Construction and identification of recombinant eukaryotic vector for expression of human scFv-Fc

Objective: To construct the anti-human IL-33 scFv-Fc recombinant vector and transfection of Chinese hamster ovary cells( CHO),then express and identify the fusion protein. Methods: The IgG1 Fc was amplified and inserted into eukaryotic expression plasmid pcDNA3. 1 to construct recombinant vector pcDNA3. 1 / Fc. The signal peptide( SP) was synthesized and inserted into plasmid pcDNA3. 1 / Fc to construct recombinant vector pcDNA3. 1 / SP-Fc. Finally,the human anti-IL33 scFv which was selected in our previous work was inserted into plasmid pcDNA3. 1 / SP-Fc to construct recombinant vector pcDNA3. 1 / SP-scFv-Fc. After sequencing,the CHO cell was transfected by the correct recombinant plasmid pcDNA3. 1 / SP-scFv-Fc and the level of transcription and translation of target SP-scFv-Fc was identified by RT-PCR and Western blot. Results: The sequencing results showed that the recombinant vector pcDNA3. 1 / SP-scFv-Fc was successfully constructed and the size of inserted SP-scFv-Fc was about 1 560 bp. The RT-PCR results showed that the target SP-scFv-Fc was successfully transfected into CHO cells and the Western blot results indicated that the expressed protein had the specific binding reactions with goat anti human IgG1 Fc antibody. Conclusion: the SP-scFv-Fc eukaryotic expression vector was successfully constructed and could be used for the protein expression in CHO cells.