摘要
依据本室获得的人TPO模拟肽序列 ,合成了该模拟肽的DNA序列 ,分别连接至 4种不同长度的人IgG1Fc基因片段的 5′端 ,并克隆至质粒表达载体pET2 8a(+) ,转化大肠杆菌BL2 1(DE3) ,筛选获得了 4种重组工程菌 ,其中3种分别高效表达了 3种不同长度的融合蛋白 ,而第 4种工程菌未表达。表达的 3种融合蛋白的分子量分别约为2 8kD、12kD和 12kD ,表达量约占菌体蛋白总量的 30 %左右 ,纯化获得了 3种TPO模拟肽融合蛋白。 3种融合蛋白均有较好的体外活性 ,维持TPO依赖细胞Ba F3-mp1生长的EC5 0分别为 :13、10、10nmol L。用血小板减少症小鼠动物模型 ,测定了它们的体内活性 ,3种融合蛋白均有升高血小板和缩短血小板恢复时间的功能 ,分别比TPO模拟肽活性提高了 18、8、8倍 ;而对白细胞及红细胞无显著影响。分别用 3种融合蛋白免疫BALB c小鼠 ,均未刺激小鼠产生抗TPO模拟肽抗体 ,并显示了较好的应用潜力。
Many antineoplastic agents can cause myelosuppression and thrombocytopenia. Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels. We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo. For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments. The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5′ terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5′ 648bp; Fc2: Fc 5′ 270 bp; Fc3: Fc 5′ 267 bp; Fc4: Fc 5′ 90 bp), and cloned into expression plasmid pET28a(+) for constructing four recombinant plasmids. By tranforming the four recombinant plasmids into E.coli. BL21(DE3) respectively, we got 3 kinds of engineered E.coli which express TPO+Fc chimeric proteins(28kD TPO+Fc1, 12kD TPO+Fc2 and 12kD TPO+Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF colume. The bioactivities of the expressed chimeric proteins(TPO+Fc1, TPO+Fc2 and TPO+Fc3 ),TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo , the EC50 on Ba/F3-mp1cells were 13, 10, 10 , 50, and 25nmol/L respectively, all of them can increase circulating platelet counts. Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO+Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc. These studies have laid basis for production of TPO mimetic peptide by genetic engineering .
出处
《生物工程学报》
CAS
CSCD
北大核心
2002年第4期424-430,共7页
Chinese Journal of Biotechnology
基金
全军"九五"重点攻关课题基金资助 (No .Z95 110 6 )~~
