Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a – Escherichia coli DH5α system

Aim This study aims at cloning, sequencing, and phylogenetic analysis of a partial CDS of ligA gene in pET-32a - Escherichia coli DH5α system, with the objective of identifying the conserved nature of the ligA gene in the genus Leptospira. Materials and Methods A partial CDS (nucleotide 1873 to nucleotide 3363) of the ligA gene was amplified from genomic DNA of Leptospira interrogans serovar Canicola by polymerase chain reaction (PCR). The PCR-amplified DNA was cloned into pET-32a vector and transformed into competent E. coli DH5α bacterial cells. The partial ligA gene insert was sequenced and the nucleotide sequences obtained were aligned with the published ligA gene sequences of other Leptospira serovars, using nucleotide BLAST, NCBI. Phylogenetic analysis of the gene sequence was done by maximum likelihood method using Mega 6.06 software. Results The PCR could amplify the 1491 nucleotide sequence spanning from nucleotide 1873 to nucleotide 3363 of the ligA gene and the partial ligA gene could be successfully cloned in E. coli DH5α cells. The nucleotide sequence when analyzed for homology with the reported gene sequences of other Leptospira serovars was found to have 100% homology to the 1910 bp to 3320 bp sequence of ligA gene of L. interrogans strain Kito serogroup Canicola. The predicted protein consisted of 470 aminoacids. Phylogenetic analysis revealed that the ligA gene was conserved in L.interrogans species. Conclusion The partial ligA gene could be successfully cloned and sequenced from E. coli DH5α cells. The sequence showed 100% homology to the published ligA gene sequences. The phylogenetic analysis revealed the conserved nature of the ligA gene. Further studies on the expression and immunogenicity of the partial LigA protein need to be carried out to determine its competence as a subunit vaccine candidate.


Introduction
Epidemics of leptospirosis continue to occur in tropical developing countries, due to poor standards of sanitation and hygiene that leave people and animal at risk to the disease [1]. Leptospirosis is caused by a Spirochaete of the genus, Leptospira, which comprises about 22 genomospecies, further divided into about 300 antigenically different serovars [2,3]. Recently, 12 new species of Leptospira have also been identified [4].
Currently available whole-cell inactivated vaccines provide only serovar-specific, short-term immunity and can afford little cross-protection against the different leptospiral serovars. Genus-specific leptospiral proteins that are conserved throughout the different serovars of Leptospira which are immunogenic and uniquely expressed during acute infection may help in the development of an effective vaccine for leptospirosis as well as aid in studies on its pathogenesis [5]. Recombinant DNA technology aids in the production of purified recombinant genus-specific proteins in bulk quantities and hence helps in the development of subunit vaccines for various infections.
This study aims at cloning of a highly immunodominant region of the ligA gene of Leptospira in the pET32 vector -E. coli DH5α system followed by sequencing and phylogenetic analysis of the partial ligA gene.

Ethical approval
All the procedures have been carried out in accordance with the guidelines laid down by the Bulk PCR (100 µL) was performed in 50 µL reaction mixture containing 34 µL nuclease-free water, 5 µl 10 X PCR buffer, 1 µl of 10 mM dNTP mix (200 µM), 2 µl (20 pmol) of each of the forward and reverse primers, 5 µL of suitably diluted DNA template, and 1 µl of Jumpstart Taq DNA polymerase. The PCR amplification cycle comprised an initial denaturation cycle at 96°C for 30 s followed by 35 cycles of denaturation (96°C for 15 s), annealing (55°C for 30 s) and extension (68°C for 2 min), followed by a final extension cycle of 68°C for 5 min. The amplified PCR product of 1491 bp was eluted from the agarose gel using GeneJET ™ Gel extraction kit, Thermo Scientific ™ , to obtain the purified DNA.

Cloning of partial CDS of ligA gene
The 1491 bp PCR product obtained from L. interrogans serovar Canicola and pET-32a DNA was eluted from the agarose gel using GeneJET Gel extraction kit, Thermo Scientific. Gel-purified ligA DNA and gel-purified pET-32a DNA were digested with restriction enzymes, NcoI and XhoI (MBI, Fermentas). The digested products were again gel purified and subjected to ligation reaction using T4 DNA Ligase (MBI Fermentas). The ligation reaction mixture was incubated for 1 h at 22°C and further kept at 4°C overnight. Competent cells of E. coli DH5α ™ were prepared by calcium chloride method as described by Sambrook and Russell [6] with some modifications. The pET-32a ligA DNA was transformed into competent E. coli DH5α cells by heat shock method. Five microliters of ligation mixture was mixed with 200 µL of E. coli DH5α competent cells and kept on ice for 1 h. These cells were exposed to heat shock at 42°C for exactly 90 s and immediately kept on ice for 5 min. About 800 µL of Luria-Bertani (LB) broth with ampicillin (100 mg/mL) was added to the transformed cells and incubated at 37°C for 45 min. The cells were centrifuged at 6000 × g for 8 min and the supernatant was discarded retaining 100 µL of media to resuspend the cells. The transformed cells were plated on LB agar plates containing ampicillin. Appropriate negative controls with untransformed E. coli DH5α ™ cells were also processed simultaneously and the plates were incubated at 37°C for 24-36 h. The recombinant clones were screened by colony PCR. The pET-32a ligA plasmid construct was extracted from the transformed E. coli DH5α using Thermo Scientific Gene JET plasmid Miniprep Kit and subjected to sequencing.

