Cloning And Sequence Analysis Of Human Lactoferrin (hLF) CDNA

Transferrins form a family of proteins that are involved in regulating the levels of free iron in body fluids of animals. One principal member of this family is lactoferrin (LF). LF is a secretive protein and distributes widely in breast milk of mammal, tears, saliva, sweat, bile, spermatic fluid, windpipe and rhinal cavities secretions, pancreatic juice, blood plasma, amniotic fluid, intestinal fluid, synovial fluid, neutrophil of animals and so on.In order to study the differences of nucleotide and amino acid sequence among hLFs from different individuals or tissues, and the heterogenous expression of hLF gene, white blood cells were isolated from normal human peripheral blood and total RNA was extracted. The expected 1.5 and 0.8kb cDNA fragments of lactoferrin (hLF) were amplified by RT-PCR after the first cDNA strand was synthesized with oligo (dT) as primer. Then, the two fragments were sub-cloned into pMD18-T vector and were obtained. The two fragments were recut out, and ligated into pPICZa-A vector which was used to express and secrete recombinant proteins in Pichia pastoris. The recombinant expression plasmid pPICZa-hLF contains full length hLF cDNA. The result from sequence of hLF cDNA shows that hLF cDNA consists of 2,136bp, encoding 711 amino acid residues. Comparison with hLFs fromother different tissues reveals 99% homology in nucleotide sequence and 96-97% homology in amino acid sequence. It was found that there are 21 differrent nucleotides, 14 different nucleotides nucleotides among which cause amino acid mutation.Bio-softwares eg. Vector NTI, DNAMAN and DNA Tools were used to analyze bioinformatics of hLF. The results display: (1) the secondary structure of hLF is mainly composed of α-helix and β-pleated sheet, which are alternately arranged along whole protein, and the number of α-helix are more than that of β-pleated sheet; (2) hLF proteins are folded into two globular lobes with highly similar structure, termed the N- and C-lobes, to form tertiary structure. Each lobe is further divided into two domains, the N1 and N2 domains in the N-lobe and C1 and C2 domains in the C-lobe. Iron and carbonate binding sites are located within a deep cleft formed between the domains of each lobe and every LF molecule can bind two Fe3+and two CO32- molecules; (3) Homology analysis of LF amino acid sequences among human and 9 different mammals shows great homologous and their homologous levels descend as house mouse, goat, cow, pig, rabbit and dog; (4) Molecular weight of hLF protein is 78,414Da, and isoelectric point is 8.05; (5) hLF protein has 39.1% neutral hydrophobic and 35.7% neutral hydrophilic amino acids, 14.1% charged basic and 11.1% charged acidic amino acids, respectively; (6) N-and C-terminal of hLF protein show strong hydrophobicity and hydrophobic regions lying in interior of molecule.