The Impact Of Mutations Within Immunodominant Residue Of T Cell Epitope Region 18-34 On The Biological Activity Of Staphylokinase

Staphylokinase(Sak), a profibrinolytic protein produced by the lysogenic Staphylococcus aureus, consists of 136 amino acids in a single polypeptide chain without disulfide bridges and has a molecular weight of approximately 15,500. Clinical treatment of acute myocardial infarction research showed that Sak was a promising thrombolytic agent, which was at least equipotent to recombinant tissue plasminogen activator(rt-PA) for acute myocardial infarction and significantly more fibrin-selective. Furthermore, it is easy to be over-expressed in Escherichia coli and has relatively low production costs. But Sak could elicit high titers of neutralizing antibodies within two weeks after intravenous injection due to its heterologous property, patient’s body will produce immune response, even produce allergic reaction, A certain extent has affected the Sak in clinical applications.Using the method of site-directed mutation reconstruction Sak, Remove its antigen epitope, It is getting new low immunogenicity thrombolysis drug one of the important methods.According to report, there were six distinct immunogenic regions (17-34,44-57, 73-87,89-99,111-120,125-135) binding to multiple HLA-DR molecules.The 18-34 epitope region with a core of Y24 amino acid was crucial to Sak unite HLA-DR. Therefore, this paper mainly studies mutations of key amino acids in Sak T cells epitope region 18-34 to the influence of the biological activity, screening a fibrinolytic high activity, immunogenicity low mutants.Using QuickChang site-directed mutation PCR method, To Sak T cells epitopea regional 18-34 anchor amino acids Y24 and its adjacent sites mutations, Obtain Sak(Y24A),Sak(Y24V),Sak(Y24I),Sak(Y24L),Sak(V27A),Sak(N28A) and Sak(V29A), were successfully expressed in E. coli DH5a as a soluble cytoplasmic proteins and accounted for more than 40% of the total cellular proteins. The expressed proteins were purified by a three-step chromatographic purification process. SDS-PAGE and HPLC analyses results indicated that the purified proteins were almost completely homogeneous and the purities of Sak mutants exceeded 97%. The next step, To the Sak variants stability, fibrinolytic activity and immunogenicity systematically studied.With casein flat measure each Sak mutants fibrinolytic activity, The results show Sak(V29A) and Sak(N28A) maintains the wt-Sak quite fibrinolytic activity (80%) Sak(V27A) of fibrinolytic activity drop to 50% of wt-Sak, The rest of the fibrinolytic activity is very low.Use Sak(V27A), Sak(N28A) and Sak(V29A) immune mice, The immunoreactivity of the Sak variants to polyclonal antibody against Wt-sak was reduced determined by ELISA, The antibody compared with wt-sak were down nearly 13%,16% and 10%. The abilities of Sak(V27A), Sak(N28A) and Sak(V29A) to stimulate proliferation of T cells from BALB/c mice and to bind mouse anti-Sak polyclonal serum were significantly lower than those of Wt-sak.From constructing the seven variants, screening Sak(N28A) and Sak(V29A) These two Sak variants which decrease the immunogenicity maintained again and Wt-sak quite active, for further establishing a new low immunogenicity variants laid a foundation.