Biological Function Research Of The Toxoplasma Gondii Dense Granule Protein 1 (GRA1)

Toxoplasma gondii is a obligate intracellular protozoan.T.gondii is a widespread parasite that infects many species of animals,including mammals,birds and cold blooded animals.In addition to its successful invasion mechanism,its powerful immune evasion mechanism and ability to manipulate host cells support T.gondii has a wide range of hosts.This is the main reason leading to the toxoplasmosis difficult to prevent and control.Toxoplasma gondii dense granule proteins(GRA16,GRA24,Tg IST)can use host cell signaling pathway to enter host cell nucleus,regulate host cell expression,change host cell immune status and intracellular environment,in order to achieve immune escape and long-term parasitism.The dense granule proteins GRA17 and GRA23 can form small membrane pore on the PV membrane,which can help the host to take up host nutrition.The dense granule proteins MAF1,GRA3 and GRA5 participate in the recruitment of T.gondii to the host cell mitochondria and endoplasmic reticulum,and help T.gondii utilize host nutrition.It can be said that dense granule proteins are very important for intracellular parasitism of T.gondii.GRA1 was first discovered as a dense granule protein,but we did not know much about its biological function.Studying its biological functions can help us to understand more about intracellular parasites of T.gondii,For the future study of toxoplasmosis laid the theoretical foundation.In this study,we used CRISPR / Cas9 and Cre-LoxP system to knockout GRA1 in RH?hxgprt strain,then comparing the difference of ability of replication between GRA1 gene knockout strain and RH?hxgprt strain.BioID technology was used to capture the proteins surrounding GRA1,attempt to find out more proteins involved in immune evasion and alteration of the host cell environment.1.Prokaryotic expression of GRA1 protein and preparation of polyclonal antibody Firstly,we constructed p E-SUMO-GRA1 prokaryotic expression plasmid which can express in BL21(DE3)smoothly,then immunized animal with the purificated protein.Collecting animal sera after immunization,enzyme-linked immunosorbent assay was used to detect antibody potency.The results revealed that the polyclonal antibody has a higher level of antibody titer.2.Construction the TATi-TetO7-GRA1 strain Conditional knockout of the GRA1 gene using the Tet-off system.First,we constructed a CRISPR plasmid pSAG1 :: Cas9-U6 :: sgGRA1 which could recognize the GRA1 locus,and contained a homologously recombination template plasmid pUC19-TetO7-GRA1.Then the homologous fragments and plasmid pSAG1 :: Cas9-U6 :: sgGRA1 were transferred into the tachyzoites of TATi strain.After screening by pyrimethamine and limited dilution,single clone was selected.The results indicated that the TATi-TetO7-GRA1 strain was successfully obtained by the method of PCR.The TATi-TetO7-GRA1 strain was treated with ATc.The expression of GRA1 protein was not turned off by indirect immunofluorescence,and the growth of the strain was normal.Description Tet-off system does not apply to the loss of GRA1.3.Construction the RH?hxgprt-LoxP-GRA1 strain Cre-LoxP system was performed in conditional knockout of GRA1 gene.First,we constructed a CRISPR plasmid pSAG1 :: Cas9-U6 :: sgGRA1 which could recognize the GRA1 locus,and contained a homologously recombination template plasmid pUC19-GRA1-YFP.Then the homologous fragments and plasmid pSAG1 :: Cas9-U6 :: sgGRA1 were transferred into the tachyzoites of RH?hxgprt strain.After screening by xanthine and mycophenolic acid and limited dilution,single clone was selected.The results indicated that the RH?hxgprt-LoxP-GRA1 strain was successfully obtained by the method of PCR amplification and IFA.4.phenotype analysis of GRA1 knockout strains On the basis of the GRA1 conditional knockout strain,GRA1 gene was deleted by transferring of a plasmid pmin-e GFP-Cre which can express Cre-splicing enzyme.GRA1 deficient strain and RH?hxgprt strain were simultaneously conducted in intracellular replication.The results showed that The GRA1 knockout strain showed a significant decrease in the ability to replicate compared with the RH?hxgprt-LoxP-GRA1 strain.5.capture the proteins surrounding GRA1 by BioID technique The genetically engineered strain RH?hxgprt-GRA1-BirA* were labeled with biotin,the result showed that some specific proteins labeled with biotin were detected by western-blot after 24 hours.In order to further identify these proteins,we analysed by mass spectrometry.After analyzing the results of mass spectrometry,13 known GRA proteins were obtained in this experiment,indicating that the results are reliable,9 unknown proteins and 17 known proteins were found.Identified by the localization experiment,2 of the 9 unknown proteins are the dense granule proteins,and the functional analysis of 17 known proteins suggests that MYR1 and GRA1 may interact with each other,but the specific relationship between them also need to be verified by further experimental.In general,the Cre-LoxP conditional knockout system and CRISPR / Cas9 system were used to knockout the GRA1 gene in RH?hxgprt strain.Comparing with GRA1 gene deletion strain and RH?hxgprt-LoxP-GRA1 strain,we find that the deletion strain showed a significant decrease in the ability to replicate.At the same time,36 specific proteins were screened by BioID and LC-MS/MS.Through the localization experiment we found two new dense granule proteins.It is hoped that the above results will provide a theoretical basis for the study of Toxoplasma gondii immune evasion mechanism and change the host cell environmental mechanism.