本研究的目的是為了選殖出台灣特有種的台灣獼猴乳鐵蛋白基因，於畜牧生產方面除了可以幫助我們快速建立稀有動物基因資料庫；此外則可幫助我們將此有用之外源蛋白基因導入基因轉殖家禽系統中生產人類藥用蛋白質，以便提升台灣家禽方面的生物技術產業。
　　目前我們已經成功的自泌乳時期的台灣獼猴之乳腺細胞選殖出其乳鐵蛋白基因，經由重複定序後得知基因全長為2,133 bp，其中前面60鹼基部分為訊息胜肽鏈(signal peptide)，可供細胞辨識而送至外泌性腺體中，而基因所轉譯出的台灣獼猴乳鐵蛋白分子量與人類乳鐵蛋白相似約為80 kDa；將定序完畢的台灣獼猴乳鐵蛋白基因送至基因資料庫中進行比對，在基因與蛋白質的比對結果分別發現台灣獼猴乳鐵蛋白基因與人類乳鐵蛋白基因有高達93%的相同度(identity)，蛋白質則有88%相同度。與其他非靈長類動物的基因比對結果亦達到70 ~ 90 %的相同度；至於在乳鐵蛋白N端抗菌的胜肽鏈比對方面，發現自第41個胺基酸開始起全長達14個胺基酸的抗菌胜肽鏈中台灣獼猴與人類的抗菌胜肽鏈僅有一個胺基酸的差異性，台灣獼猴為離胺酸(Leu)，人類乳鐵蛋白為蛋胺酸(Met)，同屬中性胺基酸，因而根據軟體推測整個抗菌胜肽鏈與菌的結合效果將與人類的乳鐵蛋白相當。我們將乳鐵蛋白基因融合酵母表現載體以進行蛋白大量表現並測試重組蛋白之生化活性，我們已初步發現酵母表現的最佳時間點是在蛋白開始表現的第五天。至於在蛋白活性方面雖無溶菌素之顯著液菌效果，但添加表現乳鐵蛋白之酵母培養液之後，的確可使菌數下降。我們將台灣獼猴乳鐵蛋白基因以電穿孔基因轉染法轉染至家禽的始基生殖細胞(PGC)中，藉由乳鐵螢光蛋白的表現我們得知乳鐵蛋白的確能夠暫時性的表現於家禽細胞中；此外由西方點墨法的結果顯示家禽細胞也能夠辨識台灣獼猴乳鐵蛋白的訊息胜肽蛋白鏈，因而無論是細胞培養液或是細胞內部皆可以觀察到乳鐵螢光蛋白的表現，顯示利用家禽進行乳鐵蛋白基因的轉殖是值得開發的。

　This project mainly aims at cloning the gene encoding Lactoferrin(LF) of rare animal, the Macaca Cyclopis, in Taiwan. It helps us to establish the gene data of wildlife animal in Taiwan, moreover, it also provides the gene source of lactoferrin from primates. By transgenic poultry, it is possible to produce the lactoferrin for human pharmaceutical purpose from eggs.
　A full-length cDNA of 2,133 bp from Macaca Cyclopsis lactoferrin (mcLF) was cloned by RT-PCR, and its molecular weight was about 80Kda. Sequence analysis indicated that there were signal peptide in first 60 bp, and a high homology of LFs across mammalian species was also noticed, especially to the human neutrophile LF. Identities (%) at nucleotide and amino acid between mcLFG and hLF are 93% and 88%, respectively. The results of N-terminal alignment from the 41th to 52 th amino acid between mcLFG and hLF indicated there was only one amino acid ( Leu vs Met) difference. Since both Leu and Met belong to the neutral amino acid family, mcLF and hLF might have the similar anti-bacterial effect. The bio-function of anti-bacterial effect was also examined by subcloning the mcLF into Pachia pastoris expression vector. It was found that the optimal expression time of mcLF in Pachia pastoris started from day 5 after culture. Although the anti-bacterial effect of mcLF tested in this experiment did not show a significant difference compared to lysozyme effect, it did show an inhibitive growth after the induction of Pachia pastoris promoter by methanol.
　When the mcLF was subcloned to EGFPN1 vector and transfected into primordial germ cells of poultry embryos, a transient expression of fusion protein of GFP-LF in the PGC was obtained. Western blot showed that the PGC could recognize the signal peptide of the mcLF and also expressed the EGFP-LF extracellularly. The preliminary results indicate the production of mcLF through transgenic poultry is worth trying.

王介民, 2001. Lactoferricin II立體結構之研究, 國立成功大學理學院生物科技研究所碩士論文

王怡玲, 2001.鴨始基生殖細胞之非病毒轉染法, 國立成功大學理學院生物科技研究所碩士論文

Abramson, J.S., Parce, J.W., Lewis, J.C., Lyles, D.S., Mills, E.L., Nelson, R.D. and Bass, D.A. (1984) Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses. Blood. 64 131-8.

Aisen, P. and Leibman A. (1972) Lactoferrin and transferrin: a comparative study. Biochim Biophys Acta. 257, 314-23.

Allegretti JP, Panje WR. (2001) Electroporation therapy for head and neck cancer including carotid artery involvement. Laryngoscope. 111, 52-6

Allioli N, Verdier G, Legras C. (1997) Use of gonadal primordial germ cells (PGCs) as tools for gene transfer in chickens. Methods Mol Biol. 62, 425-32

Anderson, B.F., Baker, H.M., Norris, G.E., Rice, D.W. and Baker, E.N. (1989) Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution. J Mol Biol. 209, 711-34.

