人过敏毒素C5a反义cDNA的克隆、表达和拮抗作用

Cloning and Expression of the Antisense cDNA of the Human C5a Anaphylatoxin in E.coli and Antagonism

采用引物二聚体配对搭桥法成功扩增了人过敏毒素C5a全长的反义cNDA ,将扩增的反义cNDA克隆到原核表达载体pPROEXTM HTc上 ,与 6×His头形成融合基因 ,转化E .coliDH5α ,30℃下经IPTG诱导 ,该融合蛋白在大肠杆菌中以可溶性形式表达 ,表达量达 10 % ;利用金属螯合亲和层析一步法纯化 ,得到纯度 80 %的融合蛋白 ;烟草蚀刻病毒蛋白酶酶切超滤浓缩后 ,得到高浓度高纯度人过敏毒素C5a反义肽 ,经N端蛋白测序鉴定 ,其氨基酸序列正确 .髓过氧化物酶 (myeloperoxi dase ,MPO)为单核细胞PMN所特有 ,其活性可以间接反映C5a趋化白细胞的能力 ,C5a刺激血管内皮细胞表达胞间粘附分子 (ICAM 1)、血管间粘附分子 (PCAM 1)等 ,后者能增加对PMN的粘附 ,检测MPO活性可间接反映出C5a的功能 .还观察了纯化的C5a反义肽对C5a刺激内皮细胞胞浆[Ca2 + ]浓度变化的影响 .高浓度高纯度的C5a反义肽对C5a过敏毒素存在拮抗作用 ,说明反义肽在补体系统C5a分子中是存在的 .

To obtain the whole antisense cDNA and the antisense peptide of the human C5a anaphylatoxin the whole sequence cDNA of the human antisense C5a anaphylatoxin was cloned by PCR using the bridge primer dimer. The antisense cDNA were cloned on prokaryotic expression vector pPROEXTM HTc by fusion expression with 6×His gene in the E.coli DH5α. The fusion peptides were expressed in the soluble form by IPTG at the 30℃. The antisense peptides of the C5a could inhibit the C5a anaphylatoxin function that induced ICAM 1 expression, and could decrease the MPO activity of the adhesion PMN cell. This work establishes a foundation for investigating the interactions between the sense peptide and its antisense peptides.