doi:10.5455/vetworld.2013.193-195 Cloning and sequencing of the virulent gene LipL32 of Leptospira interrogans serovar Autumnalis

Aim: To clone the virulent gene LipL32 of Leptospira interrogans serovar Autumnalis and to analyze the sequence with LipL32 gene of other pathogenic serovars of Leptopsira. 
 
Materials and Methods: Leptospira interrogans serovar Autumnalis procured from Leptospira research laboratory, Chennai was used in the study. Polymerase chain reaction (PCR) was carried out for amplifying LipL32 gene using the reported primers of Leptospira Kirschnerii. The PCR product was cloned into TA cloning vector and the vector was transformed into E.Coli DH5a cells. The plasmid was isolated from E.Coli and sent for sequencing with universal primers. The sequence was 
submitted in genbank with accession number JQ861883. 
 
Results: The PCR product revealed an amplicon of 790 bp. The LipL32 gene sequence of Leptospira interrogans serovar Autumnalis showed 99 % similarity with most of the pathogenic Leptospires. 
 
Conclusions: LipL32 gene of Leptospira is highly conserved in most of the pathogenic Leptospires. The study concludes that this gene could be used as a target for the diagnosis of leptospirosis in animals and humans and could be tested as an important candidate antigen for vaccine production.

Harris medium (EMJH) (Difco) supplemented with field microscopy, isolation are less sensitive and bovine albumin and sub cultured at seven days interval.Microscopic agglutination test, the gold standard test E.coli DH5α was maintained in Luria-Bertani agar can be performed only in reference laboratories since it (HiMedia, Mumbai).The genomic DNA of Leptospira requires the maintenance of live cultures.The tests was extracted as per the method described [9].The employing outer membrane proteins (OMPs) showed PCR was carried out using the reported primers of high sensitivity and specificity [4,5].The results of LipL32 gene.The primer sequences were 5' -CGC recombinant OMP antigen based enzyme linked GCT GCA GTT ACT TAG TCG CGT CA G AAG-3' immunosorbant assay's (ELISA) are promising [6].and 5'-CGC GGT CGA CGC TTT CGG TGG TCT LipL32 was consistently observed in all pathogenic CTG CCA AGC -3 ' [11].The primers were diluted in leptospires and was observed to be the immunodominuclease free water so as to have a final concentration nant protein antigen recognized by the human humoral of 20 pmol/µl.The reaction was carried out in a 25 µl response during natural infections.This protein could reaction mixture, containing 10X PCR buffer with be useful as a marker of infection for laboratory case MgCl 10 mM dNTP mix, 20 pmol of each primer, 1.0 2, confirmation in the field and in differentiating lepto-U of Taq DNA polymerase, nuclease free water and spirosis from other causes of acute febrile illness [7].
template DNA.The PCR progamme was standardized Leptospiral protein p32 is the most immunodominant with one cycle of initial denaturation at 94º C for 4 min protein and could be employed for the development of followed by 30 cycles each of denaturation (94º C for novel diagnostic assays [8].The present study was 45 sec, annealing (54º C for 45 sec) and extension (72º carried out to clone and sequence the LipL32 gene of C for 45 sec) and final extension (72º C for 5 min).The Leptospira interrogans serovar Autumnalis and to analyze PCR product thus obtained was analyzed by running on the sequence relationship with other pathogenic leptospires.
1.0 % agarose gel.The gel was visualized under UV

Materials and Methods
transilluminator and the results were documented in a gel documentation system (Alpha Innnotech Corporation, Leptospira interrogans serovar Autumnalis, USA).The PCR product was gel eluted using GeneAll degree of similarity with other pathogenic serovars of DNA purification kit as per the manufacturer protocol Leptospira (Table -1).The LipL32 gene is highly (GeneAll, Korea).The cloning vector pTZ57R/T was conserved in most of the pathogenic serovars of used for cloning the purified DNA as per the method Leptospira as evidenced from the sequence analysis.described by the manufacturer (Fermentas).The cloning The obtained sequences shown 100 % similarity with vector was transformed into E. coli DH5α cells and the LipL32 gene sequences of Leptospira interrogans culture was plated on Luria-Bertani (LB) agar plates serovar Autumnalis (EU526391) and Leptospira intecontaining ampicillin (100 µg/ml), with the help of rrogans serovar Lai (AY568679) and 99% similarity sterile L-spreader and incubated at 37º C overnight.
serovar Mini (AY609333) and Leptospira kirschneri The plasmid was reconfirmed by PCR before sending (AY461917).LipL32 protein is the most abundant and for sequencing.The plasmid was sequenced using universal an important surface protein present only in pathogenic M13 forward and reverse primers (SciGenom labs Pvt. Leptospira and found to play a major role in host Ltd, Cochin, Kerala).The vector sequence was removed immunogenic response and in infection [10].The using Vecscreen tool and the trimmed sequence was LipL32 gene of Leptospira interrogans serovar Sejroe submitted to NCBI GenBank (accession no.JQ861883).
was found to be conserved in many pathogenic Further, the nucleotide sequence was subjected to nucleotide serovars [11] which is in agreement with the present BLAST to compare for sequence similarities with study.Similar findings were reported by several other other sequences available in the NCBI database (http:// workers [12].The utility of LipL32 for diagnosis of blast.ncbi.nl.nih.gov).

Results and Discussion Conclusion
The extracted genomic DNA from Leptospira The present study concludes that the similarity in interrogans serovar Autumnalis showed an Optical the sequences of the cloned product with other Density of 1.7 indicating the purity of isolated DNA.pathogenic serovars, suggests its importance in the The PCR amplification of LipL32 gene revealed an utility of LipL32 gene as an important target for the amplicon of 790 bp on 1.0 % agarose gel (Fig- 1 ).The diagnosis of leptospirosis in animals and humans as sequence was deposited in the nucleotide GenBank well as promising vaccine candidate as the protein is under the accession no.JQ861883.The sequence when reported to be immunodominant.subjected to nucleotide BLAST search revealed a high

Introduction
procured form Leptospira research centre, Tamil Nadu Veterinary and Animal Sciences University, Chennai Leptospirosis has re-emerged as an important and maintained in the Department of Veterinary Microzoonotic disease in India and is endemic in most of the biology, College of Veterinary and Animal Sciences, southern states like Kerala, Tamil Nadu and certain Mannuthy was used in the study.The organisms were parts of Andhra Pradesh [1-3].Conventional methods maintained in Ellinghausen-McCullough-Johnsonof diagnosis like demonstration of organisms by dark