• 농업과학연구
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• 제45권4호
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• pp.635-643
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• 2018

Bovine milk is widely consumed by humans and is a primary ingredient of dairy foods. Proteomic approaches have the potential to elucidate complex milk proteins and have been used to study milk of various species. Here, we performed a proteomic analysis using 2-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) to identify whey proteins in bovine milk obtained soon after parturition (bovine early milk). The major casein proteins were removed, and the whey proteins were analyzed with 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The whey proteins (2 mg) were separated by pI and molecular weight across pH ranges of 3.0 - 10.0 and 4.0 - 7.0. The 2-DE gels held about 300 to 700 detectable protein spots. We randomly picked 12 and nine spots that were consistently expressed in the pH 3.0 - 10.0 and pH 4.0 - 7.0 ranges, respectively. Following MALDI-TOF MS analysis, the 21 randomly selected proteins included proteins known to be present in bovine milk, such as albumin, lactoferrin, serum albumin precursor, T cell receptor, polymeric immunoglobulin receptor, pancreatic trypsin inhibitor, aldehyde oxidase and microglobulin. These proteins have major functions in immune responses, metabolism and protein binding. In summary, we herein identified both known and novel whey proteins present in bovine early milk, and our sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed their expression pattern.

• Applied Biological Chemistry
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• 제31권4호
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• pp.361-370
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• 1988

유청 및 대두 단백질의 상호작용을 규명하기 위하여 두 단백질용액 및 1 : 1 혼합용액에 대한 겔투과크로마토그래피와 전기영동에 의하여 열처리 중 조성단백질의 변화 및 상호작용에 관여한 조성단백질을 조사하였다. 크로마토그래피 결과 $80^{\circ}C$ 이상에서 열처리할 경우, 대두단백질 및 혼합물에서 저분자량의 단백질과 고분자량의 응집물이 증가한 것으로 나타났는데, 이는 열처리에 의하여 대두의 11S globulin이 subunit로 해리되고, 이것이 thiol기, disulfide bond 등을 함유한 유청단백질과 가용성 응질물을 형성하기 때문으로 생각되었다. 전기영동에 의하여, 가열시 유청조성단백질 중의 ${\beta}-Lactoglobulin$, ${\alpha}-Lactalbumin$ 및 proteose-peptone 3이 대두단백질과 상호작용을 일으키는 것으로 나타났다. 그리고 용액의 염환경에 따라 Bovine Serum Albumin, Immunoglobulin G(H) 및 Lactoferrin도 상호작용을 일으킬 수 있으며, 대두조성단백질 중의 11S globulin의 basic subunit와 acidic subunit, 7S globulin의 ${\alpha}'$ subunit가 유청단백질과 상호작용을 일으킬 수 있음을 추측할 수 있었다.

• Journal of Dairy Science and Biotechnology
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• 제34권3호
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• pp.193-198
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• 2016

본 연구는 젖소 초산우, 2산, 3산, 4산, 5산, 6산, 7산, 그리고 8산 초유의 분만 직후부터 5일간 전기영동을 통한 단백질 변화를 조사하였다. 분만 직후부터 5일간 전체적인 단백질 함량의 변화를 보면 초일 대부분의 단백질이 많이 함유되어 있다가 시간이 경과할수록 점점 감소하는 것을 보여주었다. 특히 면역과 감염예방에 관련되어 있는 면역 글로불린과 락토페린의 일일변화는 시간이 경과할수록 현저한 감소를 보여주었다. 면역글로불린은 분만 후 초일에 많은 함유량을 나타내고 있으나, 2일 후부터 현저히 감소하였으며, 이후 5일까지 감소하였다. 또한 락토페린도 분만 후, 초일에는 현저히 높은 함량을 나타내었다. 그러나 2일부터 면역글로불린과 같이 유의적으로 감소하는 것을 보여 주었다. 그 외, 혈액 내에서 여러 가지 기능을 하고 있는 것으로 알려져 있으나, 우유 속에서는 그 중요성이 낮은 것으로 보고된 혈청알부민은 분만 후 초일에는 함량이 높은 수준이었으나, 2일부터 함량이 급격히 감소되었다. 그 후, 3일부터 5일까지는 그 함량이 동일하게 유지되는 것을 보여주었다. 한편, 각 산차별 분만 후 일일 젖소 초유의 단백질 함량에 대하여 비교한 결과, 산차에 의한 단백질 함량의 차이는 나타나지 않았다.

