In Vitro Analysis Of EgM9 Gene Expression Of Echinococcus Granulosus

The prevalence of echinococcosis is high in China,Dogs,as the core source of infection,lack effective vaccination,and the key reason lies in the lack of clear functional vaccine target genes.We studied Echinococcus granulosus at different developmental stages by differential display and genomics,and found that EgM9 gene is a specific gene at the mature stage of adult Echinococcus granulosus,especially in the body surface and testis.After inoculation with EgM9 prokaryotic expression antigen,the number of parasitoids decreased significantly and the development of parasitoids was inhibited,suggesting that EgM9 may be a key gene in parasitoid development,especially testis development,and affect sperm production.At present,no sequence similar to that encoding EgM9 protein has been found in Gen Bank,and the function of EgM9 protein and gene expression of mature insect eggs are not very clear.EG₀9888gene is highly expressed during the formation of meiosis fertilized eggs of Echinococcus granulosus adults.Therefore,in this study,we established an in vitro model of Echinococcus granulosus to develop into adults,and morphological identification of the development status of worms at different periods in vitro and collection of worms at different periods in vitro.It can provide materials for different stages of EgM9 gene intervention to affect the development of parasite.The expression of EgM9 and EG₀9888 genes in different developmental stages of Echinococcus granulosus was determined by RT-q PCR,which provided experimental basis for exploring the function of EgM9 gene in adult development of Echinococcus granulosus,and also provided technical support for the development of effective dog vaccine against Echinococcus granulosus in the future.（1）Nutritional serum of different quality was added into three groups（group 1: 20% fetal bovine serum;Group 2: 20% newborn bovine serum at the first 15 days + 20% fetal bovine serum at the last 10days;Group 3: 20% newborn bovine serum）the survival rates of protoscoleces at different development times（3 d,10 d,17 d,25 d）were counted within 25 d of culture in vitro.The serum medium of group 2was used to screen the ingredients stimulating the ectropis of protoscoleces in 3 groups（group 1: pure water stimulation + 0.02% taurocholic salt;Group 2: 0.02% taurocholate;Group 3: 0.2% trypsin + 0.02%taurocholate）were cultured in vitro for 32 d at different developmental times（0 d,7 d,15 d,22 d,32 d）.The results showed that the Echinococcus granulosus cultured on the complete medium supplemented with0.2% trypsin and 0.02% taurocholate had the best Echinococcus granulosus evaginate rate and survival rate,and the model of Echinococcus granulosus cultured to adult was established.Under the microscope,the worms body segmented on the 20 d,producing the first segment.After 30 d and 40 d,the unmature proglottid segment gradually increased;Until 50 d is formed into mature proglottid.（2）In this study,Hematoxylin-eosin staining（H&E）was conducted to observe morphology,extract RNA and reverse transcription into c DNA.The target fragment was amplified by common PCR.The expression levels of EgM9 and EG₀9888 were detected by RT-q PCR and β-Actin as the reference gene.The results showed that the expression levels of EgM9 and EG₀9888 genes were the highest and no significant difference was found on the 50 d cultured in vitro with 2-ΔΔCt method（P > 0.05）,significantly different from other culture days（P < 0.05）.（3）In this study,three pairs of si RNA molecules were designed and synthesized according to EgM9 gene using RNA Interference（RNAi）technology to explore the electroporation efficiency and silencing effect of Echinococcus granulosus at different stages during its development into adult larvae in vitro.The results showed that the electroporation efficiency of protoscoleces without abscess cyst was higher,and the electroporation of the larvae cultured at 3 d,15 d and 30 d could reach 90% 95%.Although the electroporation conditions and efficiency were valid,the surface type of the larvae changed to varying degrees,but RT-QPCR did not find EgM9 gene silencing effect.Fine particle in this direction of Echinococcus former head of worms to adult in vitro model of development,it is easy to study the in vitro culture in different periods of worms body growth and the expression of genes and intervention,to fine grained Echinococcus immunology,biological development,provide the basis for a specific development of functional genomics and vaccine development,At the same time,it also provides a certain experimental platform for Echinococcus granulosus gene intervention technology.