Cloning and characterization of goat enteric _Ò-defensin cDNA.
2010
Kumar, Ashish | Sharma, Ambika | Kumar, Ashok | Dev, Kranti
The mRNA from distal ileum of Indian goat was cloned and characterized after purification. cDNA was synthesized using goat ileal epithelial RNA, omniscript and sensiscript reverse transcriptase and amplified by Hotstart Taq DNA polymerase with primers designed by taking conserved regions of cattle enteric _Ò-defensin, cattle lingual antimicrobial peptide(LAP) and goat _Ò-defensin-2 sequences. The amplified cDNA of 253bp was, ligated to linearised TA cloning vector and transformed into XLblue strain of E.coli which was grown overnight at 37oC in a LB plate containing ampicillin, IPTG and X-Gal. The recombinant plasmid was isolated and digested with NcoI. The white colonies showed a release of 253bp insert. The sequence analysis showed 26, 16 and 5-nucleotide substitution having 85.6%, 91.3%, 97.4% homology with reported cattle EBD, buffalo EBD and goat BD2 mRNA respectively. The deduced amino acid sequence encodes for a 64 amino acid precursor peptide showing 12,18 and 4 amino acid substitution having 80%, 70.8%, 93.8% homology with buffalo EBD, cattle EBD and goat BD2 peptide respectively. Both nucleotide and amino acid sequence homology showed that the cloned sequence was closer to goat BD2.
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