简介：

简介：

简介：InordertoimprovethefunctionalaffinityofthehumanizedVHsingledomainantibodyagainsthumanlungcancer,thegenescodingthehomogenousdimersdihu3D3V_Handtetramerstehu3D3V_HwereconstructedbyfusingtheSVS-Cysshortpeptideandp53tetramerizationstructuraldomaingenetohu3D3V_HgeneviarecombinantPCRtechnique,respectively.Then,thedihu3D3V_Handtehu3D3V_HgeneswereclonedtotheprokaryoticexpressionvectorpET-22b(+)andexpressedinE.coliBL21(DE3).TheproteinsexpressedwerepurifiedthroughNi~(2+)-affinitychromatographiccolumn.Meanwhile,thehu3D3V_H,dihu3D3V_Handtehu3D3V_HproteinswerelabeledwithFITC,andtheirreactivitywithan-tigenandspecificitywereanalyzedbyimmunofluorescenceassay.Astotheirfunctionalaffinities,itwasanalyzedandcomparedbyflowcytometry.Theresultsindicatedthatthesetwogeneswereexpressedasmonomersandmainlyasinclusionbodies.Afterpurificationandrenaturation,therewereabout50%ofdimersand70%oftetramerremainingintheproteinsolution.Inaddition,thedihu3D3V_Handte-hu3D3V_Hproteinsstillremainedthereactivitywithantigenandspecificityofhu3D3V_Hprotein,andtheirfunctionalaffinitieswereincreasedabout60%or100%respectively,comparedwiththoseofhu3D3V_Hprotein.Itisevidentthatthefunctionalaffinityofhu3D3V_Hproteincanbegreatlyimprovedbyincreasingitsbindingvalency.