• Journal of Microbiology and Biotechnology
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• 제31권8호
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• pp.1175-1182
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• 2021

We investigated the effect of bovine lactoferricin (Lfcin-B), a peptide derived from bovine lactoferrin, on activation of intestinal epithelial cells in IEC-6 intestinal cell, and protection against in vivo rotavirus (RV) infection. Treatment with Lfcin-B significantly enhanced the growth of IEC-6 cells and increased their capacity for attachment and spreading in culture plates. Also, Lfcin-B synergistically augmented the binding of IEC-6 cells to laminin, a component of the extracellular matrix (ECM). In the analysis of the intracellular mechanism related to Lfcin-B-induced activation of IEC-6 cells, this peptide upregulated tyrosine-dependent phosphorylation of focal adhesion kinase (FAK) and paxillin, which are intracellular proteins associated with cell adhesion, spreading, and signal transduction during cell activation. An experiment using synthetic peptides with various sequences of amino acids revealed that a sequence of 9 amino acids (FKCRRWQWR) corresponding to 17-25 of the N-terminus of Lfcin-B is responsible for the epithelial cell activation. In an in vivo experiment, treatment with Lfcin-B one day before RV infection effectively prevented RV-induced diarrhea and significantly reduced RV titers in the bowels of infected mice. These results suggest that Lfcin-B plays meaningful roles in the maintenance and repair of intestinal mucosal tissues, as well as in protecting against intestinal infection by RV. Collectively, Lfcin-B is a promising candidate with potential applications in drugs or functional foods beneficial for intestinal health and mucosal immunity.

• 농업과학연구
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• 제28권2호
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• pp.92-98
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• 2001

락토페리신은 다양한 생리활성을 나타내는 락토페린(약 80kD)에서 유래된 항균성펩타이드 분획물(5kD)이다. 마우스HC11유선상피세포에서 인체 락토페리신의 발현은 bovine beta-casein을 promotor로 하고 인체 락토페리신 cDNA를 삽입하여 제작한 pBL1-cin발현벡타를 이용하였다. 이 발현벡타를 이용하여 인체 락토페리신 발현여부를 RT-PCR, northern blot, dot blot분석을 통하여 확인하였다. pBL1-cin 발현백타를 HC11세포에 transfection 하여 얻은 RNA를 이용하여 RT-PCR를 한 결과 150bp의 크기로 확인되었고 Northern blot 분석결과는 약 2.3 kb의 크기로 확인되었다. 인체 락토페린 polyclonal항체를 이용하여 dot blot한 결과 인체 락토페리신이 분비됨을 확인하였다.

• 한국축산식품학회지
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• 제27권4호
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• pp.522-530
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• 2007

본 실험은 홀스타인 젖소의 초유에 함유된 lactoferrin, IgA, $IgG_1,\;IgG_2$의 함량과 병원성미생물의 항균효과, 유산균증식효과를 알아보기 위하여 수행하였다. 홀스타인종 젖소 초유의 lactoferrin, IgA, $IgG_1,\;IgG_2$의 함량은 여름철의 경우 분만 후 첫째 날에 각각 0.30 mg/mL, 0.37 mg/mL, 4.00 mg/mL, 0.37 mg/mL, 겨울철의 경우 분만 후 첫째 날에 각각 1.16 mg/mL, 2.60 mg/mL, 13.35 mg/mL, 1.30 mg/mL로 측정되었다. 무열처리한 초유와 $65^{\circ}C$에서 30분간 열처리한 초유에서 배양한 병원성 미생물의 변화는 E. coli 균주에 대하여 초유의 항균활성이 나타났다. 초유 첫째 날, 둘째 날, 세째 날의 유산균수 성장촉진 효과는 B. longum, L. acidophilus S. themophilus의 상업용 혼합균주와 L. acidophilus, L. casei, L. bulgaricus, L. lactis subsp. cremoris에서 모두 정상유보다 균수가 증가하였다. 초유 첫째 날, 둘째 날, 세째 날의 pH는 배양 15시간 후 B. longum, L. acidophilus S. themophilus의 상업용 혼합균주와 L. acidophilus, L. casei, L. bulgaricus, L. lactis subsp. cremoris에서 각각 4.97-5.22 및 5.96-6.47로 나타났으나, 정상유에서는 각각 4.89, 6.50-6.81이었다. 초유 첫째 날, 둘째 날, 세째 날의 산도는 B. longum, L. acidophilus S. themophilus의 상업용 혼합균주와 L. acidophilus, L. casei, L. bulgaricus, L. lactis subsp. cremoris에서 각각 0.75-0.88% 및 0.29-0.48%로 나타났으나, 정상유에서는 각각 0.70%, 0.20-0.25% 이었다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.85-85
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• 2003

