Expression, antibody production and species specificity of SpaA pilin subunit from Lactobacillus rhamnosus

Abstract:

[Objective] To obtain the polyclonal antibody against SpaA pilin subunit from Lactobacillus rhamnosus, and study its species specificity. [Methods] The spaA of Lactobacillus rhamnosus GG (LGG) was amplified by PCR and cloned into plasmid pET-28α(+). The resultant plasmid was transformed into Escherichia coli BL21(DE3), and the recombinant SpaA was expressed by IPTG induction and purified by Ni-NTA column. The purified protein was used to immunize the BALB/c mice for raising polyclonal antibody. The SpaA presence in 18 Lactic acid bacteria (LAB) strains belonging to 12 species was detected by indirect whole-cell ELISA, Western and Dot-blot assays. [Results] The recombinant SpaA was obtained with molecular mass of 36 kD as expected. The antibody against SpaA was generated with titer of 1:12 800. Western analysis showed that the antibody can specifically react with the native SpaA. Among 18 LAB strains, all of Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus paracasei strains showed positive reactions in the spaA-PCR and RT-PCR detection, while only 3 strains of Lactobacillus rhamnosus showed positive reactions with SpaA antibody in whole-cell ELISA and Dot-blot, and none of the other strains had obvious cross reaction with SpaA antibody. [Conclusion] Although SpaA encoding gene was highly homologous among Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus paracasei species, SpaA protein was uniquely expressed and cell surface-exposed in Lactobacillus rhamnosus. The SpaA antibody obtained in this study provide a useful tool for further immunomagnetic separation and pilin function investigation of highly adhesive Lactobacillus rhamnosus strains.

Key words:Lactobacillus rhamnosus GG, Pilin, SpaA subunit, Expression, Antibody