• BMB Reports
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• v.28 no.1
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• pp.57-61
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• 1995

The expression of a recombinant human lactoferrin is reported in mouse HC11 mammary epithelial cells. Expression of human lactoferrin (hLF) was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To improve the hLF expression level in a cell culture system, two artificial introns were also introduced to construct expression vectors. One intron was a hybrid-splice signal consisting of bovine ${\beta}$-casein intron 1 and rabbit ${\beta}$-globin intron II. The other intron was a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. The hybrid intron moderately elevated hLF expression, whereas intron 8 alone did not express any detectable amount of hLF as judged by Northem and Western blot analyses. When the two introns were used together they contributed to a synergistic elevation of hLF expression. These data indicate that artificial introns on both sides of the hLF cDNA were necessary to increase expression of cDNA.

• Food Science of Animal Resources
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• v.21 no.4
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• pp.374-382
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• 2001

Lactoferrin(Lf) has the function of modulation in the host defense systems, including cytokine production and immune responses. We have tested the effect of Lf and Lf ydrolysates(Lf-H) on the productions of tumor necrosis factor-${\alpha}$(TNF-${\alpha}$) and nitric oxide(NO) in macrophage cells. Lf from Korean native cattle(K-Lf) and hydrolyzed K-Lf(K-Lf-H) increased the production of TNF-${\alpha}$ in RAW264.7 cells with dose-dependency. Bovine Lf(B-Lf), human Lf(H-Lf), and its hydrolysates did not induce either TNF-${\alpha}$ production or NO production. On the other hand those didn\`t affect on the production of TNF-${\alpha}$ in lipopolysaccharide(LPS)-stimulated RAW264.7 cells. K-Lf induced the production of NO similar to its role on the TNF-${\alpha}$ production.

• Journal of Plant Biotechnology
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• v.34 no.2
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• pp.145-152
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• 2007

Lactoferrin is an 80-kDa iron-binding glycoprotein known to exert many biological activities, such as facilitating iron absorption and having antimicrobial and anti-inflammatory effects. Rice can be a useful target for edible food plants to introduce human lactoferrin, because it has lower allergenicity and is likely to be safer than microorganisms or transgenic animals. A cDNA fragment encoding human lactoferrin (HLF) driven by the maize polyubiquitin promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was introduced into rice (Oryza sativa L. cv. Dong Jin) using the Agrobacterium -mediated transformation system. Putative transformants were initially selected on the medium containing bialaphos. The stable integration of the bar and HLF genes into transgenic rice plants was further confirmed through polymerase chain reaction (PCR) and Southern blot analyses. The expression of the full length HLF protein from various tissues such as grains and young leaves of transgenic rice was verified by Western blot analysis. Analysis of progeny also demonstrated that introduced genes were stably inherited to the next generation at the Mendelian fashion.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.153-159
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• 2008

The limitations in current technology for generating transgenic animals, such as the time and the expense, hampered its extensive use in recombinant protein production for therapeutic purpose. In this report, we present a simple and less expensive alternative by directly infusing a recombinant adenovirus vector carrying human lactoferrin cDNA into rabbit mammary glands. The milk serum was collected from the infected mammary gland 48 h post-infection and subjected to a 10% SDS-PAGE and Western blotting. An 80-kDa protein was visualized after viral vector infection. With this method, we obtained a high level of expressed human lactoferrin of up to 2.3mg/ml in the milk. Taken together, the method is useful for the transient high-level expression recombinant proteins, and the approach established here is probably one of the most economical and efficient ways for large-scale production of recombinant proteins of biopharmaceutical interest.

