ABSTRACT
<p><b>OBJECTIVE</b>To construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins.</p><p><b>METHODS</b>The fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA.</p><p><b>RESULTS</b>The human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen.</p><p><b>CONCLUSION</b>Fusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.</p>
Subject(s)
Humans , Male , Antigens, Surface , Allergy and Immunology , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Allergy and Immunology , Glutamate Carboxypeptidase II , Allergy and Immunology , Polymerase Chain Reaction , RNA, Small Interfering , Allergy and Immunology , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Single-Chain Antibodies , Genetics , Allergy and ImmunologyABSTRACT
Objective: To construct, express and purify ScFvl4/EGFP fusion proteins which containing Arg9, and to study their binding activities and internalization functions. Methods: Arg9 gene was recombined into 5' terminal, 3' terminal of ScFv/EGFP gene and between them respectively before they were cloned into the expression vector pET32a. After induced in E. coli BL21 and purified, their binding activities and internalization were respectively analyzed by indirect ELISA and indirect immunofluorescence analysis. Results: DNA sequencing and restriction endonuclease digestion proved that the four fusion genes were correctly constructed. SDS-PAGE analysis and Western blot showed that they were successfully expressed and purified. Indirect ELISA confirmed that the expressed products had antigen specific binding activities. Indirect immunofluorescence analysis revealed the fusion protein containing Arg9 at its N terminal had much better internalization function, but never internalized into the cells which do not express HBsAg. Conclusion: The four fusion genes were constructed, expressed and purified successfully. The purified fusion proteins maintained the binding activities to HBsAg and the fusion protein containing Arg9 at its N terminal had much better internalization effect.