Leptospira, a member of the order Spirochaetales, is the causative agent of leptospirosis, an important zoonosis encountered worldwide. The Leptospira interrogans serovar Sejroe was grown in EMJH medium and its DNA was isolated using standard techniques. The LipL32 gene was amplified using the reported primer of Kirschneri of LipL32. The amplified product was found to comprise 756 base pairs. This amplified gene fragment of LipL32 lipoprotein was cloned in E. coli (DH5 alpha) cells using pDrive plasmid as a vector. The recombinant cells were selected on LB agar medium containing ampicillin, X-gal and isopropyl-beta-D-thiogalactopyranoside. Plasmid was extracted from the recombinant white colonies, and restriction endonuclease (RE) analysis was carried out using PstI and SalI. On partial sequence analysis, the product exhibited 756 base pairs, corresponding to 251 amino acids. The cloned gene could be further used for expression of recombinant protein for serodiagnosis of leptospirosis.
Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. A gene homologous to that of 35-kDa antigen of Mycobacterium leprae was cloned and sequenced from Mycobacterium paratuberculosis. The database searches revealed 82.79% and 95.67% similarities of its nucleotide sequence, with those of immunodominant 35-kDa protein of M. leprae and M. avium, respectively.
Summary Full-length cDNA (582 bp) of the interleukin-18 (IL-18) gene of the Indian water buffalo (Bubalus bubalis) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequenced. The deduced amino acid sequence has 99% and 95% similarity with the IL-18 sequences of cattle and sheep, respectively. There are two amino acid substitutions at positions 132 and 182 in buffalo IL-18 compared with that of cattle. Phylogenetic analysis showed that the IL-18 sequence of fish forms a different lineage and is most divergent from that of cattle, buffalo, sheep, pig, dog, horse, human, monkey, mouse, rat and chicken.
Neutrophil beta-defensins have been identified as naturally occurring potent antibacterial cationic peptides serving as effector molecules of innate immunity that provide a first line of defence against pathogens. Considering the broad-spectrum antimicrobial activity against microorganisms and role in innate immunity of the neutrophil beta-defensins, it has been characterized in many livestock species including cattle, sheep, caprine and porcines. Here we report the isolation, cloning, sequencing and expression of precursor bovine neutrophil beta-defensin isolated from Indian water buffalo. Full-length cDNA was amplified using reverse transcription polymerase chain reaction (RT-PCR). The cDNA contained an open reading frame of 192 bp encoding a putative polypeptide of 63 amino acids. Deduced amino acid sequence of buffalo BNBD4 showed varying amino acid identity with the published sequences of related beta-defensins of other domestic ruminant species ranging from 67.18 to 79.68%. Recombinant buffalo defensin was produced in Escherichia coli as fusion protein.
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