• Korean Journal of Microbiology
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• v.38 no.1
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• pp.38-44
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• 2002

Pneumococcal surfacce protein A (PspA) is an important virulence factor and an antigenically variable surface protein of the pneumococci. To purify the PspA from S. pneumoniae KNIH1156 , a clinical isolate (type 19F), we have taken advantage of the fact that PspA is released from the surface of pneumococci into the medium by growing in a CDM-ET medium and PspA is capable of binding human lactoferrin, the iron carrier protein. PspA of S. pneumoniae KNIH1156 was purified from culture supernatant by human lactoferrin (hLf) affinity chromatography. The purified PspA was confirmed with anti-PspA antiserum and also had the binding capacity to hLf specifically. To determine whether the purified PspA could elicit protection in mice against pneumococcal inflection, we immunized the mice with purified PspA and subsequently challenged with S. pneumoniae KNIH1156. Immunization with purified PspA protected mice from 500 times the $LD^{50}$ of S. pneumoniae KNIH1156. Therefore, it has been shown that purified PspA fromS. pneumoniae KNIH1156 (type 19F) is a protective immunogen.

• Food Science of Animal Resources
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• v.25 no.1
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• pp.98-102
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• 2005

The purpose of this study was to demonstrate biochemical properties of lactoferrin (Lf) obtained from the colostrum of Korea native cow. The molecular weight of the purified Korean native cow's Lf (K-Lf) was 81kDa, the isoelectric point was 9, and the content of iron was 0.56 mg/g, which is indicated that iron saturation of the lactoferrin was 40.6%. Amino acid composition and a-helix content were different K-Lf from bovine Lf (B-Lf). Immunological cross reactivity was observed between K-Lf and B-Lf but not between K-Lf and human Lf (H-Lf) by immunodiffusion test and Western blot analysis. Out results indicate that structure of K-Lf is different from that of B-Lf although K-Lf and B-Lf were immunologically cross-reactive.

• Journal of Dairy Science and Biotechnology
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• v.14 no.2
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• pp.229-245
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• 1996

1. CM-Sephadex C-50-120 column을 사용하여 HLF를 효과적으로 정제하였다. CM-Sepha-dex 50-120 column chromatography를 수행한 결과, HLF는 NaCl 350 mM-500 mM 사이에서 용출되었으며, 용출된 분획을 SDS-PAGE를 수행해서 단일 band를 확인하였다. Anti-HLF antibody를 이용한 Western blotting 결과 nitrocellulose paper 상에 단일 band 가 나타나므로 HLF 가 효과적으로 정제되었다는 것을 알 수 있었다. 2. Con A, PWM, PWA LPS 등의 자극원으로 단핵구와 대식세포를 자극한 다음 BLF를처리한 배 양액을 IL-1 bioassy한 결과는 Con A 33%, PMA 33%, PWM 15%, LPS 35% 이고, HLF로 처리하여 IL-1 bioassay를 한 결과는 Con A 15%, PMA 22%, PWM 10%, LPS 5%로 감소된 것으로 나타났다. 3. K-562 세포를 이용한 colony forming assay에서 BLF가 10 g/ml일 때는 30%, 30 g/ml일 때는 35%, HLF는 10 g/ml일 때는 5%,30 g/ml일 때는 30%의 저해를 나타냈다. 4. Lactoferrin의 면역증강효과를 알아보기 위하여 hapten인 VCR-BSA를 투여 한 후, 생성되는 항체생성능력을 ELISA로 비교하였다. 그 결과 HLF 및 BLF 투여군에서 대조군에 비하여 adjuvant 효과를 관찰할 수 있었다. 또한 이때 macrophage 수는 대조군에 비하여 HLF와 BLF를 투여한 군이 증가됨을 알 수 있었다. 이러한 결과로 미루어 볼 때 LF는 macrophage를 활성화 시켜서 항체 생성 능력을 증가시키는 효과가 있음을 관찰할 수 있었다. 5. Balb/c mouse의 thymus로부터 분리한 CD4- CD8- 세포를 BLF로 처리하여 24 시간 배양한 후 CB4$^-$ CD8$^-$ 세포의 분화를 측정한 결과, CD4$^-$를 CD4$^+$ 로 분화하였다. 그리고 HLF로 처리하여 24 시간 배양 후 CD4$^-$ CD8$^-$ 세포의 분화를 측정한 결과, CD4$^-$ CD8$^-$를 CD4$^+$ CD8$^+$ 로 분화하였다. 6. Lactoferrin이 T cell의 IL-2 production에 미치는 영향은 PMA 처리군, PMA+OKT3처리군, LF 단독 처리군 보다 PMA+OKT3+LF를 처리한 군이 IL-2 생성에 영향을 미쳤다. 7. Lfcin B의 단일크론항체에 의해 인식되어지는 Lfcin B의 항원결정기는 ‘QWR’로 밝혀졌다.

