Abstract:

Objective To construct a recombinant lentivirus expression vector containing Flag-tagged sequence of Toxoplasma gondii ROP16, and test its expression in nerve cells and its effect on apoptosis of brain nerve cells in mice.Methods The Flag-tagged ROP16 gene was amplified by PCR and cloned into lentiviral vector to construct the recombinant plasmid pCDH-CMV-copGFP-ROP16-Flag, which was then transformed into E.coli DH5α competent cells.The recombinant plasmids were verified with PCR, double enzymetic digestion and sequencing.Then they were packaged into virus to infect 293T cells.The green fluorescence was observed under a fluorescence microscope and the virus titer was calculated.The packaged lentivirus was then used to infect SH-SY5Y cells, accompanied by a pCDH-CMV-copGFP empty plasmid group.Expression of ROP16 was examined by Western blotting.The concentrated lentivirus was injected into the mouse prefrontal cortex M1 region by stereotactic injection.Two weeks later, the mice were sacrificed and frozen slices of the brain were prepared.Apoptosis of brain nerve cells was observed under a fluorescence microscope after TUNEL staining.Results As expected, PCR and double enzymetic digestion both resulted in a band of ~2 200 bp from the recombinant plasmid pCDH-copGFP-ROP16-Flag.Sequencing result was consistent with the ROP16 gene of Toxoplsma gondii RH strain (GenBank Accession No.GQ249093.1).Green fluorescence was seen under the fluorescence microscope with a titer of about 2E + 8 TU/ml.Western blotting analysis showed a specific band at a relative molecular weight of 120 000, suggesting that the recombinant plasmid could express ROP16 protein in SH-SY5Y cells.TUNEL staining revealed significantly more apoptotic cells in the ROP16 group than other groups.Conclusion The lentiviral vector pCDH-copGFP-ROP16-Flag expresses ROP16 protein in nerve cells and induces apoptosis of mouse brain nerve cells.

Key words: Toxoplasma gondii, ROP16, Brain nerve cell, Apoptosis