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Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A–J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine‐cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET‐15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta‐ d‐thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni‐sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines. Highlights: The versatile sequence optimization was the reason to produce a high level of rVP6.The high yield of rVP6 can apply to produce cheaper commercial kit.Polyclonal antibody was raised against rVP6.Immunoblotting and immunofluorescence assays showed the potency of VP6 antibody to detect RV. [ABSTRACT FROM AUTHOR]