Prokaryotic Expression,immunolocalization And Transcription Level Analysis Of Echinococcus Granulosus Annexin B3 And B7

Cystic Echinococcosis is a parasitic zoonoses disease caused by Echinococcus granulosus,a larval stage of E.granulosus.It is a worldwide disease,which is one of the most common areas in China and poses a serious threat to the development of public health and animal husbandry in China.Annexin is a kind of phospholipid binding protein with calcium ion binding activity,which has many functions such as cell membrane repair,membrane transport,anti-inflammatory,participation in cell proliferation,differentiation and apoptosis.In this study,two annexin protein family genes(Eg Anx B3,Eg Anx B7)were screened and from the E.granulosus transcriptional database and expressed in prokaryotes.Their molecular characteristics were described in conjunction with bioinformatics methods,and recombinant proteins were studied by immunoblotting and cross-reaction.The distribution and transcription levels of the two genes in different developmental stages of E.granulosus were analyzed by immunofluorescence localization and relative fluorescence quantitative PCR(q RT-PCR),and their secretion characteristics were preliminary analyzed.The main findings are as follows:1.Characteristic analysis and immunoreactivity of annexin B3 and B7 genes of E.granulosusIn this study,two annexin genes(Eg Anx B3,B7)of E.granulosus were successfully cloned.The size of the Eg Anx B3 and Eg Anx B7 genes were 933 bp and 1356 bp,respectively,corresponding to the encoding of 310 and 451 amino acids.Bioinformatics analysis showed that Eg Anx B3 and Eg Anx B7 are conservative and all contain calcium binding sites.Immunoblotting showed that both r Eg Anx B3 and r Eg Anx B7 can be specifically recognized by the serum samples from E.granulosus-infected sheep,which have good immune reactivity.The results of this study indicate that Eg Anx B3 and Eg Anx B7 proteins may be inseparable from the regulation of calcium ions during the development of E.granulosus.2.Transcription and tissue localization of annexins B3 and B7 of E.granulosus.Based on the above research,this study analyzed the transcription level and tissue localization of annexin B3 and B7 of E.granulosus.The results of fluorescence quantitative PCR showed that both Eg Anx B3 and Eg Anx B7 had transcription in the protoscoleces and in the adult worm stage of E.granulosus(28d),and the transcription level was higher in the adult stage.The results of immunofluorescence localization showed that Eg Anx B3 and Eg Anx B7 were distributed in the germinating layer of E.granulosus,protoscoleces,tegument and parenchymatous tissue of adult(45d),suggesting that these two annexins play an important role in the growth and development of E.granulosus.In addition,Eg Anx B3 and Eg Anx B7 are also distributed at the junction of the sheep hydatid E.granulosus cysts and healthy liver tissue,indicating that these two annexins can be secreted out of the cell through special secretory pathways to participate in physiological activities.Meanwhile,both r Eg Anx B3 and r Eg Anx B7 can be recognized by the positive serum of other tapeworms and cannot be used as diagnostic candidate antigens.