[Construction of phage antibody library with predetermined CDR3 gene and screening of humanized Fab of anti-human integrin alphanubeta3 monoclonal antibody].

Aim: To construct phage antibody library with predetermined CDR3 and to screen humanized Fab of anti-human integrin alphanubeta(3) monoclonal antibody (mAb) by epitope guided selection.

Methods: LCDR3 gene of mAb E10 was inserted into human light chain variable region gene library. Hybrid phage antibody library was constructed by cloning E10 chimeric Fd gene and human light chain variable region gene into pComb3.

[Construction and expression of anti-human integrin alphavbeta3 scFv].

Aim: To construct single chain antibody (scFv) gene of mAb E10 against human integrin alphavbeta3.

Methods: The VH and VL genes were amplified from hybridoma cells secreting mAb E10 by RT-PCR and connected with the use of linker (Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into prokaryotic expression vector pTIG-TRX and expressed in E.

[Construction of eucaryotic expression plasmid carrying the BMP7 gene and expression in mesenchymal stem cells].

Objectives: To construct an eucaryotic expression plasmid carrying the BMP7 gene and express in MSCs.

Methods: The BMP7 gene was cloned into the eucaryotic expression vector pcDNA3.1.

In vitro assay for HCV serine proteinase expressed in insect cells.

Aim: To produce the recombinant NS3 protease of hepatitis C virus with enzymatic activity in insect cells.

Methods: The gene of HCV serine proteinase domain which encodes 181 amino acids was inserted into pFastBacHTc and the recombinant plasmid pFBCNS3N was transformed into DH10Bac competent cells for transposition. After the recombinant bacmids had been determined to be correct by both blue-white colonies and PCR analysis, the isolated bacmid DNAs were transfected into Sf9 insect cells.

Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single-chain protease.

Aim: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors.

Methods: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing.