重组pET30a-EgM9蛋白高免兔血清对体外培养细粒棘球绦虫发育的影响

Effects of High Immune Rabbit Serum against Recombinant pET30a-EgM9 Protein on Development of Echinococcus granulosus in Vitro

探究egM9基因在细粒棘球绦虫性成熟发育过程中的调控机制。利用重组pET30a-EgM9蛋白高免血清对细粒棘球绦虫进行体外干预试验,观察干预后虫体的发育情况。在体外培养模型中,用pET30a-EgM9蛋白高免兔血清作用于体外培养30 d的虫体,通过Western blot检测虫体蛋白的免疫反应性,RT-qPCR分析虫体中EgM9基因的表达水平。显微观察表明经高免血清干预后虫体明显向成囊方向发育。Western blot直接法表明高免兔血清可以与虫体蛋白形成复合物,间接法证明EgM9多克隆鼠血清未与上述复合物发生抗原抗体特异性结合。RT-qPCR验证EgM9高免兔血清可刺激虫体中的EgM9基因的表达。说明通过高免血清体外干预,可促进EgM9在基因和转录水平上的表达,为筛选细粒棘球绦虫终末宿主疫苗候选基因提供理论依据。

To explore the regulatory mechanism of EgM9 gene during sexual maturation of Echinococcus granulosus,pET30a-EgM9 recombinant protein high immune serum was used to interfere with Echinococcus granulosus in vitro,and the development of Echinococcus granulosus was observed after intervention.Rabbits were immunized with pET30a-EgM9 protein to obtain high-valence serum.In vitro,the serum was applied to the worms,which was cultured for 30 days.Western blot was used to detect the immunoreactivity of the EgM9 proteins.RT-qPCR was used to analyze the expression level of EgM9 gene.The microscopical observation showed that the worm developed obviously in the direction of cyst formation after intervention with high immune serum.The Western blot showed that the serum of rabbits could form complex with worm protein,and the indirect method showed that the mouse serum of the EgM9 protein did not bind to the above complexes specifically.RT-qPCR analysis showed that the rabbit serum against EgM9 protein could stimulate the expression of EgM9 gene in worms.The expression of EgM9 gene can be promoted by in vitro intervention with high immune serum,which provided a theoretical basis for screening the candidate genes of the final host vaccine of Echinococcus granulosus.