Molecular Characterization of Gene Encoding Outer Membrane Protein loa22 in Pathogenic Leptospira Serovars in Iran

Abstract

The loa22 protein is highly conserved among pathogenic Leptospira serovars and it is expressed during both acute and chronic infections. The aim of this study was to clone and sequence of the loa22 protein-encoding gene of Leptospira serovars. In this study, 23 pathogenic Leptospira serovars and two nonpathogenic Leptospira serovars were used. These serovars were obtained from the microbial culture collection of Leptospira Reference Laboratory, Department of Microbiology, Razi Vaccine and Serum Research Institute, Karaj, Iran. Three serovars, including L. Sejroe Hardjo-bovis, L. Grippotyphosa, L. Canicola, are used in the preparation of the trivalent vaccine. The loa22 gene was amplified by specific primers and the PCR products were then purified using kit and were cloned into a pTZ57R/T vector and transformed in competent E. coli DH5α cells. The cells were then plated onto LB agar containing ampicillin and recombinant colonies subjected to colony PCR to confirm the presence of the Leptospiral gene. Positive colonies plasmid vector was isolated from cells by High Pure Plasmid Isolation Kit. The loa22 gene was detected in all 23 pathogenic serovars, while this gene was not observed in nonpathogenic L. biflexa. It was determined that the similarity percentage of the sequenced pathogenic serovars is between 95.5% and 100%. The results concluded that the loa22 gene was highly conserved among various pathogenic Leptospira serovars and can be used to develop an effective recombinant vaccine.

Keywords: cloning; leptospirosis; loa22 gene; pathogenic serovars; sequencing.

Figure 1

PCR results of seven pathogenic and two nonpathogenic of Leptospira studied samples. (1) 100 bp size marker (Bio-Rad); (2) L. autumnalis (RTCC 2802); (3) L. Canicola (RTCC 2805); (4) L. Grippotyphosa (RTCC 2808); (5) L. icterohaemorrhagiae (RTCC 2812); (6) L. pomona (RTCC 2815); (7) L. icterohaemorrhagiae (RTCC 2823); (8) positive control (L. icterohaemorrhagiae [RTCC 2837]); (9): L. biflexa (RTCC 2819); (10): L. biflexa (RTCC 2828); (11) Negative control; (12): 100 bp size marker (Bio-Rad).

Figure 2

Evaluation of the cloned loa22 gene in E. coli (DH5α) by PCR colony. (1) 100 bp size marker (Bio-Rad); (2) loa22 cloned gene; (3) negative control.

Figure 3

Purified plasmid electrophoresis of L. interrogans serovar Hardjo-bovis (2810). (1) 100 bp plus size marker (Bio-Fact); (2) plasmid containing loa22 gene; (3) control 2 or positive control kit; (4) control 1 or negative control kit; (5) PCR fragment control; (6) PCR product (671 bp).

Figure 4

Percentage of similarity and divergence between different sequenced serovars in the present study and Leptospira serovars registered in NCBI for loa22 gene, based on sequence analysis of loa22 gene using MegAlign software.

Figure 5

Dendrogram of the similarity of 18 serovars sequenced in the present study with three serovars registered in the NCBI based on the sequence analysis of the loa22 gene using MegAlign software.