Effect of antiserum against recombinant EGr_09888 on the in vitro development of Echinococcus granulosus protoscoleces.

Abstract

To investigate the effects of Echinococcus granulosus sensu stricto (E. granulosus s.s.) reproduction-associated protein EGr_09888 on the development of E. granulosus s.s. protoscoleces (PSCs) in vitro. SWISS-MODEL, Netphos3.1, and PyMOL bioinformatics analysis software, along with subcellular localization prediction, were used to analyze the structure, phosphorylation sites, epitopes, and distribution of the EGr_09888 protein. The recombinant plasmid pET30a-EGr_09888 was constructed for protein expression and purification, and New Zealand white rabbits were immunized with the recombinant protein. An indirect ELISA method was used to determine serum IgG levels, through Western blotting to verify immune specificity and different stages worm protein extracts EGr_09888 protein expression. The distribution of EGr_09888 in PSCs, adult worms and gravid segments was analyzed by immunofluorescence staining. PSCs cultured in vitro for 3, 6, and 10 weeks were treated with rabbit anti-EGr_09888 high-titer immune serum for 6 days and then their survival rate and the gene expression of EGr_09888 in the PSCs was analyzed. The result revealed that the EGr_09888 protein has diverse secondary-structure amino acid proportions, and its SWISS-MODEL-predicted tertiary structure is reliable, containing 54 potential phosphorylation sites. It has B- and T-cell epitopes at amino acids (aa) 25-44 and 166-185, shows immunogenic potential, and is localized mainly in the cytoplasm. Induced expression of the recombinant EGr_09888 protein (27 kDa) resulted in rabbit anti-EGr_09888 serum with a potency greater than 1:160000. Immunoblotting revealed that the rabbit anti-EGr_09888 serum could specifically bind to the recombinant EGr_09888 protein. And the expression level of EGr_09888 protein was higher in adult worm protein extracts (P ≤ 0.001), Immunofluorescence staining analysis revealed that EGr_09888 was localized mainly within the uterus of the gravid segment of E. granulosus s.s adult worms. After 6- and 10-week-old PSCs were treated with rabbit anti-EGr_09888 serum, the survival rate and relative PSC EGr_09888 expression level were significantly reduced (P < 0.0001). This study revealed that EGr_09888, a reproduction-related protein in E. granulosus s.s., has potential as a candidate vaccine for definitive hosts.