High-level expression of Yersinia pestis F1-V fusion protein in Escherichia coli and its identification

Objective To construct pET-llc-Fl-V fusion expression plasmid in order to get fusion protein with immunogenicity. Methods Fl gene and V gene of Yersinia pestis were cloned and inserted into pGEM-T vector. After sequence correction, the recombinant expression plasmid pET-11c-F1-V was constructed by inserting the DNA fiagment of Yersinia pestis F1 gene and V gene into pET-11c and transformed into E. coli BL21 (DE3) cell. The positive clones were selected by PCR and enzymes digest and, the expression product of Fl gene induced by IPTG was detected by SDS-PAGE, while the expression protein immunogenicity was detected by Western-blot. Results Sequence analysis revealed that DNA sequence of Fl gene was as same as that of Gene-bank record (X 61996). DNA sequence of V gene was mutanted at the site of 546 bp, but encoded same ammo acid. An expression band about Mr 58 000 was found by SDS-PAGE. The aim protein, most of it was in soluble form, was about 25% of total cell protein. While the immunogenicity of expression protein was detected by Western-blot. Conclusion pET-llc-Fl-V is successfully constructed and its high-level expression in Escherichia. coli is attained. The aim protein has immunogenicity .