Cloning And Expression Of Three Calcium Dependent Protein Kinases Of Toxoplasma Gondii And Their Regulations On Mouse Macrophages Function

Toxoplasma.gondii（T.gondii）is one intracellular parasitic protozoa which can infect almost all warm-blooded animals,causes toxoplasmosis in animals and human.T.gondii is much popular all around the world,especially in Europe,America and Africa.The higher infection rates of T.gondii causes abortion,stillbirth in pregnant women and other animals,bringing great theart to those immunosuppressed patients,patients with AIDS and organ transplantation recipients also greatly threatened.Toxoplasmosis really influences animal husbandry development.T.gondii is of great significance in both veterinary medicine and public health.Much work has been performed upon prevention and control of T.gondii.Explored drugs and vaccines got great progress,which made a solid foundation for further research.In this article a variety of the preparation of T.gondii calcium dependent protein kinases and cultivation of mice macrophages were performed under in vitro conditions to assess the effects of different proteins on host macrophages,which will provide reference to a better understanding of T.gondii pathogenic mechanism.1.TgCDPK3,TgCDPK4A,TgCDPK9 gene cloning and protein expressionRT-PCR amplification gained TgCDPK3（GenBank accession:XM₀02370317.2.）,TgCDPK4A（GenBank accession:XM₀18782284.1.）,TgCDPK9（GenBank accession:XM₀18779775.1.）genes.After purification of gene products,recombinant plasmids were constructed and proteins were expressed.The ORF of TgCDPK3 gene containing 1614 bp,encodes protein of 60.4 kDa which contains 537 amino acids;the ORF of TgCDPK4A gene containing 1632 bp,encodes protein of 60.9 kDa which contains 543 amino acids;while the ORF of TgCDPK9 gene containing 2001 bp,encodes protein of 75.6 kDa which contains 666 amino acids.Western blot results proved that those three kinds of recombinant proteins were of good antigenicity.2.Binding assay of rTgCDPK3,rTgCDPK4A,rTgCDPK9 to macropahage and their effects on the proliferation activity of macrophageThis test demonstrated that three kinds of recombinant proteins binded to macrophages in vitro.CCK-8 test found that rTgCDPK3,rTgCDPK9 inhibited cell activation only in relatively high concentration;low concentration of rTgCDPK4A（5μg/mL）promoted cell proliferation,but inhinbited proliferation in higher concentration.3.Effects of TgCDPK3,rTgCDPK4A,rTgCDPK9 on phagocytosis function and apoptosis of macrophageData showed that the low concentration of rTgCDPK3 stimulation improved phagocytosis,and higher concentrations inhibited cell phagocytosis;for the different concentrations of rTgCDPK4A and rTgCDPK9 protein both reinforced the phagocytosis of macrophage cell.Cell apoptosis rate got up by three kinds of recombinant proteins.4.Effects of rTgCDPK3,rTgCDPK4A,rTgCDPK9 on the productions of different cytokines and NO synthesis of macrophageResults showed that the rTgCDPK3 had a role in promoting secretions of TNF-a,TGF-β1,IL-1β and IL-10,and inhibitory effect on TGF-β1 secretion;rTgCDPK4A increased the secretion of cytokines;rTgCDPK9 protein stimulation promoted secretions of TNF-a and IL-1β,and inhibited the secretion of TGF-β1 and IL-10.All of three recombinant proteins were found to be important in the enhancement of the production of NO of macrophages.