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 .
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
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）
