Construction, Expression, Activity Identification And The DNA Loading Capacity Of Anti-HER2His6H520C9scFv/tP Fusion Proteins

The traditional means of treatment for malignant tumor includeoperation, radiotherapy and chemotherapy, are difficult to achieve thedesired effect. In recent years, with the rapid development of geneticengineering techniques, antibody in diagnosis, treating malignant tumoris becoming a research hotspot. Single chain antibody (ScFv) because ofits antigen binding activity, small molecular weight, strong penetratingpower, short circulating half-life in vivo and exclusion of Fc segmentinduced immune complex reaction, and become a new tumor therapeutictargeting carrier, show broad prospects in the field of geneticengineering antibody research. Protamine is a strong alkaline proteinwith strong DNA binding capacity. But the protamine with large molecularweight, and poor tissue penetration, most of the current research focuseson truncated protamine (TP), which both with the DNA binding capacity andthe small molecular weight. The purpose of this study is to combine theHER2-positive-ScFv and TP by recombinant methods, constructing a doublefunction fusion protein (ScFv/tp), which can identify the surface of tumorcell specific binding sites, and combine with the nucleic acid. Objectiveto identify the fusion protein and study its function; at the same timefor the development of the antibody-mediated tumor targeted drugsprovides a platform.Part1：AIM: Construction of anti-HER2-H520C9ScFv/tP fusion proteinMETHODS: Using pcDNA3.1-His6-H520C9ScFv plasmid as template, whichcarrying the corresponding restriction sites, amplified it by PCR. The ScFv will be obtained from cutting by restriction enzyme digestion (HindIII and EcoR I). The truncated protamine, which encoding by22amino acids,has two oligonucleotide sequences (forward and reverse).Combining the tpwith ScFv cDNA fragment, and then cloning the recombinant fragment intothe eukaryotic expression vector pcDNA3.1,so the eukaryotic expressionvector pcDNA3.1-His6-H520C9ScFv/tp was constructed. Transforming therecombinant Plasmid into the competent Escherichia coli DH5α, screeningpositive clone. The recombinant gene sequence was identified by Hind IIIand Xho I restriction enzyme digestion analysis and agarose gelelectrophoresis. Target gene was sent to sequencing.RESULTS and CONCLUSION: Restriction endonuclease digestion analysisand DNA sequencing proved that the amplified ScFv/tP sequence was correct,and the eukaryotic expression vector pcDNA3.1-His6-H520C9ScFv/tP wasconstructed successfully.Part2：AIM: Expression, and Purification of scFv/tP fusion proteinMETHODS: The recombinant eukaryotic expression vectorpcDNA3.1-His6-H520C9ScFv/tP was transfected into293cells Bylipofectamine LTX and PLUS, and screened transfected cells by G418, inorder to obtain the293cell system, which stably expression of ScFv/tPfusion protein. Culturing293cells which were screened, a large numberof ScFv/tP fusion proteins was obtained after extracting. The expressionand transcription of the fusion protein in293cells will be detected byWestern-blot, and purified with Ni-affinity chromatography.RESULTS and CONCLUSION: The Western-blot showed that293cells whichtransfected by the recombinant plasmid can express the ScFv/tP fusionprotein, which containing His tags. The ScFv/tP fusion protein was at36kDa tested by SDS-PAGE analysis.Part3： AIM: Activity identification and the DNA loading capacity of scFv/tPfusion proteinMETHODS: The human ovarian cancer cells (SKOV-3ip1) was chosen to testthe ability of the ScFv/tP fusion protein compared to the human hepatomacells (SMMC-7721). DNA binding ability was verified by Gel shift assay,and the internalization function was confirmed by indirectimmunofluorescence staining.RESULTS: Indirect immunofluorescence staining revealed that theScFv/tP fusion protein was able to internalize into SKOV-3ip1cells butnot SMMC-7721cells. Gel shift assay assured that the ScFv/tP fusionprotein had DNA binding activity. The observation by the fluorescencemicroscope showed that the ScFv/tP fusion protein could successfullydeliver the exogenous DNA with fluorescence into SKOV-3ip1cells but notSMMC-7721cells.CONCLUSION: The ScFv/tP fusion protein obtained DNA binding activitiesand was able to internalize into SKOV-3ip1cells successfully. It couldspecifically deliver exogenous DNA into SKOV-3ip1cells.