低出血倾向、低免疫原性葡激酶mSAK(K130T,K135R)的表达、纯化与活性测定

为了有效降低葡激酶应用的副作用,构建低出血倾向、低免疫原性葡激酶突变体,高效表达纯化后进行活性鉴定,以野生型重组葡激酶基因为模板,PCR法引入突变位点(K130T,K135R),并将该片段克隆测序鉴定后,克隆至表达载体pBV220,构建低免疫原性葡激酶突变体.表达后的蛋白用Q-SepharoseFF柱与SephacrylS-200进行纯化,纤维蛋白溶圈法进行活性测定,体外抗体中和试验与豚鼠免疫试验测定突变体蛋白的免疫原性,同时进行动物体内出血倾向观察.测序结果表明,相应位点获得突变,无非特异性突变,将突变后的片段连接pBV220导入大肠杆菌热激诱导获得了高效表达,表达产物占菌体总蛋白的40%~50%.产物主要以可溶性形式存在,经两步纯化后的蛋白的纯度可达98%以上.活性测定试验表明,该突变体的活性较野生型葡激酶稍低,体外中和抗体试验和豚鼠免疫试验证明免疫原性大为下降,豚鼠的皮肤出血以及肺部病理切片均显示该突变体蛋白引起的出血倾向明显降低.

Constuction, Over-expression, Purification and Activity Analysis of mSAK (K130T,K135R) with Reduced Immunogenicity and Lower Bleeding Tendency

To construct mSAK mutant with reduced immunogenicity and lower bleeding tendency, and identify its biological activity after purification, mSAK (K130T, K135R) gene fragment was amplified by PCR and inserted into the prokaryotic expression vector pBV220 with P_RP_L promoters after confirmed by DNA sequencing. The expression plasmid pBV220-mSAK (K130T, K135R) was constructed, and then was transformed into E.coli DH5α. After temperature induction, the mutant staphylokinase was over-expressed and much of the protein was in the supernate of lysates, which was about 40%～50% of total protein in the host cells. The protein was isolated and purified in Q-Sepharose FF and Sephacryl S-200 HR, high purity protein was obtained with purity 98%. The thrombolysis activity of the mSAK protein was 8.77×10 4 U/mg by fibrin plate assay, which was slightly lower than that of wild type, and antiserum titers in guinea pigs were much lower than those of wild-type SAK with ELISA assay. Its immunogenicity, which was determined by both antibody neutralizing assay in vitro and guinea pig in vivo, was sharply reduced comparing to that of wild-type staphylokinase. Meanwhile, all guinea pigs in wt-SAK immunogenicity assay have body surface bleeding while the bleeding was not observed in mSAK group, and this was confirmed by the lung pathological section. These results provided the basis for further research of mSAK with lower bleeding tendency.