LIN Xiaoyun,HOU Bailong,XIONG Yirong, et al. Prokaryotic expression of the Z domain of Staphylococcus protein A and preparation of its polyclonal antibody[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2015, 45(1): 1-.
Abstract：Objective: To prepare the Z domain of Staphylococcus protein A(SpA-3Z) expressed in prokaryotic cell and its polyclonal antibody, and dentify the specific activity of the antibody. Methods: The tandem repeats (SpA-3Z) were formed by three SpA-Z domains which linked with flexible linker peptide (GS). After prokaryotic codon optimized, the whole tandem repeats gene sequences were synthesized and then cloned into an expression vector pET21a(+) and constructed the recombinant plasmid pET21a(+)/SpA-3Z. After sequencing, the recombinant plasmid was transformed to E.coli BL21 (DE3) and induced expressed by Isopropyl β-D-1-Thiogalactopyranoside (IPTG), the recombinant protein (SpA-3Z protein) was purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western Blot analysis. Then the rabbits were immunized with the SpA-3Z protein, the sera samples were collected before and after immunization, in order to assay the response and titer of the serum polyclonal antibody by ELISA, and the combining specificity to natural SpA protein by Western Blot analysis. Results: The pET21a(+)/SpA-3Z recombinant plasmid was successfully constructed, and the SpA-3Z recombinant protein was expressed in prokaryotic expression system and the purified SpA-3Z was prepared, the molecular weight of SpA-3Z protein was about 21 kDa, which conform to its theoretical molecular weight, indicated by SDS-PAGE and also confirmed by Western Blot analysis. The specific serum polyclonal antibody IgG was induced by immunization with SpA-3Z in the rabbits, and its titer was up to 1:40 000. The Western Blot analysis showed that the single band appeared in the molecular weight of about 42 kDa position (natural SpA protein), indicated that the rabbit serum polyclonal antibody could specifically recognize the natural SpA protein. Conclusion: The SpA-3Z recombinant protein shows strong immunogenicity and antigenicity. The rabbits serum polyclonal antibody IgG shows high titer and good specificity, and can be used in the further study of SpA-Z related biological and immunological function.
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