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期刊论文

Ppic9-Fc: A Vector System for the Production of Single-Chain Fv-Fe Fusions in Pichina pastoris as Detection Reagents In Vitro

魏东芝Jianglan Liu Dongzhi Wei* Feng Qian Yuxun Zhou Wang Yushu Ma and Zeguang Han

J. Biochem. 134, 911-917 (2000),-0001,():

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摘要/描述

Reconmbinatn antibodies, especially ScFv fragments can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibodyenzyme conjugates. For ScFv fragments there is no such universal system available up to now. A vector ystem was onstructed based on pPIC9-Fc, in which the hinge, CB2 and CB3 domin (Fe fragment) of mouse IgG1 and HIS-tag wee eloned into the Pichia expression vectro pPIC9. A model SeFv was introduced into pPIC9-Fc. which can bind Gluathione-S-transfrease (GST) from Schistsoma Japonicum to yield the fusion was expreessed at high levels in the methylotrphic yeast Pichia Pastoris, secreted as a dimeric form in the culture, ad purified by Ni2+-NTA column chromatography. The expression yield can reach 10-30mg/liter of culture medium. The SeFv-Fc fusio retains the bioglogyical binding ability of the parent Scfv, and can be applied as anti-GST antibodies for the detection of GST and GST-fusion proteins. Furthermore. the successful expression and maintenance of the binding activitv verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.

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