Functional Studies On Neospora Caninum Tyrosine Kinase-like Protein 1

Neospora caninum is an intracellular parasitic protozoan with canids as its final host and cattle and sheep as its intermediate hosts.N.caninum has a worldwide distribution and has been reported in over 70 countries.N.caninum infections can cause abortions,stillbirths and neurological disorders in newborn animals,seriously affecting the development of the livestock industry in China.However,there is a lack of effective vaccines and drugs to prevent and control N.caninum.The lack of detailed studies on the function of N.caninum genes is one of the main reasons limiting the development of drugs and vaccines against N.caninum.The tyrosine kinase-like protein family has commonalities with the serine/threonine kinase family in terms of catalytic activity.Studies on Plasmodium falciparum and Toxoplasma gondii tyrosine kinase-like proteins have found associations with invasion,virulence and immune response.In contrast,the function of N.caninum tyrosine kinase-like proteins is not known.Therefore,in this study,we addressed this issue by observing the subcellular localization of N.caninum tyrosine kinase-like protein 1(Nc TKL1)protein through indirect immunofluorescence,constructing Nc TKL1 knockout strains(ΔNc TKL1)using CRISPR-Cas9 gene editing technology,and analysing the invasion,proliferation and pathogenicity ability of ΔNc TKL1 strains by detecting the Nc TKL1 The effect of Nc TKL1 on N.caninum and the immunoprotective effect of the ΔNc TKL1 strain against wild-type strain infection were further investigated to determine the function of the TKL1 gene of N.caninum and the immunoprotective effect of the ΔNc TKL1 strain,and to provide a candidate vaccine for the prevention of N.caninum.1.Expression and subcellular localization of Neospora caninum tyrosine kinase-like protein 1According to the gene sequence of Nc TKL1 of N.caninum,the recombinant protein of Nc TKL1 was expressed in vitro and purified,and the anti-Nc TKL1 polyclonal antibody was prepared after immunization of BALB/c mice,and the parasite subcellular localization of Nc TKL1 was performed by indirect immunofluorescence.The results showed that the recombinant Nc TKL1 protein was an inclusion body expressed protein with a size of 39 k Da;the Western Blot results indicated that the recombinant protein was successfully induced;the anti-Nc TKL1 polyclonal antibody had a potency of 1:128 000;and the Nc TKL1 protein was localized in the nucleus of tachyzoites.2.Construction and function of a Neospora caninum tyrosine kinase-like protein 1 knockout strainThe Nc TKL1 gene was knocked out by CRISPR-Cas9 gene editing technology and identified by PCR and immunofluorescence assay.The effects of Nc TKL1 gene on the invasion,proliferation,in vitro growth and pathogenicity of N.caninum were investigated by invasion assay,proliferation assay,spot-eating assay and mouse virulence assay.The results showed that the Nc TKL1 knockout strain was successfully constructed;the invasion rate(P<0.0001),proliferation rate(P<0.001)and phagocytic area of the ΔNc TKL1 strain were significantly lower(P<0.0001)compared with the wild-type strain;all mice infected with the wild-type strain died at d 9 while the survival rate of mice infected with the ΔNc TKL1 strain was 100%.The body weight of mice infected with the wild-type strain continued to decrease while that of mice infected with the ΔNc TKL1 strain fluctuated less;IL-6(P < 0.01),TNF-α(P < 0.01)and IFN-γ(P <0.05)were significantly lower in the serum of mice infected with the ΔNc TKL1 strain,while IL-12 did not change significantly.The fluorescence quantification PCR results showed that compared with the wild-type strain,the heart,kidney,liver,spleen and lung tissues of mice infected with ΔNc TKL1 were significantly lower(P < 0.05),while the brain tissues were not significantly different;the heart,liver,spleen,lung,kidney and brain lesions caused by the ΔNc TKL1 strain were less severe.3.Protective effect of Neospora caninum tyrosine kinase-like protein 1knockout strains against wild-type strainsAfter intraperitoneal injection of the ΔNc TKL1 strain into C57BL/6 mice,they were infected with the wild-type strain at different times on the 10 th,20th,30 th and40th d.The immunoprotective effects of the knockout strain on mice against infection with the wild-type strain were evaluated by detecting cytokine changes,tissue and organ load and survival rate,with the wild-type strain-infected group and normal mice as the control group.The results showed that compared with the wild-type strain control group,the secretion of serum IL-6 and IFN-γ decreased significantly(P<0.05)and the secretion of IL-12 and TNF-α increased significantly(P<0.001)in the 10 th d ofΔNc TKL1 immunization;the secretion of serum IFN-γ decreased significantly(P<0.0001)and the secretion of IL-12 and TNF-α increased significantly(P<0.001)in the 20 th d.The secretion of serum IFN-γ decreased significantly(P < 0.001)and IL-12 increased significantly(P < 0.0001)in both the 30 th and 40 th d post-immunization groups.100% of the mice infected with the wild-type strain of Nc TKL1 survived after the 10 th d post-immunization.Fluorescence quantitative PCR showed a significant decrease(P < 0.05)in heart,liver,spleen,lung,kidney and brain tissue charge in mice infected with the wild-type strain on the 10 th day of immunisation;pathological sections showed the least pathological damage to heart,liver,spleen,lung,kidney and brain.The survival rate of the mice infected with the wild-type strain decreased gradually from d20 to d40,and the number of parasites and lesions were heavier.These results suggest that the ΔNc TKL1 strain is effective in activating the immune response in mice and has a good immune protection against the wild-type strain after 10 d of immunisation.In summary,the Nc TKL1 protein was localized in the nucleus of N.caninum tachyzoites;the ΔNc TKL1 strain was successfully constructed;the invasion,proliferation,in vitro growth and pathogenicity of the ΔNc TKL1 strain were reduced compared to the wild-type strain;and the ΔNc TKL1 strain provided better immune protection against the wild-type strain in mice at 10 d after immunization.TheΔNc TKL1 strain is a candidate for N.caninum vaccine.