Objective: To evaluate the effect of Ficolin-A, a lectin complement against Plasmodium berghei in mice model.
Methods: The Mr 19,000 fragment of merozoite surface protein-1 of P. berghei (MSP1(19)) was cloned and then subcloned into the vector pGEX-KG. The recombinants of pGEX-KG-Ficolin-A and pGEX-KG-MSP1(19) were transformed into Escherichia coli BL21, and followed by expression of the protein induced by 1 mmol/L IPTG. The fusion protein was purified by affinity chromatography using Glutathione Sepharose 4B, and then identified by SDS-PAGE and Western-blotting. Five mouse model groups were treated with PBS, GST, Ficolin-A, MSP1(19), or Ficolin-A+MSP1(19), respectively. Each group had eight mice. Mice in Ficolin-A or MSP1(19) groups were injected with 20 microg Ficolin-A or MSP1(19) protein each time, respectively. Mice in Ficolin-A+MSP1(19) group were injected with 20 microg Ficolin-A and 20 microg MSP1(19) each time. Mice in control groups were injected with 200 microl PBS or 20 microg GST, respectively. All the mice received four immunizations at 2-week intervals. Two weeks after the last immunization, all the mice were inoculated with 300 microl Plasmodium berghei-infected red blood cells. On day 2, 4, 6, 8, and 10 post-infection, blood samples were collected from three mice of each group, and the Giemsa stained-blood films were microscopically examined. Density of malaria parasites was calculated. The survival rate was evaluated on day 20 post-infection.
Results: The recombinant vectors of pGEX-KG-Ficolin-A and pGEX-KG-MSP1(19) were constructed. Purified fusion proteins, Ficolin-A-GST and MSP1(19)-GST, were obtained. Western blotting analysis indicated that the relative molecular mass of fusion proteins Ficolin-A-GST and MSP1(19)-GST was about Mr 69,000 and Mr 41,000. Animal experiments showed that on day 10 after infection, the parasite density in Ficolin-A+MSP1(19) group [(22.2 +/- 1.7)%] was slightly lower than that of the groups MSP1(19) [(33.4 +/- 2.7)%], Ficolin-A [(36.2 3.1)%], GST [(43.8 +/- 4.8)%] and PBS [(45.3 +/- 3.6)%], but the difference was not statistically significant (P > 0.05). No mouse survived in PBS group on day 20 after infection. There was no significant difference in number of survival mice between Ficolin-A group (3 mice) and GST group (2 mice). Six mice survived in Ficolin-A+MSP1(19) group, which was significantly more than that of GST group (P < 0.05).
Conclusion: Ficolin-A cannot significantly suppress parasite density. However, Ficolin-A+MSP1(19) can increase the survival rate of Plasmodium berghei-infected mice.