Expression Characteristics Of Four Serine Protease Inhibitors From Trichinella Spiralis

Trichinosis is a foodborne parasitic zoonoses which spreads all over the world and does a great damage.The main cause of this disease is that the host had eaten raw or not cooked meat with infectious cyst,severe infection can cause death.Almost all mammals can be infected.In recent years,several parasites have been isolated and identified in addition to the serine protease inhibitor family.Studies have shown that serine protease inhibitors have unique enzyme inhibitory activity,which can protect the parasite to resist the digestion of the host digestive juice,participate in the parasite migration process within the body of the host,facilitate the development of parasitic nematodes and resist immune response of the host.We designed the quantitative real-time fluorescent primers based on the published serotonin serine protease inhibitors Tsp Ad5,TS11-1,Tsp03044,Tsp03548?their gene sequence accession numbers: EU263307,AF231948.1,XM₀03379333,XM₀03379851?on NCBI,and applied molecular biologic techniques to ligate cloned target fragments and p MD-18 T cloning vector,respectively transformed into the cloned competent cells DH5?.After microbial PCR identification and sequence by sequencing company,sequence analysis proving the cloned sequence is correct,then prepare the corresponding standard substances,and establish the corresponding quantitative real-time fluorescence standard curve.In order to elucidate the transcription expression quantity of four Ts-SPI from Trichinella spiralis at different development stages,total RNA obtained from collected Trichinella spiralis was analyzed by RT-PCR for the expression quantity difference of four Trichinella spiralis genes at mRNA level at different development stages.The results showed that Tsp Ad5 and TS11-1showed a transcription peak on the day 3 after Trichinella infection,and the expression quantity was significantly higher than that of the day 1 and day 5?p <0.01?,and slightly lower on the day 5compare to NBL(p <?P <0.05?.Another transcription peak appeared in the muscle larvae on the day 38 after being infected,and the difference was significant?p <0.05?.The expression quantity of Tsp03044 at the adult stage on the day 3 after infection was evidently higher than that of other stages with significant difference?p <0.01?.At the pre encysted larvae stage,the expression quantity was the lowest on the day 18?p <0.05?,and transcription peak appeared on the day 22 with significant difference?P <0.01?.On the day 3,the expression quantity of Tsp03548 wasslightly higher than that of the day 1 and day 5?p <0.05?,and slightly lower than that of day 5compare to NBL with significant difference?p <0.05?.At the pre encysted larvae stage,the transcription peak appeared on the day 18?p < 0.01?.At the muscle larvae stage,the expression quantity showed significant difference?p <0.05?.In this study,immunofluorescence technique was used to study the body distribution of four Ts-SPIs at different development stages of Trichinella spiralis,and the interaction between SPI and the host.Immunofluorescence localization showed that a large number of Tsp Ad5 and TS11-1were expressed in the epidermis and body of the adult and muscle larvae,and were also expressed in newborn larvae and pre encysted larvae body.The expression of Tsp03044 in the epidermis and body of the newborn larvae is less,while the red fluorescence effect in the body of other development stages are clearly visible.Tsp03548 became evident in the epidermis and body of the pre encysted larvae,also can be seen in the epidermis of the adult,the epidermis and body of newborn larvae,but the whole red fluorescence effect was very weak;in the body and epidermis of the muscle larvae are also expressed.In this study,the transcription expression quantity and distribution of four Ts-SPI in the body of Trichinella spiralis at different development stages were studied.Using quantitative real-time fluorescence PCR and immunofluorescence localization techniques to analyze the different transcription expression quantity and location of Tsp Ad5,TS11-1,Tsp03044,Tsp03548 at different developmental stages,that will lay the foundation for elucidating the transformational mechanism of the Trichinella spiralis at different developmental stages and the immune escape mechanism in the process of attacking the host,also lay a solid foundation for the research of new type anti-Trichinella and related parasite drugs.