDS-PAGE.Results The protein contents of 3 lots of purified SPA were 4.7,4.4 and 5.4 mg/ml,and their yields were 2.70,2.64 and 3.78 g/100 g wet bacteria respectively.All the titers of the 3 lots determined by double immunodiffusion test with human IgG were 1:64.The purified SPA showed a single precipitation line with each of normal human,guinea pig,rabbit and mouse sera,however,it showed no precipitation line with chick or sheep serum.Non-reduced SDS-PAGE showed a single protein band with relative molecular mass of about 160 000-180 000,while reduced SDS-PAGE showed two protein bands with relative molecular masses of about 67000 and 34000 respectively.Conclusion A novel procedure for separation and purification of SPA was successfully developed.