In Vitro Expression of Recombinaat Buffalo Lactoferrin Gene and its Antibacterial Activity
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Date
2013
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Birsa Agricultural University, Kanke, Ranchi, Jharkhand
Abstract
Lactoferrin (Lf) is an iron binding glycoprotein having many biological
activities, such as facilitating iron absorption and having antimicrobial and
anti-inflammatory effects. Therefore, the present study was undertaken to
characterize the buffalo Lf at the genic level and express N-terminal of buffalo
Lf in E. coli system to see its antibacterial activity. For that, total RNA was
isolated from the mammary gland of lactating buffalo and reverse
transcription-polymerase chain reaction (RT-PCR) was carried out for first
strand cDNA synthesis. Buffalo lactoferrin gene of 2.2 kb was amplified using
gene specific primers and cloned into pTZ57R/T cloning vector using DH5α
strain of E. coli. The positive-negative selection of colony was done based on
differential colour development by LacZ selection. Positive clones of buffalo Lf
gene cDNA were sequenced and were analyzed with the help of laser gene
software and submitted to NCBI GenBank (Acc. No. JF825526). Sequencing
of the selected representative clones revealed that nucleotide sequence of
buffalo Lf contained an ORF of 2127 bp encoding a precursor peptide of 708
amino acids with a molecular weight of 77.65 kDa and isoelectric point of
7.979. Comparative study of buffalo Lf with other reported livestock available
in GenBank showed that buffalo Lf had higher homology both at nucleotide
and amino acid level with cattle. The phylogenetic analysis also indicated that
buffalo Lf had a close relationship with that of cattle.
To see the antibacterial activity of in vitro expressed buffalo Lf, 150 bp of Nterminal
of buffalo Lf was amplified and cloned in pTZ57R/T cloning vector.
Then it was further sub-cloned into the expression vector pQE30 and
expressed in E. coli strain (SG13009). After induction with IPTG, the target
fusion protein was successfully expressed and identified by SDS-PAGE and
Western blotting. The protein purified using His-tag had a molecular weight of
7.0 kDa. Further the expressed buffalo N-Lf protein displayed bactericidal
activity after activation by enzymatic proteolysis using porcine pepsin against
S. aureus and E. coli comparable to commercially available bovine Lf. The
successful expression of functionally active and intact buffalo N-Lf in this
study can be used to study biochemical antimicrobial interaction and has the
potential to be used as an immunomodulator in future, particularly against
infectious diseases like mastitis, thus having significant impact on buffalo
productivity.
Description
In Vitro Expression of Recombinaat Buffalo Lactoferrin Gene and its Antibacterial Activity
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