| Toxoplasma gondii（T.gondii,Tg）is a member of a large family of apicomplexan parasites that exclusively parasitize nucleated cells and belong to parasitic protozoa,which can not only infect common mammals in life,but also infect birds and even some cold-blooded animals.T.gondii invades host cells,unlike passive invasion of bacteria and viruses by endocytosis,T.gondii invasion is a continuous and complex active process that involves the allocation of a variety of organelles and proteins.Pinnatifida parasites do not only have one pathway to achieve the purpose of invading host cells,and one common pathway is the sialic acid pathway and the other is the aminoglucan pathway.As the most widely distributed sugar receptor in the outer membrane of host cells,sialic acid plays an important role in the recognition and invasion of host cells by T.gondii.However,there are relatively few studies on the interaction between T.gondii invasion-related proteins and sialic acid molecules.Vesicular transport in eukaryotes requires the participation of a crowd of proteins in collaboration,with SNAP proteins playing a key role.The SNAP-25 subfamily is commonly localized downstream of the Golgi apparatus or Golgi action,and this subfamily can couple to Ca2+sensors,or by driving receptor transport,it is also seen to participate in the secretory pathway and ultimately complete the mission of membrane fusion and exocytosis.However,the vesicular transport mechanism of T.gondii as a eukaryote is not clear.Therefore,our group previously conducted a relevant proteomics analysis of the proteins that can bind to sialic acid molecules for T.gondii,and finally selected the T.gondii putative SNAP protein with unknown function and gene number TGGT＿1218760,or Tgp SNAP（putative SNAP protein）,from the 59 up-regulated proteins for topic research through the search and review of a large number of literatures and a comprehensive analysis of bioinformatics.In this study,we first downloaded the amino acid sequence of Tgp SNAP for multi-angle bioinformatics analysis.The protein was found to be well conserved,and the results of phenotypic value analysis predicted that the protein may be essential for T.gondii.The Tgp SNAP protein was then cloned,expressed,purified and subcellular localized,and its function was preliminarily studied.First,we successfully constructed expression vectors of two fusion tags with the gateway system and successfully purified two target proteins with different tags,namely GST-Tgp SNAP protein and His-Tgp SNAP protein.Freshly purified His-Tgp SNAP protein was dialyzed and immunized to healthy experimental animals with adjuvant,and the specific Ig G in animal serum was obtained after the titer reached the standard.Furthermore,the localization of Tgp SNAP in T.gondii was observed by indirect immunofluorescence assay（IFA）,and the results showed that Tgp SNAP localized above the T.gondii nucleus,which may be located in the Golgi apparatus;subsequent sialic acid binding,inhibition experiments,and cell adhesion experiments were performed,and we successfully demonstrated that GST-Tgp SNAP recombinant protein could specifically bind to sialic acid molecules and host cells（DC and Vero cells）to mediate parasite invasion.In order to investigate whether the invasion site on the surface of T.gondii or host cells is affected after blocking,we used recombinant protein and Ig G antibody to analyze the inhibition of parasite invasion.The results showed that incubation of cells with recombinant protein gradient concentration resulted in a decrease in the gradient of parasite invasion rate,and Ig G against Tgp SNAP could also play a role in inhibiting parasite invasion after incubation of parasites.Finally,we verified that Tgp SNAP and Toxoplasma SNARE protein,MIC1 protein interact by in vitro biointeraction technique.This provides a scientific basis for the involvement of T.gondii vesicle transporters in the regulation of invasion systems. |