Sequencing and analysis of the partial CDS of ligA gene
The partial ligA gene insert of pET-32a ligA plasmid was submitted to DNA sequencing facility at SciGenom Services, Kakkanad, Cochin, for nucleotide sequencing using the SP6 and T7 promoter primers by dideoxy chain-termination method [7]. The nucleotide sequences obtained were aligned with the published ligA gene sequences of other Leptospira serovars, using nucleotide BLAST, NCBI.

Phylogenetic analysis
The phylogenetic analysis involved 8 nucleotide sequences. The sequence alignment was done by ClustalW method in the BioEdit software. Evolutionary analyses were conducted in MEGA 6.06 software [8]. The evolutionary history was inferred using the maximum likelihood method based on the Tamura-Nei model. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting the topology with superior log-likelihood value.

Results
The genomic DNA isolated from L. interrogans serovar, Canicola, using QIAamp DNA Mini Kit (Qiagen) was of high purity and required concentration (69.1 ng/µL). Primers were designed for the 1491 bp fraction corresponding to nucleotides 1873-3363 of the complete CDS of ligA gene, with addition of primer tag regions and RE sites for NcoI and XhoI. The annealing temperature for the ligA primers was determined as 55°C. The PCR could amplify the 1491 The pET-32a-transformed E. coli DH5α plated on LB agar with ampicillin yielded 25-30 colonies following 36 h of incubation at 37°C. Control plates inoculated with untransformed E. coli DH5α did not yield any colonies. Colony PCR using LigAF and LigAR primers revealed bands at 1491 bp size (Figure-

Phylogenetic analysis
The optimal tree constructed with 8 nucleotide sequences with the highest log likelihood (−3438.4275) is shown in Figure-3.

Discussion
The Lig (leptospiral Ig-like) protein is a family of surface-exposed lipoproteins found only in pathogenic Leptospira and expressed during acute infection and hence is thought to play a role in the pathogenesis of leptospirosis. The LigA, a 130 kDa protein encoded by the ligA gene, belongs to the Lig family of proteins comprising of LigA and LigB that possess a series of 90 amino acids tandem repeats homologous to the bacterial immunoglobulin-like (Big) domain. The recombination patterns and sequence variations of ligA, ligB, and ligC genes, studied in 10 pathogenic strains of five Leptospira species, revealed that ligA might have been created by partial gene duplication of ligB involving two steps. The amino-terminal domains of LigB and LigA proteins, of strains possessing both genes, were found to be identical having 98.5±0.8% mean identity [9]. The LigA proteins are predicted to be lipoproteins as they possess a 17 amino acid N-terminal signal peptide and a lipoprotein signal peptidase cleavage site. The ligA gene encoding this protein reportedly comprise of 3675 bp with 12 tandem repeats. It has been reported that cloning of full-length ligA gene in pET-22b plasmid and expression in E. coli produced very low levels of rLigA, which was attributed to the high toxicity of the protein [10]. Several studies have demonstrated the cross-reactive immunoprotective effect of recombinant LigAN1 protein, a truncated form of LigA comprising a carboxy-terminal repeat domain unique to the LigA [11][12][13]. In this study, we have amplified a 1491 bp fraction of the ligA gene, within the LigAN1 region, encoding hydrophilic amino acids from 624 to 1121. The amplified DNA sequence was cloned on to pET-32a expression vector using host E.coli DH5α. The pET vectors are considered to be one of the most powerful vector systems developed for cloning and expression of bacterial genes in E. coli [14]. The pET-32a (+) vector series is designed for expression of peptide sequences fused with the 6X histidine tags. Target genes cloned in pET plasmids are placed under the control of a strong bacteriophage T7 promoter and expression is induced by providing a source of T7 RNA polymerase in the host cell. The T7 RNA polymerase is highly selective and active and helps in targeting almost all of the cells' resources on the expres sion of the specific gene. In a study, the parasporin 1 gene of Bacillus thuringiensis was amplified by PCR and cloned into pGEM-vector. The predicted protein encoded by the 2371 nucleotides' long gene sequence was composed of 789 amino acids with an estimated molecular weight of 84 kDa [15]. Vector pRham-SUMO was used to clone and sequence the nagH gene of C. chauvoei [16]. In another study, P67 gene of Mycoplasma leachii was cloned in pRham N-His SUMO Kan vector and transformed into competent Escherichia cloni 10G cells [17]. In this study, the pET-32a ligA construct was transformed into E. coli DH5α, an expression host that lacks T7 RNA polymerase gene. E. coli is the most preferred host in recombinant DNA technology because its genome is well studied, is relatively cheap and has short generation time [18]. The phylogenetic tree revealed that the ligA gene of the tested strain of Leptospira species occupied the same position in the phylogenetic tree as other reference leptospiral strains of the particular species. This confirmed that the ligA gene was conserved in the genus Leptospira and hence indicated that the LigA protein was a probable candidate for subunit vaccines. The truncated recombinant LigA protein for vaccine studies may be expressed by subcloning the pET-32a ligA construct into E. coli BL21 (DE3) expression system and induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) [19].

Conclusion
The results of the study reveal that the partial ligA gene can be cloned into pET-32a -E. coli DH5α system. The phylogenetic analysis revealed that the partial ligA gene was conserved in the genus Leptospira. Hence, further studies need to be conducted on the immunogenicity and vaccine properties of the protein.