Objective To achieve prokaryotic expression of the tomato (Solanum lycopersicum) SlHSP21-1/2 gene and to prepare and characterize its polyclonal antibody, providing a foundation for further studies on its biological function.
Method The coding sequences of SlHSP21-1/2 genes were cloned from tomato and subjected to bioinformatic analyses to predict their physicochemical properties and subcellular localization. Gene-specific primers were designed for amplification of the target fragments, which were then inserted into a prokaryotic expression vector. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) expression strain, and recombinant protein expression was induced by IPTG. The expressed proteins were analyzed by SDS-PAGE and purified using Ni-NTA affinity chromatography. The purified proteins were used as antigens to immunize New Zealand rabbits. Polyclonal antibodies were obtained through multiple immunizations, and their specificity were evaluated.
Result Bioinformatics analysis indicated that SlHSP21-1/2 proteins possess contain typical features of small heat shock protein, including a conserved α-crystallin domain and highly conserved N- and C-terminal regions. Subcellular localization predictions suggested that these proteins are localized in chloroplasts. SDS-PAGE analysis confirmed that the SlHSP21-1/2 fusion proteins were successfully expressed, predominantly as inclusion bodies. Following purification by Ni-NTA affinity chromatography, high-purity recombinant proteins were obtained, providing reliable antigens for immunization. Polyclonal antisera raised in New Zealand rabbits using the recombinant proteins specifically recognized the target protein bands in Western blot assays, demonstrating that the prepared antibodies possess strong specificity.
Conclusion The SlHSP21-1/2 gene was successfully expressed in a prokaryotic system, and polyclonal antibody with good specificity was obtained.
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