Expression And Purification Of Human Lactoferrin In Silkworm And Its Bioactivity Study

Human lactoferrin (hLF) is a multifunctional iron-binding glycoprotin. In this study, recombinant human lactoferrin (rhLF) was expressed successfully using Bombyx mori nuclear polyhedrosis baculovirus expression system. We cloned the hLF cDNA by PT-PCR from normal human mammary gland. A BLAST search revealed that the N-terminal sequence of rhLF was determined as GRRRRSV which was similar to that of the native hLF, indicating the correct processing of the signal peptide and identical posttranscriptional processing of the signal sequence.The recombinant plasmid pBacPAK-hLF was co-transfected with linearized Bombyx mori nuclear polyhedrosis virus Bm-BacPAK₆ DNA into BmN cells, then homologous recombination occurred inside the cells. The recombinant virus vBm-hLF harboring hLF gene was purified by three round of plaque isolation and identified by Southern blotting. The recombinant virus was used to infect BmN cells and the larvae of silkworm to express the rhLF. The immunoreactivities of expression products was determined by Western blotting. A band with a molecular weight of ⁷8 kDa was found in both BmN cells and the silkworm larvae, lower than that of the hLF standard(⁸2kDa), which might be due to incomplete glycosylation or protein degradation.Around 13.5 μg rhLF was purified from 10⁵ BmN cells infected with vBm-hLF and the rhLF was proved to be biologically active. The results indicated that rhLF and hLF standard were bacteriostatic to E. coli. and the bacteriostatic activity of rhLF was slightly stronger than that of hLF standard. It was most likely attributable to the distinct glycosylation which were essential to the protein functions. In this study, rhLF is expected to be useful as a natural antibacterial material in general and clinical foods, drugs, and cosmetics. Further studies concerning the mechanism of this activity are now in progress.A high expression level of functional rhLF in silkworm hemolymph (192 mg/liter of silkworm hemolymph) was reported. The production level of the recombinant protein was maximal at around 120 h postinfection and then decreased rapidly.