Construction, Expression and Internalization Study of Human Anti-HBsAg Single Chain Antibody/EGFP Fusion Proteins Containing Arg9 / 中国生物工程杂志

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.

Investigation of Apoptosis of the SGC7901 Cells Induced by the Expression of the Recombinant Gene of anti-HER2 ScFv/tBid / 中国生物工程杂志

Objetive: To investigate whether apoptosis of SGC7901 cells can be induced by the expression of the recombinant gene of anti-HER2 ScFv/tBid. Methods: The recombinant anti-HER2 ScFv/tBid gene was cloned into vector pCMV and the recombinant plasmid was transfected into SGC7901 cells. The gene expression was detected by RT-PCR and immunofluorescent staining. Cell counting was carried out to show the effect of the gene transfection on cell growth. At the same time, significant apoptotic peak was detected by flow cytometry in recombinant anti-HER2 ScFv/tBid gene transfected cells. Results: The fusion protein of anti-HER2 ScFv/tBid was observed in the cytoplasm of transfected SGC7901 cells. The transfected cells displayed typical cell growth inhibition and apoptosis. Conclusion: Fusion protein of anti-HER2 ScFv/tBid can induce apoptosis of SGC7901.