PCR amplification, molecular cloning, DNA sequence analysis and immuno/protection in BALB/C mice of the 33 kDa endoflagellar protein of L. interrorgans serovar lai.
A pair of oligonucleotide primers were designed to amplify the endoflagella gene of L. interrogans serovar lai. An approximately 840 bp fragment was generated with PCR and inserted into plasmid pUC8, after the fragment and pUC8 were digested respectively with BamHI and PstI. A recombinant plasmid (designated as pLF1) was obtained. SDS-PAGE analysis indicated that a 33 kDa was expressed in E. coli JM103 harboring pLF1 and the expression level of the protein was 11% of the total bacterial soluble proteins. Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflegella (Axial filament) of Leptospira interrogans serover lai. Nucleotide sequence data showed an open reading frame encoding 282 aminoacids residues, corresponding to a protein of molecular weight 33.6 kDa. Comparison of the deduced endoflagellar subunit protein (flaB) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Treponema pallidum flaB proteins. Immunization/protection experiment was performed on the model of BALB/C mice and showed that there was higher survival rate in the group JM103-pLE1 than in the group JM103-pUC8.
Research Unit of Leptospirosis, Department of Pathophysiology, West China University of Medical Sciences, Chengdu 610044.
B
B
Dai
Z
Z
Chen
H
H
Yan
H
H
Zhao
S
S
Li
D000595:Amino Acid Sequence; D000818:Animals; D001428:Bacterial Vaccines; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D007921:Leptospira interrogans / Q000737:chemistry*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D019444:Vaccines, DNA; D014895:Weil Disease / Q000517:prevention & control
D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D019444:Vaccines, DNA