Zn^2＋及其谷氨酸结合位点突变对金黄色葡萄球菌肠毒素 C2超抗原活性的影响  

Effects of Zn^2＋and Zn^2＋-coordinating glutamic acid residues on the superantigenic activities of staph-ylococcal enterotoxin C2

作　　者：刘彦礼[1] 牛荣成[1] 徐明恺[2] 李旭[2] 孙健[2] 张惠文[2] 

机构地区：[1]新乡医学院生命科学技术学院,453003 [2]中国科学院沈阳应用生态研究所,110016

出　　处：《中华微生物学和免疫学杂志》2014年第6期442-446,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家科技重大专项重大新药创制（2012ZX09102301-013）;新乡医学院博士科研启动基金（505033）

摘　　要：目的：研究金黄色葡萄球菌（金葡菌）肠毒素C2（SEC2）中与Zn^2＋结合相关的谷氨酸残基突变及Zn^2＋自身对其超抗原活性的影响。方法应用定点突变技术将SEC2中与Zn^2＋结合相关的谷氨酸残基突变成丙氨酸残基，分离纯化后获得突变蛋白rSEC2（E71A）、rSEC2（E80A）及rSEC2（E71A/E80A）；突变蛋白的超抗原活性及Zn^2＋对SEC2超抗原活性的影响通过MTT法检测其刺激小鼠脾淋巴细胞的增殖来验证，而结构稳定性通过对胃蛋白酶及胰蛋白酶的抵抗能力来检测。结果蛋白酶酶切结果与脾淋巴细胞增殖实验结果表明参与第2个Zn^2＋结合的71位及80位谷氨酸残基突变对SEC2的酶解稳定性及超抗原活性没有显著影响；但微量Zn^2＋（10μmol/L）却能显著增强rSEC2的超抗原活性，并且在EDTA存在下，仅有Zn^2＋在一定浓度下才可完全恢复其超抗原活性。结论SEC2分子中的Zn^2＋结合位点的突变对其自身结构稳定性和超抗原活性无显著影响，但SEC2超抗原活性发挥过程中Zn^2＋可能通过其他途径间接影响其超抗原活性。Objective To investigate the role of Zn^2＋and Zn^2＋-coordinating glutamic acid residues （ 71Glu and 80Glu ） in the superantigenic activities of staphylococcal enterotoxin C2 （ SEC2 ） .Methods Over-lap PCR was used to amply genes encoding recombinant mutant proteins rSEC 2 （ E71A）, rSEC2 （E80A） and rSEC2 （E71A/E80A）.The mutant proteins were expressed in E.coli BL21 （DE3） and purified by affinity chromatography .The differences of biological activities between rSEC 2 and its mutants were compared in vitro.The effects of Zn^2＋on the superantigenic activities of rSEC 2 were evaluated by analyzing the proliferation of splenic lymphocytes in the presence or absence of ethylenediaminetetraacetic acid （ EDTA） .Results The substitution of glutamic acid residue at position 71 and 80 by alanine residue had no significant effects on the superantigenic activities and the conformational stability of rSEC 2 mutants.However, traces of Zn^2＋（10μmol/L） could significantly enhance rSEC2-induced proliferation of splenic lympho-cytes, and only certain amount of Zn^2＋could completely restore rSEC2-induced proliferation in the presence of EDTA.Conclusion This study indicated that mutations at Zn^2＋-coordinating glutamic acid residues had no significant effects on the conformational stability and the superantigenic activities of SEC 2.Zn^2＋might play a role in regulating the superantigenic activities of SEC 2 through some indirect ways .

关 键 词：ZN^2+ 金黄色葡萄球菌肠毒素C2 超抗原 T淋巴细胞 

分 类 号：R155.31[医药卫生—营养与食品卫生学]