摘要
以葡激酶突变体质粒mSAK(K130T ,K135R) pBV2 2 0为模板,PCR重叠引物延伸法引入突变位点,并将该片段克隆至载体pBV2 2 0 ,构建了RGD mSAK pBV2 2 0质粒,转化大肠杆菌后热激诱导获得了高效表达,表达产物占菌体总蛋白的5 0 %以上,且主要以可溶性形式存在,所获蛋白依次用Q SepharoseHP柱、SephaycrylS 2 0 0HR柱和SP柱进行纯化,纯化的蛋白的纯度可达98%以上,纤维蛋白溶圈法体外溶栓活性测定结果表明,所获RGD mSAK蛋白溶栓活性与野生型葡激酶相当,豚鼠体内免疫试验证明突变体的免疫原性也有所降低,血小板聚集试验分析突变体蛋白的抗血小板聚集能力,RGD 葡激酶突变体具有一定的抗血小板聚集能力。
In order to construct RGD-mSAK mutant with reduced immunogenicity, and identify its biological activity after purification, mSAK gene fragment was amplified by over-lapping extension PCR. Then the gene was inserted into the prokaryotic expression vector pBV220 with P RP L promoters after confirmed by DNA sequencing; the expression plasmid pBV220-RGD-mSAK was constructed, and then was transformed into E. coli. DH5α. After temperature induction, the mutant Staphylokinase was over-expressed and much of protein was in the supernate of lysate, which is over 50% of total protein in the host. The protein was isolated and purified in Q-Sepharose FF, Sephacryl S-200 and SP, high purity protein was obtained and its purity was over 98%. The thrombolysis activity of the RGD-mSAK protein is 1.68×10 5u/mg by fibrin plate assay, which is slightly higher than that of the wild-type, and antiserum titers raised against this protein in guinea pigs were much lower than those of wild-type SAK, determined by ELISA. In anti-platelets aggregation assay in vitro, the RGD-mSAK protein has obvious inhibition activity of platelet aggregation in low concentration comparing to the control group and wild-type SAK group. So the RGD-mSAK protein is a low immunogenicity, bi-function molecular with both thrombolysis activity and anti-embolism activity. It provided the basis for further research of RGD-SAK.
出处
《生物工程学报》
CAS
CSCD
北大核心
2005年第3期456-460,共5页
Chinese Journal of Biotechnology
基金
北京市自然科学基金 (No.7992 0 3 0 )
军事医学科学院新药开发基金 (No .2 0 0 10 80 41)资助项目~~
关键词
葡激酶
RGD
免疫原性
staphylokinase, immunogenicity, RGD
