Abstract:

 Objective To construct the RSepitope-HPV16L1 fusion protein using the dominant epitope of ROP2-SAG1 antigen from Toxoplasma gondii (RSepitope) and late structural protein 1 of HPV type 16 (HPV16L1), and verify its expression in eukaryotic cells. Methods The recombinant plasmid pcDNA3.1/RSepitope was constructed after optimizing the RSepitope gene sequence. The gene fragment of HPV16L1 was amplified from the plasmid T-HPV16L1 preserved in our lab, and the recombinant plasmid pcDNA3.1/RSepitope-HPV16L1 was constructed. The recombinant plasmid was verified by enzymatic digestion, PCR amplification and sequencing, and transfected into the COS-7 cells. After 48 h of culture, cells were collected to extract RNA. RT-PCR was performed to amplify target genes. Meanwhile, Western blotting and indirect immunofluorescence test were performed to detect the expression of target proteins RSepitope and RSepitope-HPV16L1 in cells.  Results After amplification and enzymatic digestion, a 282-bp fragment was obtained from pcDNA3.1/RSepitope, and a 1 500 bp fragment was obtained from pcDNA3.1/RSepitope-HPV16L1. RT-PCR using cDNA of the recombinant plasmid-transfected cells as the template produced specific bands of 282 bp and 1 500 bp, respectively. Western blotting analysis showed that when the RSepitope anti-serum was used as the primary antibody, specific bands with relative Mr of 10 000 and 65 000 were seen for RSepitope and RSepitope-HPV16L1, respectively.  When anti-HPV16L1 monoclonal antibody was used as the primary antibody, a specific band with relative Mr of 65 000 was seen for RSepitope-HPV16L1. Indirect immunofluorescence analysis using RSepitope anti-serum as the primary antibody produced green fluorescence in cells transfected with pcDNA3.1/RSepitope and pcDNA3.1/RSepitope-HPV16L1. When the HPV16L1 monoclonal antibody was used as the primary antibody, green fluorescence was seen only in cells transfected with pcDNA3.1/RSepitope-HPV16L1.  Conclusion The RSepitope-HPV16L1 fusion is constructed using RSepitope and HPV16L1 and could be expressed in eukaryotic cells.

Key words:  Toxoplasma gondii, Dominant epitope, Late structural protein of human papillomavirus 16, Expression