葡萄球菌肠毒素B突变体的纯化及其活性  

Purification and Characterization of Staphylococcal Enterotoxin B Mutants

作　　者：俞红[1] 钱利生[1] 熊思东[2] 

机构地区：[1]复旦大学基础医学院微生物学教研室,上海200032 [2]复旦大学基础医学院免疫学教研室,上海200032

出　　处：《复旦学报（医学版）》2001年第3期187-190,共4页Fudan University Journal of Medical Sciences

摘　　要：目的　从基因重组菌中纯化葡萄球菌肠毒素B(SEB)突变体及其野生型蛋白 ,研究其毒力及抗原性。方法　表达野生型SEB蛋白 (SEB N)和SEB突变体 (SEB M2 3、SEB 1 50 )的 3株重组菌经IPTG诱导 ,反复冻融后 ,制备细菌裂解液。将初步纯化的抗SEB抗血清与CNBr活化的Sepharose 4B作共价偶联 ,制成亲和层析柱。分柱纯化SEB N、SEB M2 3和SEB M1 50蛋白。用SDS PAGE和酚试剂改良法对纯化蛋白作纯度鉴定和定量 ;用双向免疫扩散试验和ELISA法作抗原性鉴定 ;用小鼠致死性试验作毒力鉴定。结果　SDS PAGE和双向免疫扩散试验表明 ,3种纯化蛋白均为单一条带 ,且都能与抗SEB抗体形成明显的沉淀线。ELISA表明SEB N、SEB M2 3、SEB M 1 50与抗SEB抗体有相似的结合力 ,提示 2 3位突变和 1 50位突变并未改变SEB的抗原表位。小鼠致死性试验表明 ,LPS激发的 3组小鼠 ( 6只 /组 ) ,分别用上述纯化蛋白攻击 ( 1 0 μg/只 ) ,SEB N组死亡率为 5/6;SEB M1 50组死亡率为 4 /6;SEB M2 3组死亡率为 0 /6;即使SEB M 2 3剂量增至 2 0 0 μg ,小鼠仍无死亡。结论　获得了SEB N、SEB M2 3和SEB M1 50 3种纯化蛋白 ,其中SEB突变体都保留了抗原性 ;SEB M 2 3与野生型SEB相比 ,毒力明显下降。为进一步研究其作为新型淋巴细胞激?Purpose: To purify staphylococcal enterotoxin B(SEB) mutants (SEB-M23 and SEB-M150) and SEB wide-type(SEB-N) from recombinant strains and to study the toxicity and antigenicity of purified SEB proteins. Methods: The bacterial cultures of three recombinant strains were frozen and thawed three times, centrifuged, filtered(O.45 μm) and SEB-containing lysates were prepared. SEB-N, SEB-M23 and SEB-M150 were purified by three columns containing Sepharose 4B coupled with anti-SEB respectively. The purified proteins were identified and quantified by SDS-PAGE and Lowery method. The antigenicity were identified by double agar immunodiffusion and ELISA. The toxicity were identified by mouse lethality assay. Results: SDS-PAGE manifested that three purified proteins showed a single band at 28 000. Double agar immunodiffusion showed three purified proteins could produce obvious precipitin line with anti-SEB. ELISA indicated that SEB-N, SEB-M23 and SEB-M150 were serologically identical with polyclonal anti-SEB and suggested that the mutation at residue 23 or 150 did not grossly distort the SEB molecule and that the mutant proteins share common epitopes with wild-type SEB. Mouse lethality assay demonstrated that among 6 LPS-potentiated mice, 5 mice died when given 10 μg SEB-N; 4mice died when given 10 μg SEB-M150; no mouse died when given 10 μg, even 200 μg SEB-M23. Conclusions: We obtained purified SEB-N, SEB-M23 and SEB-M150 proteins. SEB-M23 mutant protein showed normal antigenicity but low toxicity. These results have laid the foundation to further study SEB-M23 as novel immunoactivator or superantigen vaccine.

关 键 词：葡萄球菌肠毒素B 突变性 纯化 毒力 抗原性 

分 类 号：R378.11[医药卫生—病原生物学] Q75[医药卫生—基础医学]