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Published on: February 28, 2019
[Expression pattern and level of cytotoxic T lymphocyte-associated antigen-4 targeted anti-caries plasmids in
Ji-hua Guo1, Ming-wen Fan, Rong Jia
1Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School of Stomatology, Wuhan University, Wuhan 430079, China.
Objective:
To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells.
Methods:
The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants.
Results:
Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX.
Conclusions:
CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine.
