葡萄球菌蛋白A的纯化新工艺

A Novel Procedure for Purification of Staphylococcal Protein A

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。

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 67 000 and 34 000 respectively. Conclusion A novel procedure for separation and purification of SPA was successfully developed.