【摘要】 PCSK9是机体内调节低密度脂蛋白胆固醇(LDL-C)代谢的重要蛋白之一,抑制PCSK9能够显著地降低血清LDL-C的水平。本研究的目的是构建靶向PCSK9的重组蛋白疫苗,为降低胆固醇疫苗药物的开发提供实验基础。本课题通过重叠PCR方法将白喉毒素跨膜结构和人PCSK9催化结构域基因串联,构建重组基因片段DTT-h PCSK9,在大肠杆菌中表达后经GST亲和纯化制备重组蛋白DTT-h PCSK9。用该蛋白免疫Balb/c小鼠后通过ELISA方法检测抗体应答水平,并测定小鼠体内胆固醇含量。结果显示,在小鼠体内DTT-h PCSK9引起了抗人PCSK9免疫反应,抗血清效价为1:10 000,并且该疫苗能够显著降低小鼠体内血清胆固醇含量(p<0.05)。更多还原

【Abstract】 Proprotein convertase subtilisin-like/kexin type 9(PCSK9) is an attractive therapeutic target for cholesterol lowering. Inhibit the function of PCSK9 can significantly reduce serum levels of LDL-C. This research is to design a recombinant protein vaccine targeting PCSK9 and provide an experimental basis for the development of cholesterol-lowering vaccine drugs. Firstly, human PCSK9 catalytic domain was fused with the transmembrane domain of diphtheria toxin(DTT) by overlap PCR. After prokaryotic expression and purification by GST affinity chromatography recombinant protein vaccine DDT-h PCSK9 was obtained. After immunization with the protein in Balb/c mice, antibody response levels were detected by ELISA, and cholesterol levels in mice were measured. ELISA results show that DTT-h PCSK9 induced anti-h PCSK9 immune response in mice, the titer is about 1:10 000.Furthermore, DTT-h PCSK9 could elicit anti-m PCSK9 immune response, and lowers total serum cholesterol greatly(p <0.05). This study is the first time to develop a PCSK9-targeting protein vaccine DTT-h PCSK9, and the protein may become a protein vaccine that can treat hypercholesterolemia.更多还原