Cloning and sequencing of the cDNA for bovine lactoferrin : this thesis is submitted to Massey University as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
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Date
1992
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Massey University
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Abstract
Bovine lactoferrin isolated from colostrum was partially sequenced by tryptic mapping and automated peptide sequencing. Homogeneous lactoferrin was used to raise polyclonal antibodies in rabbits. Specific anti-lactoferrin antibodies were isolated from the total rabbit gamma-globulin fraction by affinity chromatography on bovine lactoferrin Sepharose. These antibodies were used to quantify lactoferrin in various solutions (by electroimmuno-diffusion assay) and to demonstrate the de novo synthesis of lactoferrin in involuting bovine mammary tissue. RNA was isolated from mammary tissue biopsies that were synthesizing lactoferrin. The presence of lactoferrin messenger RNA was verified by northern blot analysis. Complementary DNA (cDNA) was prepared from RNA samples and ligated into either the bacteriophage vector λ-gt11 or the plasmid vector pGEM-2. Recombinant clones with cDNA inserts coding for bovine lactoferrin were identified by hybridisation to radiolabelled human lactoferrin cDNA. Several clones were isolated and characterised by restriction map analysis and DNA sequencing. The overlapping nucleotide sequence from these clones encoded most of the mature protein sequence for bovine lactoferrin. Nucleotide sequence encoding the 5' end of the lactoferrin messenger RNA was isolated by enzymatic amplification of homopolymeric-tailed first strand cDNA. Specific oligonucleotide primers were used to direct the synthesis of lactoferrin-specific sequences by the polymerase chain reaction (PCR). Double-stranded products were produced by the inclusion of an oligonucleotide that would prime DNA synthesis from the homopolymeric tract on the 3' end of the first strand cDNA. The nucleotide sequence of the PCR products overlapped the 5'-most sequence of the cDNA clones and extended to encode the initiation codon for bovine lactoferrin. The combined nucleotide sequence of the cDNA and PCR clones overlapped to encode the entire coding region for bovine lactoferrin and included 5' and 3' untranslated flanking sequences. The deduced amino acid sequence of the mature protein concurred with the amino acid sequence of the tryptic peptides prepared from bovine colostrum lactoferrin.
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Keywords
Lactoferrin, Molecular cloning, Bovine lactoferrin