Partial Biological Functions Of Toxoplasma Gondii ROP7 Gene And Effect Of HSP21 And HSP60 On The Function Of Mouse Macrophages

Toxoplasma Gondii(T.gondii)is a kind of nucleocytoplasmic protozoa that widely parasitic on human and many thermostatic animals,which could cause toxoplasmosis.T.gondii may cause Toxoplasma encephalitis,peritonitis,abortion and other diseases in the population.In addition,T.gondii can cause abortion and stillbirth through vertical infection of female animals,resulting in serious economic losses.The rhoptries proteins(ROPs)of T.gondii play an important role in the invasion of host cells.It can not only regulate the proliferation of the parasite in the host cells,but also the virulence-related gene of the parasite.ROP7 is one of the members of T.gondii ROPs family,but its effect on the growth and reproduction of T.gondii is not clear.Heat shock protein(HSP)is a kind of stress protein,which widely exists in eukaryotes and has many functions,such as molecular chaperone,anti-heat stress,regulated immune response,but its effect on the function of mouse macrophages is not clear.1 Construction of Toxoplasma gondii 14-3-3 and ROP7 gene deletion strainsThe specific CRISPR/Cas9 vector and DHFR homologous recombinant vector resistant to pyrimethamine targeted at 14-3-3 gene,ROP7 gene and positive control gene calcium dependent protein kinase 3 were constructed.The two successfully constructed plasmids were co-transfected into T.gondii,and after being cultured with pyrimethamine,the genome of the transfected T.gondii was extracted and verified by PCR.The verification results showed that the CDPK3 gene in the positive control group and the ROP7 gene in the experimental group were successfully knocked out,while the 14-3-3 deletion strain was not successfully obtained.The independent experiment was repeated five times,and the same results were obtained.The results showed that 14-3-3 gene was necessary for the growth and development of T.gondii.2 The effect of Toxoplasma gondii ROP7 gene deletion strain on the reproduction and pathogenicity of Toxoplasma gondiiIn order to investigate the effect of ROP7 gene on the reproduction and pathogenicity of the parasites,a large number of ROP7 gene-deleted strains were cultured.Cell adhesion and invasion experiments showed that the deletion of ROP7 gene would reduce the ability of T.gondii to adhere to cells and weaken its ability to invade host cells.The plaque and reproduction experiments demonstrated that ROP7 gene had little effect on the reproduction of T.gondii in vitro.Furthermore,the virulence experiment in vivo showed that the mice infected with ROP7 knock out parasites could prolong the survival time of mice.In conclusion,the ROP7 gene of T.gondii had little effect on the reproduction speed of T.gondii,but it can reduce the adhesion and pathogenicity of T.gondii.3 The effect of Toxoplasma Gondii HSP21on mouse macrophagesThe expression vector of HSP21 of T.gondii was successfully constructed and the recombinant protein was obtained.Western blot experiment showed that the recombinant HSP21(rTgHSP21)had good antigenicity.Immunofluorescence showed that rTgHSP21 could combine with mouse macrophages in vitro.It was found that rTgHSP21 with concentrations of 5 and 10 μg/mL significantly promoted the proliferation of macrophages.Flow cytometry was used to detect the effect of recombinant protein on phagocytosis and apoptosis of macrophages.It was found that rTgHSP21 significantly enhanced the phagocytic capacity of macrophages at concentrations of 5,10,20,40 and 80 μg/mL.rTgHSP21 at the concentration of 10 μgh/mL significantly promoted early apoptosis of Ana-1 cells,while other concentrations had no significant effect on the early apoptosis of Ana-1 cells.rTgHSP21 could promote the late apoptosis of Ana-1 cells at the concentration of 10 μg/mL,while it significantly promoted the late apoptosis of Ana-1 cells at the concentrations of 20,40 and 80 μg/mL.The results of total NO test kit showed that 80μg/mL of rTgHSP21 could significantly promote the secretion of nitric oxide(NO)in macrophages.The results showed that the expression of interleukin-6(IL-6)and tumor necrosis factor-a(TNF-α)in macrophages increased significantly after treated with rTgHSP21,but the secretion of interleukin-12(IL-12)was inhibited at concentrations of 10,20,40 and 80 μg/mL.In addition,the cell chemotaxis was detected by Transwell cell compartment.The results showed that rTgHSP21 significantly inhibited the migration of mouse macrophages.The results showed that rTgHSP21 could regulate some functions of macrophages in vitro.4 The effect of Toxoplasma Gondii HSP60 on mouse macrophagesHSP60 protein of T.gondii was successfully cloned and expressed.Western blot showed that the recombinant TgHSP60(rTgHSP60)could be recognized by the serum of rats infected with T.gondii,while the polyclonal antibody obtained from rats immunized with rTgHSP60 could recognize the natural protein of T.gondii tachyzoites.Immunofluorescence analysis showed that rTgHSP60 could bind to mouse macrophages.After incubated with rTgHSP60,the proliferation of mouse macrophages was inhibited at high concentration.However,the results of flow cytometry showed that the phagocytic capacity of macrophages was significantly enhanced,and the apoptotic ratio of macrophages and the molecular expression of Toll like receptor 4(TLR4)on the cell surface were also significantly increased.In addition,the secretion of NO,IL-6 and TNF-αin macrophages increased significantly,and the chemotaxis of macrophages were inhibited significantly.The results showed that TgHSP60 could affect some functions of macrophages in vitro.