[Identification on the immunogenic activity of recombinant rLTB/CTB-LipL41/ 1 to Leptospira interrogans and detection of lipL41 gene with its production].
OBJECTIVE
To construct prokaryotic expression systems of ltB/ctB-lipL41/1 fusion genes, identify immunogenic and adjuvant activities of the products as well as to understand the frequencies of lipL41 gene that carrying and expressing in L. interrogans wild strains and specific antibody levels in sera from patients with leptospirosis.


METHODS
Polymerase chain reaction (PCR) with linking primer was applied to construct the fusion genes ltB-lipL41/1 and ctB-lipL41/1. By routine molecular biological techniques, prokaryotic expression systems of the two fusion genes were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine expression of the target recombinant proteins rLTB-rLipL41/1 and rCTB-rLipL41/1. Both western blot and Ganglioside-enzyme linked immunosorbent assay (GM-ELISA) were used while immunogenic and adjuvant activities of rLTB-rLipL41/1 and rCTB-rLipL41/1 were measured. PCR and MAT were performed to detect lipL41 gene and expression of the gene in 97 wild strains of L. interrogans, respectively. Antibodies against product of lipL41 gene in serum samples from 228 leptospirosis patients were detected by ELISA.


RESULTS
In comparison with reported corresponding sequences, the similarities of ltB-lipL41/1 and ctB-lipL41/1 fusion genes to nucleotide and putative amino acid sequence were 99.6%-99.9% and 99.8%-100%, respectively. Expression outputs of both rLTB-rLipL41/1 and rCTB-rLipL41/1, mainly presenting with inclusion body, consisting approximate 10% of the total bacterial proteins. Both rLTB-rLipL41/1 and rCTB-rLipL41/1 could combine rabbit anti-rLipL41/1 serum as well as bovine GM1, respectively. 87.6% of the L. interrogans wild strains(85/97) having lipL41 gene while 84.5% (82/97) of the wild strains with rLipL41/1 or rLipL41/2 antiserum were positive for MAT with titers of 1:4-1:128. 84.6% (193/ 228), 78.5% (179/228) from the patients' serum samples were positive for rLipL41/1 and rLipL41/2 antibodies, respectively.


CONCLUSIONS
ltB-lipL41/1 and ctB-lipL41/1 fusion genes and their prokaryotic expression systems were successfully constructed in this study. The two expressed fusion proteins showed qualified immunogenic and adjuvant activities. lipL41 gene was extensively distributed and frequently expressed in different serogroups of L. interrogans. rLTB-rLipL41/1 or rCTB-rLipL41/1 seemed to have had good potential to serve as an antigen in L. interrogans genus-specific vaccine.
Ruan
Ping
P
Department of Medical Microbiology and Parasitology, Medical School of Shaoxing University, Shaoxing 312000, China.
Yan
Jie
J
Mao
Ya-fei
YF
Peng
Hui-qin
HQ
Zhou
Xiao-hui
XH
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007921:Leptospira interrogans / Q000235:genetics* / Q000502:physiology; D007922:Leptospirosis / Q000276:immunology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D017422:Sequence Analysis, DNA
D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; C538745:LipL41 protein, Leptospira interrogans; D011993:Recombinant Fusion Proteins