Molecular characterization of Lingual antimicrobial peptide in the female reproductive tract of Buffalo

Bubalus bubalis (Ruminantia: Bovidae, Bovinae) is an economically important animal of many Asian countries, making significant contribution to milk and meat production. Sub clinical infection of the reproductive tract is one of the important causes for reduced reproductive efficiency in dairy herd of buffaloes. Antimicrobial peptides are component of innate immune system which helps in augmenting the resistance to infection at epithelial surfaces e.g reproductive tract epithelium. In this study we have identified a ß-defensin called Lingual Antimicrobial Peptide (LAP) in buffalo reproductive tract. Interestingly the gene was 100 % identical to the LAP isolated from the tongue epithelium of Bos taurus. The 195 bp cDNA of LAP codes for 64 amino acids and of which 50% are cationic amino acids. Phylogenetic studies indicate that LAP of reproductive epithelium of buffalo is different from other beta defensins isolated from the various tissues of same species, but all beta defensin were found to have the same progenitor gene. It is concluded that buffalo reproductive tract epithelium lining contains LAP.


Introduction
with 1X phosphate buffered saline of pH 7.4 to remove blood and other cells adhering to the epithelium.Antimicrobial peptides are important Total RNA was isolated separately from 200 mg constituents of the innate immune defense of all life of epithelial scrapings lining the lumen of vagina, forms including mammals, amphibians, insects and cervix, uterus and oviduct using 'Total RNA isolation plants.Lingual antimicrobial peptide (LAP) was first system' (Promega, USA).The integrity of the RNA isolated from the tongue epithelium of bovine was confirmed by 1% agarose gel electrophoresis.The (Schonwetter et al., 1995).Subsequent investigation purity of the RNA was checked by spectrophotometer, revealed the expression of LAP in a wide variety of measuring the A260/A280.Qiagen (Germany) RTtissues like trachea, intestine, mammary gland, female PCR Kit was used to produce cDNA from the total reproductive tract of bovine and also in buffalo tongue RNA template, following the manufactures protocol.and mammary gland.LAP has broad spectrum Complete cDNA sequence of LAP including start and antibacterial activity, antifungal activity and immunostop codon was obtained by RT-PCR using the modulatory activity which is important in limiting the following primers: forward 5'-CGG CAC CGA CAG establishment of infection on epithelial surfaces CAT GAG G-3' and reverse 5'-GCC ACG TCT TCG (Stolzenberg et al., 1997).The present study reports CCT TCT TT-3.Both primers were designed based on the expression pattern of LAP along the female bovine LAP sequence published in NCBI (Accession reproductive tract, its cDNA sequence, the deduced No: S76279).The annealing temperature was amino acid sequence, degree of identity of the LAP standardized (Mastercycler, Eppendrof, Germany) with other defensin gene and its phylogenetic relation.
and the annealing temperature of 560C for 1 minute

Material and Methods
was found to be optimum.For RT-PCR the initial denaturation was carried out at 950C for 15 minutes, The female reproductive tract sample of a followed by 30 cycle of amplification with buffalo was collected from local abattoir within 30 denaturation at 940C for 1 minute, primer annealing at minutes after their slaughter.Six female reproductive 560C for 1 minute and elongation at 720C for 45 tracts were used for the study.The clinical history of the animal was unknown.The animals were 5-6 years second.The final extension was done at 720C for 10 of age.The reproductive organ was washed thoroughly minutes.The concentration of RNA in the reaction mixtures were 20 ng.A negative control (no samples was positive for the 230bp cDNA sequence.template) was also included to test for non specific The DNA sequence of the cloned LAP gene and the deduced amino acid is shown in Fig. 1.The gene amplification.
sequence was submitted to Genbank and was given the A single band of RT-PCR product was visualized accession No: EF 630358.The open reading frame after electrophoresis using 1.5% agarose gel with (ORF) of buffalo LAP cDNA sequence showed 100 %, ethidium bromide and the size of the product was 87.2% and 100% similarity with Bos taurus LAP, confirmed using Gene ruler, Fermentas.The RT-PCR tracheal antimicrobial peptide (TAP) and Bubalus product was further cloned into the pTZ57RT-cloning bubalis udder LAP respectively.The degree of vector (Ins T/A clone TM PCR product cloning Kit, similarity and phylogenetic relationship of the Fermantas).The positive clones were selected based reproductive tract LAP in comparison to other ßon blue and white screening and confirmed by plasmid defensin and LAP sequences reported in NCBI is PCR.Four positive clones each from vagina, cervix, shown in Fig. 2 and Fig. 3 respectively.In the present uterus and oviduct were stab cultured in LB agar study we have found the expression of LAP in vaginal, containing ampicillin (100mg/ml) and sent for DNA cervix, uterine and oviduct epithelium based on its sequencing to University of Delhi, South Campus, nucleotide sequence.New Delhi.The gene was sequenced by Sanger's The ORF of the gene was 195bp.The ORF dideoxy method in an automated DNA sequencer.To extends from the 15th adenine base to 208th adenine analyze the cDNA sequence, LAP gene expressed in base.Russell et al.,(1996) reported increased the reproductive tract was aligned with other reported expression of LAP mRNA in mucosal surfaces sequence of ß-defensin from different species using exposed to bacterial lipopolyssacride and tumor clustal method of 'Meg Align' programme of Laser necrosis factor alpha.When the nucleotide sequence gene software (DNA star Inc; USA).The sequences of reproductive tract LAP was compared to the lingual used for comparison were collected from NCBI LAP sequence from Bos taurus, 100 % similarity was website.
noticed in the ORF, while 87.2 % similarity was

Results and Discussion
reported with Goat LAP sequence.This high degree of similarity demonstrate remarkable inter species On RT-PCR of RNA isolated from vagina, conservation of this gene.cervix, uterus and oviduct using LAP primers we were The high level of homology in Bubalus bubalis, able to visualize a single 230 bp product in each lane Bos taurus and Ovis aries also predicts that the by running a 1.5% agarose gel stained with ethidium bromide.The total RNA collected from all the six functions of these peptides are important in disease TGC AGG AGG AAG TAA aag aag gcg aag acg tgg cca g resistance in these animals throughout the million strategies, including resistance and virulence of these years of independent evolution.Crovella ( 2005) microbes might have helped to drive diversity in all reported substantial variation in antimicrobial biological kingdoms (Yount et al., 2006).On peptides among mammalian species.Such variation comparison of buffalo reproductive LAP with other ßwas also noted with buffalo reproductive tract LAP defensin nucleotide sequences reported from the sequence and other ?-defensins.For example, LAP has same species, 95.9 % similarity was seen with enteric 88.7% sequence similarity with goat ß-defensin-2, ß-Defensin (EBD) and 87.5% similarity was seen with while only 52.8% with monkey beta defensin and Bovine Neutrophil ß-Defensin 4 (BNBD4).These 33.8% with human ß-defensin (Fig. 2).phylogenetic analysis data indicated that LAP, EBD and BNBD4 has evolved from a common ancestor ß-The rapid co-evolution of host defense peptide, defensin gene (Fig. 3).When BNBD4 and EBD gene as well as of the corresponding microbial counter

Figure- 1 :
Figure-1: The cDNA sequence of buffalo reproductive tract LAP along with deduced peptide sequence.

Figure- 2 :
Figure-2: Percent divergence of nucleotide sequence of buffalo reproductive tract LAP with LAP and other beta defensin gene from different species.NCBI accession number of the gene is also mentioned for each species.