论文部分内容阅读
AIM:A single-chain antibody fragment,ND-lscFv,againsthuman colorectal carcinoma was constructed and expressedin E.coli,and its biodistribution and pharmacokineticproperties were studied in mice bearing tumor.METHODS:V_H and V_L genes were amplified from hybridomacell IC-2,secreting monoclonal antibody ND-1,by RT-PCR,and connected by linker(Gly_4Ser)_3 to form scFv gene,whichwas cloned into expression vector pET 28a(+)and finallyexpressed in E.coli.The expressed product ND-1scFv waspurified by metal affinity chromatography using Ni-NTA,itspurity and biological activity were determined using SDS-PAGE and ELISA.ND-1scFv was labeled with ~(99m)Tc,and theninjected into mice bearing colorectal carcinoma xenograftfor phamacokinetic study in vivo.RESULTS:SDS-PAGE analysis showed that the relativemolecular weight of recombinant protein was 30kDa withpurity of 94 %.ELIAS assay revealed that ND-lscFv retainedthe immunoactivity of parent mAb,being capable of bindingspecifically to human colorectal carcinoma cell line expressingassociated antigen.Radiolabeled ND-lscFv exhibited rapidtumor targeting,with specific distribution in mice bearingcolorectal carcinoma xenograft observed as early as 1 hfollowing injection.In vivo pharmacokinetic studies alsodemonstrated that ND-1scFv had very rapid plasma clearance(T_(1/2)α of 5.7 min,T_(1/2)β of 2.6 h).CONCLUSION:ND-1scFv shows significant immunoactivity,and better pharmacokinetic and biodistribution characteristicscompared with intact mAbs,demonstrating the possibilityas a carrier for tumor-imaging.
AIM: A single-chain antibody fragment, ND-lscFv, againsthuman colorectal carcinoma was constructed and expressed in E. coli, and its biodistribution and pharmacokinetic properties were studied in mice bearing tumor. METHODS: V_H and V_L genes were amplified from hybridomacell IC- secreting monoclonal antibody ND-1, by RT-PCR, and connected by linker (Gly_4Ser) _3 to form scFv gene, which was cloned into expression vector pET 28a (+) and finallyexpressed in E. coli. expressed product ND- 1 scFv waspurified by metal affinity chromatography using Ni-NTA, its purity and biological activity were determined using SDS-PAGE and ELISA. ND-1 scFv was labeled with ~ (99m) Tc, and then intojected into bearing bearing colorectal carcinoma xenograft for phamacokinetic study in vivo .RESULTS: SDS- PAGE analysis showed that the relative molecular weight of recombinant protein was 30 kDa withpurity of 94% .ELIAS assay revealed that ND-lscFv retained the immunoactivity of parent mAb, being capable of bindingspecifically to human colorectal carcinoma cell line expressingassociated antigen. Radiolabeled ND-lscFv shows rapid tumor targeting, with specific distribution in mice bearingcolorectal carcinoma xenograft observed as early as 1 hfollowing injection. In vivo pharmacokinetic studies alsodemonstrated that ND-1scFv had very rapid plasma clearance (T_ (1/2 ) α of 5.7 min, T 1/2 (1/2) β of 2.6 h). CONCLUSION: ND-1 scFv shows significant immunoactivity, and better pharmacokinetic and biodistribution characteristicscompared with intact mAbs, demonstrating the possibilityas a carrier for tumor-imaging.