| Trichinellosis is a kind of zoonotic parasitic disease caused by Trichinella spiralis(T.spiralis),which can cause food safety issues and economic losses.Trichinella spiralis is a parasite that can complete a complete life cycle within the same host,mainly divided into the adult stage,newborn larval stage,and muscle larval stage.NBL invade the muscle cells and develop into ML gradually.ML can parasitize for a long period of time in the host and form capsule structure.Although many studies have explored the mechanism of capsules,the secreted protein components of Trichinella spiralis are complex and diverse,and there is currently no research to prove what proteins are involved in regulating the formation of capsules.In previous studies,it was found that the Ts-NBL1 protein can be localized on the capsule,and through the overexpression experiment of Ts-NBL1,it was found that TsNBL1 can upregulate collagen protein expression level in myoblasts.Therefore,in this study,we used CRISPR-Cas9 to knock out Ts-NBL1 to determine the influence of the Ts-NBL1 gene on capsule formation.This study screened for efficient targeting sequences of Ts-NBL1 through in vitro Cas9 digestion,and used homologous recombination technology to connect the targeted sequence sg RNA to the knockout vector p DD-EGFP.The Ts-NBL1 targeted knockout vector p DD-Ts-EGFP was constructed for Trichinella spiralis,and the knockout vector was transferred into Trichinella spiralis using an electroporator.The transfection effect of the knockout plasmid was determined by observing fluorescence.q PCR and Western blot experiments were used to detect m RNA and protein expression levels of Ts-NBL1.It was found that the expression levels of Ts-NBL1 in the knockout group was significantly reduced.In gene alignment,changes in the gene sequence of the knockout group were also found,indicating that the knockout vector works normally in the insect T.spiralis and the construction of the knockout vector was successful.The establishment of knockout methods can be applied as a reference in subsequent gene editing research.By observing the diaphragm of mice infected 35 days later and collecting ML,it was found that the number of offspring ML decreased and the number of capsules decreased.However,it was found that knocking out Ts-NBL1 did not affect NBL through fecundity experiments and no significant differences in the morphology of NBL,indicating that Ts-NBL1 plays a role in the formation of capsules.To confirm this speculation,we compared the effects of NBL crude extract protein from the knockout group and the normal group on C2C12 myoblast cells through in vitro experiments.Through cell apoptosis experiments,cell cycle experiments,and expression of differentiation markers for myoblasts,we found that Ts-NBL1 can protect cells and reduce the number of apoptosis cells,promote cell proliferation,upregulate collagen expression,and inhibit cell differentiation.These indicate that Ts-NBL1 promotes the formation of capsules by upregulating collagen and promoting cell proliferation.Collagen is the main component of the caspule structure of Trichinella spiralis,and the molecular mechanism by which Ts-NBL1 affects the expression of collagen is unknown.Therefore,we attempted to identify proteins on C2C12 that can interact with Ts-NBL1 to elucidate.In this study,protein expression vectors were constructed using Gateway,and Ts-NBL1 was used as a bait protein to perform with the c DNA library by yeast two hybrid and 14 proteins were discovered could interact with Ts-NBL1.ERVK3-1,KCTD10,and vimentin have the highest expression levels.We conducted pull down validation on three interacting proteins and found the interaction effect between vimentin and Ts-NBL1 was best.Transfecting HEK-293 T cells with Ts-NBL1 showed that Ts-NBL1 significantly affects the expression of vimentin.To investigate the effect of vimentin on C2C12,we established a vimentin knockdown cell line for validation.The results showed that collagen I expression downregulated in the knockdown group.Knocking down vimentin affected cell differentiation and significantly downregulated cell differentiation markers.However,after stimulation with Ts-NBL1,there was significant downregulation of type I collagen expression but the other groups showed no significant changes compared to the control group.By observing the cell immunofluorescence of C2C12 differentiation cultured,it was found that the cell differentiation in the vimentin knockdown group was slower than that in the normal group.This indicates that vimentin plays a role in the invasion of NBL into muscle cells,and Ts-NBL1 may influence the expression of type I collagen through vimentin.In summary,this study is the first to perform CRISPR-Cas9 on Trichinella spiralis and establish a knockout vector for Trichinella spiralis,which will contribute to the subsequent study of gene function.The influence of Ts-NBL1 on capsules formation was determined through in vitro experiments,and determined that vimentin can interact with Ts-NBL1 to influence myoblast differentiation.These studies contribute to the subsequent study of the mechanism of Trichinella spiralis capsules formation and provide new research ideas. |
