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Design and Functional Analysis of a Novel Staphylokinase Variant with Reduced Immunogenicity

Author: Wang
Tutor: XuDongGang
School: PLA Military Academy of Medical Sciences
Course: Genetics
Keywords: staphylokinase mutant epitope thrombolysis immunogenicity
CLC: R91
Type: Master's thesis
Year: 2007
Downloads: 33
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Abstract

Staphylokinase (SAK) is a protein of single chain polypeptide secreted by a lysogenic strain of Staphylococcus aureus. SAK of M.W. 15.5kD consists of 136 aminoacid residues containing 45 charged aminoacid residues, without either cysteine or disulfide bond.Staphylokinase is a fibrin-selective thrombolytic agent of important potency as a thrombolytics, which can mediate efficiently the lysis of platelet-rich clots and has excellent fibrin specificity. Thus, its application to thrombolysis would be extensive.Being a heterologous protein, SAK is immunogenic to human body, which can elicit a high titer of neutralizing antibodies about 2 weeks after intravenous injection, moreover, SAK activity will be affected by these antibodies.Reconstuction of the epitop of SAK has become a main way to reduce its immunogenicity. In this paper we use the Biosun analysis system to analyze the epitopes of SAK and substitute the major aminoacid residues in its epitopes with other aminoacid residues according to the results of prediction. 10 mutants of SAK(SAK1-SAK10) were constructed, 4 out of which had been espressed in the supernate. After expression, purification and bioassays of these mutants (SAK1,2,7,8), the mutant SAK2 was selected for further study. SAK2 was prepared in a large scale; the PcAb of wtSAK and SAK2 were prepared; and the change of immunogenicity of SAK2 was analyzed. The main results are as follows:1 Construction, expression and activity analysis of series mutants of staphylokinaseThe gene of wtSAK was amplified by overlap extension PCR. The mutant gene was ligated with expression vector pBV220. After induction, SAK1-10 were all expressed as inclusion body, and the mutants (SAK1, SAK2, SAK7andSAK8) with more mutant sites had been solubly expressed. After purification, these proteins were tested for their activity. The results indicated that all the specific activities of the mutants (SAK1, SAK7andSAK8) were decreased, but that of SAK2 was corresponding to wtSAK.2 Technologies for large scale preparation of SAK2, PcAb and Comparation of reactivity between antibodiesThe engineering bacteria strain of SAK2 was fermented in large scale (20L volume). The purity of SAK2 was over 95% monitored by using anion-exchange and gel filtration chromatography. The fibrin plate assay indicated that the specific activity of SAK2 was (3. 5±1. 4)×10~4AU/mg, in comparison with wtSAK (4. 1±0. 8)×10~4AU/mg.The polyclonal antibody against wtSAK and SAK2 was prepared by immunizing rabbit. The antisera reached a titer as high as 1×10~7. The immunoreactivity of SAK2 to polyclonal antibody against wtSAK was sharply reduced to a very low level determined by ELISA and fibrin plate assay, which indicated that some epitopes of wtSAK had been deleted. The antisera were purified through a DEAE chromatography.3 Analysis of antibody induced in rhesus monkey by wtSAK and SAK2Rhesus monkey was injected with wtSAK and SAK2 on alternate days within 2 weeks at a same dose of 0.02mg/kgBW, and then this procedure was repeated after ten weeks. The observation was lasted for about three weeks from the end of second procedure. The whole process contained two procedures of administration, and lasted for about six months. The sera at 15 different time points of the whole process were obtained. The level of antibody against wtSAK and SAK2 were tested by using ELISA. The result suggested that the level of antibody against SAK2 was obviously lower than that of wtSAK. The neutralizing activity of antibody against wtSAK was obviously higher than that of SAK2, which was displayed by fibrin plate assay. After being incubated with each antibody, only 3.8% thrombolysis activity was kept in wtSAK, but that in SAK2 was 75.3%.4 The effect of this injection on blood coagulation system and fibrinolysis systemIn the six months, some indexes were tested such as FIB, PLG,α2-PI and PT, APTT, TT.The results indicated that the injections of both two proteins would not cause a notable change of the index in safety aspect.

Design and Functional Analysis of a Novel Staphylokinase Variant with Reduced Immunogenicity

Abstract

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