Human lactoferrin (hLF) is an 80 kDa iron-binding glycoprotein that is expressed in high concentration in milk and in lesser amount in the secondary or specific granules of neutrophils and in plasma, LF is classically considered to be related to the binding, transport, and storage of iron. The transgenic mice carrying the human hLF gene in conjunction with the bovine $\beta$-casein promoter produced the human hLF in their milk during lactation. To screen transgenic mice, PCR was carried out using chromosomal DNA extracted from tail or toe tissues. In this study, stability of germ line transmission and expression of hLF were monitored up to generation Fl7 of a transgenic line. When female mouse of generation F9 was crossbred with normal male, generation F9 to Fl7 mice showed similar transmission rates ($66.0 \pm 12.57%, 42.0 \pm 14.98%, 72.2 \pm 25.45%, 50.0 \pm 16.70%, 65.7 \pm 6.45%, 48.6 \pm 14.65%, 54 1 \pm 18 11%, 57.8 \pm 16.16% and 48.6 \pm 20.66$, respectively), implying that the hLF gene can be transmitted stably up to long term generation in the transgenic mice For ELISA analysis, hLF expression levels were determined with an hLF ELISA kit in accordance with the supplier's protocol. Expression levels of human hLF from milk of generation F9 to Fl3 mice were $ 3.2 \pm 0.69 mg/ml, 3.1 \pm 0.81 mg/ml, 4.6 \pm 1.38 mg/ml, 3.1 \pm 0.42 mg/ml, and 4.5 \pm 1,48 mg/ml$, respectively. These expression levels were lower than that of founder (6.6 mg/$m\ell$) mouse. We concluded that transgenic mice faithfully passed the transgene on their progeny and successively secreted target proteins into their milk through several generations.

• Reproductive and Developmental Biology
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• 제32권4호
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• pp.255-261
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• 2008

The early diagnosis of bovine pregnancy is an essential component of successful reproductive planning on farms, because lack of bovine pregnancy over the long term results in reproductive failure and low milk yield-the latter of which is a special concern on dairy farms. This study was designed to identify early pregnancy-specific whey proteins in bovine, by comparing milk samples collected from cattle during pregnancy (Days 30 and 50) and from non-pregnant cattle. In this study, differentially expressed proteins in five pregnant and five non-pregnant Holstein dairy cattle were investigated and compared, using proteomics analysis. The first dimension was applied to a pH $3.0{\sim}10.0$ strip, by loading a 2-mg milk protein sample. After the second-dimension separation was performed, the gels were stained with colloidal Coomassie brilliant blue. The stained gels were scanned and the images were analyzed, to detect variations in protein spots between non-pregnant and pregnant cattle milk protein spots, using ImageMaster, this was followed by analysis with MALDI TOF-MS. Analysis of the 2-DE gel image resulted in a total of approximately $500{\sim}600$ protein spots, of which 12 spots were differentially expressed, six spots were up-regulated, and four spots were down-regulated; two spots were identified as pregnancy-specific proteins. These proteins were identified as lactoferrin, NA-DH dehydrogenase subunit 2, albumin, serum albumin precursor and transferrin. Our results via 2-D PAGE analysis revealed composite profiles of several milk proteins related to early bovine pregnancy, implying the possible use of these milk proteins in the early detection of bovine pregnancy.

• Journal of Dairy Science and Biotechnology
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• 제23권2호
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• pp.155-160
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• 2005

전세계 많은 나라들은 조제분유를 특수 영양식품으로 분류하여 관리에 각별히 신경을 쓰고 있으며, CODEX에서는 첨가되는 거의 모든 성분을 모유에 들어 있는 것이어야 하며, 또한 과학적으로 안전성이 입증된 것만을 넣을 수 있도록 규정하고 있다. 이 때문에 분유 메이커들은 제품 개발을 위해 많은 모유 및 우유 성분에 관한 연구를 바탕으로 하여 모유의 조성에 맞추는 노력을 하고 있다. 국내산 분유는 외국 제품에 비해 면역기능 향상, 항-알레르기 능력 향상, 뇌와 소화기관의 발달 등을 돕는 성분이 강화되어 있다. 현재 생명과학기술의 진보와 각종 신기술의 도입으로 모유 및 우유속의 극미량 성분까지 분리정제가 가능함에 따라 다양한 기능성을 갖는 성분들이 상품화되고 있으며, 이를 이용해 조제분유를 개발하고 있다. 두뇌 발달 성분인 DHA, 아라키돈산 등과 면역기능 강화 성분인 뉴클레오타이드, 락토페린 등의 첨가 및 정장기능을 보이는 올리고당 등의 성분강화에서 이제는 뼈 성장 발달인자, 아토피 및 알레르기의 감소를 위해 가수분해물의 이용 등의 기능을 보강한 다양한 제품이 개발되어지고 있는 중이다.

• Animal Bioscience
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• 제35권1호
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• pp.126-137
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• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.