Cloning And Identification Of Echinococcus Multilocularis Sox2Gene

Echinococcus multilocular larvae,which spread to organ surfaces in vivo in a tumor-like way, could cause a dangerous zoonotic parasitic diseases. The growth and development of Echinococcus multilocular larvae depend on germinal layer cells which have strong capacities of self-renewal and differentiation.Germinal layer cells producing all kinds of alveolar hydatid cells, belong to neoblast.Sox2is one of the key transcription factors in mammal embryonic stem cells, and it plays a pivotal role in maintaining the multipotency of stem cell.In this study, we acquired EmSOX2gene, which encoded EmSOX2, by RACE technology. We analyzed the gene structure using bioinformatics techniques. Next, several experiment procedures including the construction of prokaryotic expression vector, the preparation of polyclonal antibodies, immunoblotting, RT-PCR, and immunohistochemistry were adopted to locate the expression of EmSOX2. Finally, we researched the function of the EmSOX2in maintaining the multipotency of the stem cells by iPS induced technology.The following achievements are concluded:(1) The1422bp EmSOX2gene is composed of1185bp code reading frame (30-542bp,681-1352bp),138bp intron,70bp3’UTR and29bp5’UTR; It encoded394amino acids. In addition, EmSOX2has a high similarity reaching91%with other organisms’ Sox2protein in HMG box conservative functional domains.(2) The IHC results indicated that EmSOX2protein was mainly expressed in the nucleus of the germinal layer cells. In vitro, EmSOX2protein was expressed in the nucleus of293T cells.RT-PCR and WB results showed that EmSOX2expression level in protoscolex period was significantly higher than that in vesica period for Echinoccus multilocular.(3) The results that utilizing iPS technology to induce mouse fibroblasts indicated that mouse fibroblasts could be induced to iPS cells when EmSOX2was confected with mouse Oct4, c-Myc, and Klf. The results of iPS induction show that EmSOX2can replace mouse Sox2A11in a word, we obtained full-length sequences of EmSOX2by RACE technology from Echinococcus multilocularis; EmSOX2protein, which played a pivotal role in maintaining the multipotency of the stem cell, was mainly expressed in the nucleus of the germinal layer cells.This study will contribute to realize the endogenous factor and the regulatory mechanism in maintaining the multipotency of the cell of Echinococcus multilocularis germinal layer. Besides, the results could also provide some theoretical evidences for new drug targets and even new drugs against Echinococcus multilocularis.