多房棘球绦虫干细胞相关基因Nanos-1/2的克隆、原核表达及其在虫体内的定位分析OA

The objective was to investigate the molecular characteristics,protein expression,and localization of the stem cell-associated genes Nanos-1/2 of Echinococcus multilocularis(Em-Nos1/2)in the parasite.Bioinformatic analysis of Em-Nos1/2 was performed.Prokaryotic expression vectors pET28a-Em-Nos1/2 were constructed,and the recombinant proteins were induced,expressed,and purified.Polyclonal antibodies were generated by immunizing rabbits with the purified recombinant proteins.Western blot and immunofluorescence assays were employed to assess antibody specificity and determine the localization of the native proteins in the parasite,respectively.Phylogenetic analysis of Em-Nos1/2 from E.multilocularis revealed that these two genes were highly conserved within the genus Echinococcus,although minor differences in their three-dimensional structures were observed.High-purity recombinant Em-Nos1/2 proteins were obtained following construction of pET28a-Em-Nos1/2 vectors,induction of expression,and protein purification.The generated rabbit polyclonal antibodies exhibited high titers,reaching up to 1∶204 800.Western blot analysis confirmed that the prepared polyclonal antibodies specifically recognized the native Em-Nos1/2 proteins from E.multilocularis.Immunofluorescence localization demonstrated that both Em-Nos1/2 proteins were specifically expressed in the germinal layer of E.multilocularis larvae(metacestode).In this study,polyclonal antibodies capable of specifically recognizing the native Em-Nos1/2 proteins were successfully generated,and their specific localization to the germinal layer of E.multilocularis metacestodes was confirmed.These findings can provide crucial tools and a foundational basis for further investigations into the biological functions of Em-Nos1/2 in the stem cells of E.multilocularis.