The Properties Of Recombinant Proteins Incorporating Melittin Or Tat On Membrane

Liver targeted therapy is designed to deliver a substance preferentially to the organ in order to increase the accumulation, improve the therapeutic effect and reduce toxicity to other organs. The aim of selective targeting is to deliver a substance to a specific cell type in the liver. A variety of vehicles have been designed and further modified for selective targeting of therapeutics to the liver. However, the efficiency of these gene transfer methods is not sufficient partly due to the size of the ligand and the barrier of endosome.In this project, we describe the isolation of two scFv fragments directed against the recombinant carbohydrate recognition domain of the H1 subunit of human asialoglycoprotein receptor (rCRDH1) by selection from a large phage-display antibody library. Then we constructed, expressed recombinant protein scFv-Melittin and then explored its binding capacity to liver cells or pore formation ability on human erythrocytes; we also exploited the cell membrane translocating ability of the fusion protein which TK fused to an 11-amino-acid peptide from the basic domain of the H1V-1 Tat protein (Tat 11). this research will make a basis for future study how to improve the transfect efficiency of non-viral vectors in biological therapy of liver diseases.Objective: To identify the scFv antibody fragments specific for asialoglycoprotein receptor by biopanning based on recombinant proteins rCRDH1 from a large human naive scFv phage display library. Through the expression of a recombinant protein in which anti-ASGPR scFv fused to Melittin and study of its targeting or membrane disrupting functions, Through the expression of a recombinant protein in which HS V1 -TK fused to Tat and study of its transmembrane property. Methods: According to the gene sequence of H1, the primers were designed. The full length cDNA encoding CRDH1 amplified f(?)om pET3CRDHl by PCR were subcloned into an preukaryotic expression vector (pET-32c) and induced by IPTG. The recombinant CRDH1 was purified with Ni²⁺ chelating HiTrap HP column. Its immunoresponse was evaluated with Western-blot. After four rounds of biopanning, individual colonies were picked and selected by ELISA using rCRDH1as antigen, further to be excluded by Trx/His tag; Each selected phage infected HB2151 and which is than induced to give soluble expression of antibody fragments, detection of bound scFv to fCRDH1 by ELISA and positive ones were sequenced. Synthesize two single oligonucleotide chains of Melittin and 30% nondenaturing polyacrylamide gel electrophoresis analysis of the annealing result; then fused to the full length gene of selected anti-ASGPR scFv by subcloning into an preukaryotic expression vector (pGC). The expressed recombinant C1M was purified with Ni²⁺ chelating HiTrap HP column. Its antigen-binding ability was evaluated with immnunohistochemistry, its cytolytic activity was analysed by hemolysis test; Synthesize two single oligonucleotide chains and amplify HSV1-TK gene, then subclone into an preukaryotic expression vector (pET-32c), induced to express Tat11-TK recombinant protein in E.coli and purified with Ni²⁺ chelating HiTrap HP column. Its intercellular translocation ability was evaluated with immnunohistochemistry and Western-blot.Results: The recombinant CRDH1 protein about 35.0kDa was expressed in E.coli as inclusion body, rCRDH1 was prepared with Ni²⁺ column purification. The result of western-blot showed that CRDH1 could be recognized by rabbit anti-ASGPR H1 serum. Tweenty-four colonies were obtained after four rounds of biopanning, there were 14 colonies can react with Trx/His tag, Two of the rest 10 colonies could be induced to produce soluble scFv antibody. Comparison with known coding sequences for antibodies revealed that the heavy chain coding sequences belongs to the VH3 family, while the light chain sequence used a V segment of the VKI subgroup. The recombinant protein C1M about 29.4 kDa was expressed in E.coli as soluble style; 0.6 mg/ml C1M was prepared with Ni²⁺ column purification; The result o immnunohistochemistry showed that C1M could bind to the cell surface and hemolysi test showed that C1M kept the hemolytic activity; The recombinant CRDH1 protei(?) about 60.7 kDa was expressed in E.coli as inclusion body; Tat11-TK was preparec with Ni²⁺ column purification; The result of immnunohistochemistry showed tha Tat11-TK could bind to the cell surface and western-blot showed that it also could b(?) transducted into the HepG2.Conclusions: The human naive scFv phage library biopanning through recombinant protein permits identification of specific antibody fragments for human asialoglycoprotein receptor expressed on the cell membrane surface of HCC and successful expression of C1M was achieved in E. coli, C1M recombinant protein confers targeting and membrane-disrupting capacity; Tat11-TK recombinant protein confers trafficking capacity to the enzyme.