• Journal of Veterinary Clinics
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• v.24 no.2
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• pp.197-200
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• 2007

The purpose of this study was premature induction of lactation in the transgenic calf to confirm the expression of human lactoferrin gene in the milk as early as possible. We performed the induction of lactation in 6 normal 1-month old calves and 3 normal and 1 transgenic 5-month old calves. In order to induction of lactation, 1-month old calf was injected with estradiol benzoate (0.2 mg/kg), $17-{\beta}$ estradiol cypionate (0.1 mg/kg) and progesterone (0.25-0.5 mg/kg) in every other day by 21 days. After 10 days cessation of administration, dexamethasone (0.028 mg/kg) was injected intramuscularly, however, lactation was failed. In the 5-month old calves, estradiol benzoate (0.2 mg/kg) and progesterone (0.5 mg/kg) were injected individually every other day during 21 days. After 10 days cessation of administration, dexamethasone (0.1 mg/kg) was injected intramuscularly and milk was lactated in the next day. Lactation was successfully induced in the 5-month old normal and transgenic calves and we confirmed the lactoferrin gene in the milk of transgenic calf. In conclusion, confirmation of human lactoferrin gene expressed in the milk of the transgenic calf was possible in 20 months earlier than in normal condition.

• Proceedings of the Korean Society of Crop Science Conference
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• 2002.05a
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• pp.48-50
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• 2002

A strategy for development of a functional rice in proved with human lactoferrin and enhancement of nutrient compounds was planned. For the purposes we have cloned and characterized a human lactoferrin cDNA from human mammary gland cDNA library A endosperm storage vacuole targeting sequence and the cDNA fragment was linked to endosperm specific glutelin promoter. The fusion gene fragment was inserted into a binary vector containing MAR gene. In addition a new ${\beta}$-galactosidase gene from Bifidobacterium of human was used as a reporter gene in the vector system, Rice plants showing a high concentration of amino acids in the endosperm cells were developed by using a biochemical mutation and bred for the transformation with the binary vector system Finally we have established a transformation method for the rice endosperm cells.

• Food Science of Animal Resources
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• v.36 no.4
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• pp.487-493
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• 2016

The antimicrobial activity of bovine lactoferrin hydrolysates (bLFH) was measured against Pseudomonas strains (P. syringae and P. fluorescens) in vitro. To compare susceptibility to bLFH, minimal inhibitory concentration (MIC) values were determined using chemiluminescence assays and paper disc plate assays. Antimicrobial effect against P. fluorescens was not observed by either assay, suggesting that bLFH did not exhibit antimicrobial activity against P. fluorescens. However, a significant inhibition of P. syringae growth was observed in the presence of bLFH. The addition of bLFH in liquid or solid medium inhibited growth of P. syringae in a dose-dependent manner. Furthermore, a bLFH peptide with antimicrobial activity toward P. syringae was isolated and identified. The N-terminal amino acid sequences of thus obtained antimicrobial bLFH peptides were analyzed by a protein sequencer and were found to be Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala and Phe-Lys-Cys-Arg-Arg-Trp-Gln-Trp-Arg-Met. The latter peptide sequence is known to be characteristic of lactoferricin. Therefore, in the present study, we identified a new antimicrobial peptide against P. syringae, present within the N-terminus and possessing the amino acid sequence of Leu-Arg-Ile-Pro-Ser-Lys-Val-Asp-Ser-Ala.