重组抗人活化血小板单链抗体-尿激酶原导向溶栓剂的基因构建及表达

为了获得特异性高的导向溶栓药物,应用PCR技术,得到抗人活化血小板单抗(SZ-51)的Fab′基因片段。再用酶切方法,将Fab′中CH1基因片段替换成合成的连接分子(linker)基因,构建成单链抗体基因,并插入到人尿激酶原分泌肽基因及低分子量单链尿激酶(scu-PA-32k)之间,最终构建成重组抗人活化血小板单链抗体-尿激酶原融合蛋白基因。此融合蛋白基因在昆虫细胞中得到表达。纯化的表达产物SDS-PAGE鉴定,其分子量约为60kD,与预期值相符。其比活为9000IU/mg蛋白。ELISA法初步证明此重组的融合蛋白具有与活化血小板抗原结合特异性。

Construction and Expression of a Thrombolytic targeted Molecule with Single Chain Antibody against Activated Human Platelet

In order to obtain a thrombolytic targeted protein,a chimerical molecule,scFv UK32,containing the low molecular weight form of Pro urokinse(scu PA 32k) and the single chain form(scFv)of the monoclonal antibody SZ 51 against the glycoprotein GMP 140 on activated platelet membrane was designed and constructed.The cDNA of scFv of SZ 51 was generated by PCR in which SZ 51 Fab′ cDNA served as the template and a flexible linker (Gly 4Ser) 2Gly 3 was introduced between VH(variable region of heavy chain) and VK(variable region of light chain).Scu PA 32k DNA was generated by PCR in which the pro UK cDNA served as the template.In the transferring vector pBacPAK8/Fv UK32,scFv was inserted behind the Pst Ⅰ site after the secret peptide,and scu PA 32k was located on the C terminal of the entire chimerical molecule.pBacPAK8/Fv UK32 was cotransfected into insect cells Sf9 together with the linear virus AcNPV DNA(BacPAK6/ Bsu 36 Ⅰ digest).The fibrin plate assay showed the thrombolysis activity of the supernatant of cotransfected cells was 20 IU/ml.The affinity column of monoclonal antibody against B chain of pro UK was used to purify the expression product.SDS PAGE showed that the molecular weight of the chimerical protein was about 60 kD and the scanning of the SDS PAGE showed the purity was 82%.The thrombolysis activity of the lyophilized powder was about 9 000 IU/mg protein.The ELISA assay demonstrated that the chimerical molecule possessed the activity of binding to the activated platelet as well.