Abstract:

Objective To investigate the effect of leptin transgenic Plasmodium yoelii on mouse body weight. Methods To construct the leptin gene-containing CRISPR/Cas9 recombinant plasmid which had the 5′UTR and 3′UTR of MIF（macrophage migration inhibitory factor） of Plasmodium yoelli 17XNL strain at two ends, the exogenous mouse leptin gene was inserted downstream of MIF coding region through homologous recombination, resulting in the PYC-MIF-Leptin recombinant plasmid. The recombinant plasmid was then electroporated into P.y 17XNL mature schizonts, and the transgenic schizonts were used to infect a Kunming mouse via tail vein injection. The trangenic P.y clone was screened by pyrimethamine selection and identified by PCR. The trangenic or wild-type P.y was used to infect a C57BL/6 mouse respectively. Blood sample was collected through eye ball and tail vein, and immunofluorescence and RT-PCR were performed to determine the expression of leptin protein in the parasites. Finally, PBS （200 μl） containing trangenic or wild-type P.y （1 × 10⁴） was injected through the tail vein into C57BL/6 mice（n = 5 respectively）. The negative control received a same volume of PBS. The changes of parasitemia and body weight were recorded every two days. Results The leptin-expressing recombinant plasmid PYC-MIF-Leptin was constructed successfully. Results of DNA sequencing of transgenic parasites confirmed the integration of leptin gene at the downstream of MIF gene and successful transcription. Immunofluorescence results indicated successful expression of mouse leptin protein. The weight loss was significant in mice infected with transgenic parasites on day 17（17.26 ± 1.40）g, decreased by 10.7%, but not in the other two groups. Both transgenic and wild-type parasites began to decline when parasitemia reached about 10%, but the transgenic parasites proliferated more rapidly. Both disappeared at 23 days. Conclusion Infection with leptin transgenic parasites decreases the body weight of the infected mice.

Key words: CRISPR/Cas9 technique, Transgenic, Plasmodium yoelli 17XNL, Leptin, Weight loss