【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?更多还原

【Abstract】 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(0.45 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.更多还原