Study Of Transcription Regulatory Mechanisms Of Virulence Factor ROP5 Of Toxoplasma Gondii Using CRISPR/Cas9 Technology

Toxoplasma gondii(T.gondii)is a specialized intracellular parasite of the subspecific subgate,and it is a world-wide distributed opportunistic protozoa.The pupolation infection rate of T.gondii in the world is about 25%to 30%.Infection of T.gondii can cause severe toxoplasmosis,such as Toxoplasma peritonitis,Toxoplasma encephalitis and even death in the most extreme cases.Furhermore,the infection of T.gondii is closely related to aristogenesis fine rearing.Primary infection of T.gondii during pregnancy can infect the fetus through the placental barrier and further endanger the development of fetus and even it may lead to fetal damage and stillbirth.The newborns often suffer from serious sequelae such as congenital malformation and mental dysplasia because of congenital infection.In addition,the widespread transmission of T.gondii between domestic animals,cats and dogs not only seriously endangers the production and development of animal husbandry,but also becomes an important source of human infection.Toxoplasmosis has become a serious public health problem in the world.Because of the diversity of the clinical symptoms of toxoplasmosis,it has brought much inconvenience to the prevention and clinical diagnosis and treatment.At present,there is no very effective therapeutic drug against toxoplasmosis,the sulfadiazine and pyrimidine are the most common chemotherapeutic agents in the clinical practice,but they can not be against cysts of Toxoplasma gondii.Therefore,the development of effective prevention and treatment against T.gondii would be of great value for the effective control of T.gondii infection in human and animals,the development of safe and effective vaccines against T.gondii is a major trend.In recent years,researchers have used positive and reverse genetics to make some progress in the identification and regulation of virulence factors of T.gondii with the difference in protein expression and gene polymorphism of different genotypes of T.gondii.Rhoptry protein 5(ROP5),as an important virulence factor of T.gondii,has different expression levels in different types of T.gondii(type ? and ?),which may be one of the reasons for the virulence difference of T.gondii(type ? and ?).In this study,the CRISPR/Cas9 system was used to edit the upstream regulation sequence of the rop5 gene of T.gondii.And then further observe variation of rop5 gene expression level and phenotype of T.gondii in cell combined with transient transfection,luciferase report system,real-time PCR,Western-blot.PurposeCRISPR/Cas9 system was used to edit the rop5-ups of the rop5 gene of T.gondii(RH,PRU),And then further observe variation of rop5 gene expression level and phenotype of T.gondii in cell combined with transient transfection,luciferase report system,real-time PCR,Western-blot.And clarify the regulation mechanism of the expression of ROP5 protein.Methods1.According to the database TOXODB of T.gondii,rop5-ups primers of RH,PRU were designed respectively,the PCR technique was used to obtain the upstream sequence of the rop5 gene of T.gondii(RH,PRU),and rop5-ups was sequenced.The regulation function of rop5-ups was detected by the luciferase reporter system.2.ROP5 monoclonal antibody was produced by monoclonal antibody technology.3.The CRISPR/Cas9 system was used to edit the rop5-ups of RH T.gondii,And then further observe variation of rop5 gene expression level and phenotype of T.gondii in cell combined with transient transfection,real-time PCR,Western-blot.Results1.The specific rop5-ups of T.gondii(RH,PRU)was obtained by PCR technology.The sequence results showed that the rop5-ups of T.gondii(RH,PRU)was very different.The dual luciferase reporter system showed that-2 kb sequences of RH,PRU had different transcriptional regulatory activity.2.ROP5 monoclonal antibody with good immune response was prepared.3.CRISPR/Cas9 system plasmids targeting RH,PRU rop5-ups and rop5 genes were successfully prepared,and rop5-ups RH mutant strain were obtained by electrotransfection and monoclonal screening.The obtained RH mutants spill over from HFF cell more weakly than RH.ConclusionsSequencing analysis showed significant difference between rop5-ups of RH,PRU;the dual luciferase reporter system showed that-2 kb sequences of RH,PRU had different transcriptional regulatory activity.ROP5 monoclonal antibody with good immune response was produced;CRISPR/Cas9 system plasmids targeting rop5-ups and rop5 gene of RH,PRU were successfully constructed;The mutation in rop5-ups of RH can weeken the ability of spilling over from HFF cell.