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	Construction and expression of antibody targeted plasminogen activator
	Jiang, PC ; Changgeng, R ; Ru, BG
	2000
关键词	antibody-targeted plasminogen activator urokinase single-chain of fragments of variable region of antibody (scFv) SINGLE-CHAIN UROKINASE MOLECULAR WEIGHT FORM ESCHERICHIA-COLI RNA-POLYMERASE CLONED GENES PURIFICATION FIBRIN PROTEINS CONJUGATE BINDING
英文摘要	It has been known that antibody-mediated plasminogen activator wilt be much more specific than its parent molecular. To get a cheaper and more effective medicine for thrombolytic therapy, we used SZ51, a GMP140 specific monoclonal antibody, and a truncated single-chain urokinase to construct a novel targeted plasminogen activator. PCR was used to amplify the region of VL and VH chains from Fab of SZ51, GMP140 specific monoclonal antibody, and scu-PA-32KD(leu144-leu411) from urokinase gene, respectively. Through suitable linker and appropriate restriction sites, these fragments were joined together and inserted into the expression vector, pET-5a, via NdeI site. The recombinant protein was expressed in BL21 (DE3) plyS, a kind of E. coli. It was shown in Western-blotting and ELISA that the protein could interact with the multiple cloned antibody of urokinase. After partial purification: dialysis, Sephadex G-100, dialysis and Phenyl-Sepharose fast flow, the product had a strong fibrinolytic activity through activating plasminogen on fibrin plate. The specific activity was about 47,000 IU/mg, corresponding to 80,000 IU/mg for the part of rscu-PA-32k, and the activity could be inhibited specifically by urokinase specific antibody. Activation of plasminogen by the chimera followed Michaelis-Menten kinetics, and the K-m was 1.08 uM. (C) 2000 Elsevier Science Inc. All rights reserved.; http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000165893100005&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=8e1609b174ce4e31116a60747a720701 ; Biotechnology & Applied Microbiology; SCI(E); EI; CPCI-S(ISTP); 3
语种	英语
DOI标识	10.1016/S0141-0229(00)00296-9
内容类型	其他
源URL	[http://ir.pku.edu.cn/handle/20.500.11897/212005]
专题	生命科学学院
推荐引用方式 GB/T 7714	Jiang, PC,Changgeng, R,Ru, BG. Construction and expression of antibody targeted plasminogen activator. 2000-01-01.

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