Prokaryotic expression and vaccine potential of Mucin-6 from Rhipicephalus linnaei

Mucins are essential for tick feeding and survival, contributing to mouthpart cement formation, host immune modulation, and peritrophic membrane assembly. This study evaluates Mucin-6 from Rhipicephalus linnaei as a candidate anti-tick vaccine antigen. We predicted Mucin-6 physicochemical properties and antigenicity using bioinformatic tools. Differential gene expression was analyzed by qPCR across tick developmental stages and feeding states. The recombinant pET28a-Mucin-6 plasmid was transformed into BL21(DE3) Escherichia coli for expression. Recombinant Mucin-6 (rMucin-6) was recovered from inclusion bodies, renatured, and purified by Ni–NTA affinity chromatography. Six New Zealand White rabbits were randomly allocated to rMucin-6 ( n =3) or PBS control ( n =3) groups. The immunization group received three subcutaneous injections of 500 μg rMucin-6 with Freund's adjuvant at 14-day intervals. Serum antibody titers were monitored by ELISA. Fourteen days after the third immunization, 30 adult ticks (15 male, 15 female) were placed on each rabbit. Tick engorgement weight, egg mass weight, blood-feeding duration, and egg-hatching rate were measured. Mucin-6 is a hydrophilic, unstable protein with a predicted molecular mass of 24.9 kDa after signal peptide cleavage and abundant B-cell linear epitopes. The Mucin-6 gene was expressed across all developmental stages of R. linnaei , with significantly higher expression at the egg stage. The purified rMucin-6 migrated at approximately 41 kDa on SDS-PAGE. Rabbits immunized with rMucin-6 developed high antibody titers (1:512,000 at day 35). Compared with ticks fed on PBS-injected rabbits, ticks fed on rMucin-6-immunized rabbits showed significant reductions in engorged body weight (13.6%, P <0.05), egg mass (14.7%, P <0.05), and prolonged blood-feeding duration (8.3%, P <0.05), yielding an overall vaccine efficacy of 26.3%. Western blot confirmed that rabbit anti-rMucin-6 serum recognized native Mucin-6 in tick lysates. Prokaryotically expressed rMucin-6 elicited strong humoral immunity in rabbits, and antibodies against rMucin-6 recognized the native tick protein and reduced both engorged body weight and egg mass of ticks fed on immunized rabbits, suggesting that Mucin-6 merits further evaluation as a candidate vaccine antigen against R. linnaei .