Arnold, R.R., Russell, J.E., Champion, W.J., Brewer, M. and Gauthier, J.J. (1982) Bactericidal activity of human lactoferrin: differentiation from the stasis of iron deprivation. Infect Immun. 35, 792-9.

Arnold, R.R., Russell, J.E., Champion, W.J. and Gauthier, J.J. (1981) Bactericidal activity of human lactoferrin: influence of physical conditions and metabolic state of the target microorganism. Infect Immun. 32, 655-60.

Baker, E.N., Anderson, B.F., Baker, H.M., MacGillivray, R.T., Moore, S.A., Peterson, N.A., Shewry, S.C. and Tweedie, J.W. (1998) Three-dimensional structure of lactoferrin. Implications for function, including comparisons with transferrin. Adv Exp Med Biol. 443, 1-14.

Baker, E.N. and Lindley, P.F. (1992) New perspectives on the structure and function of transferrins. J Inorg Biochem. 47, 147-60.

Bellamy, W., Takase, M., Wakabayashi, H., Kawase, K. and Tomita, M. (1992) Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol. 73, 472-9.

Bellamy, W., Wakabayashi, H., Takase, M., Kawase, K., Shimamura, S. and Tomita, M. (1993) Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin. Med Microbiol Immunol (Berl). 182, 97-105.

Brines, R.D. and Brock, J.H. (1983) The effect of trypsin and chymotrypsin on the in vitro antimicrobial and iron-binding properties of lactoferrin in human milk and bovine colostrum. Unusual resistance of human apolactoferrin to proteolytic digestion. Biochim Biophys Acta. 759, 229-35.

Brock, J.H. (1980) Lactoferrin in human milk: its role in iron absorption and protection against enteric infection in the newborn infant. Arch Dis Child. 55, 417-21.

Brogan, T.D., Ryley, H.C., Neale, L. and Yassa, J. (1975) Soluble proteins of bronchopulmonary secretions from patients with cystic fibrosis, asthma, and bronchitis. Thorax. 30, 72-9.

Bullen, J.J. (1975) Iron-binding proteins in milk and resistance to Escherichia coli infection in infants. Postgrad Med J. 51 67-70.

Campbell, T., Skilton, R.A., Coombes, R.C., Shousha, S., Graham, M.D. and Luqmani, Y.A. (1992) Isolation of a lactoferrin cDNA clone and its expression in human breast cancer. Br J Cancer. 65, 19-26.

Carsience, R. S., M. E., Verrinder Gibbins, A. M., and Etches, R. J. (1993) Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development. 117(2), 669-75

Chang IK, Tajima A, Yasuda Y, Chikamune T, Ohno T. (1992) Simple method for isolation of primordial germ cells from chick embryos. Cell Biol Int Rep. 16, 853-7

Davidson, L.A. and Lonnerdal, B. (1989) Fe-saturation and proteolysis of human lactoferrin: effect on brush- border receptor-mediated uptake of Fe and Mn. Am J Physiol. 257, 930-4.

Davidsson, L., Kastenmayer, P., Yuen, M., Lonnerdal, B. and Hurrell, R.F. (1994) Influence of lactoferrin on iron absorption from human milk in infants. Pediatr Res. 35, 117-24.

Dubois R, Croisille Y. (1970) Germ-cell line and sexual differentiation in birds. Philos Trans R Soc Lond B Biol Sci. 259 (828), 73-89

Elass-Rochard, E., Legrand, D., Salmon, V., Roseanu, A., Trif, M., Tobias, P.S., Mazurier, J. and Spik, G. (1998) Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein. Infect Immun. 66, 486-91.

Eyal-Giladi, H., Ginsburg, M. and Farbarov, A. (1981) Avian primordial germ cells are of epiblastic origin. J Embryol Exp Morphol 65, 139-47.

Eyal-Giladi, H. and Kochav, S. (1976) From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev Biol 49, 321-37.

Eyal-Giladi, H., Kochav, S. and Menashi, M.K. (1976) On the origin of primordial germ cells in the chick embryo. Differentiation 6, 13-6.

Ellison, R.T., 3rd. (1994) The effects of lactoferrin on gram-negative bacteria. Adv Exp Med Biol. 357, 71-90.

Ellison, R.T., 3rd, LaForce, F.M., Giehl, T.J., Boose, D.S. and Dunn, B.E. (1990) Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+. J Gen Microbiol. 136, 1437-46.

Fujimoto T, Ukeshima A, Kiyofuji R. (1976) The origin, migration and morphology of the primordial germ cells in the chick embryo. Anat Rec. 185(2), 139-45

Ginsburg, M. and Eyal-Giladi, H. (1986) Temporal and spatial aspects of the gradual migration of primordial germ cells from the epiblast into the germinal crescent in the avian embryo. J Embryol Exp Morphol 95, 53-71.

Gislason, J., Iyer, S., Douglas, G.C., Hutchens, T.W. and Lonnerdal, B. (1994) Binding of porcine milk lactoferrin to piglet intestinal lactoferrin receptor. Adv Exp Med Biol. 357 239-44.

Goodman, R.E. and Schanbacher, F.L. (1991) Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland. Biochem Biophys Res Commun. 180, 75-84.

Green, M.R. and Pastewka, J.V. (1978) Lactoferrin is a marker for prolactin response in mouse mammary explants. Endocrinology. 103, 151-03.

Gutteberg, T.J., Haneberg, B. and Jorgensen, T. (1984) The latency of serum acute phase proteins in meningococcal septicemia, with special emphasis on lactoferrin. Clin Chim Acta. 136, 173-8.
Harrington, J.P. (1992) Spectroscopic analysis of the unfolding of transition metal-ion complexes of human lactoferrin and transferrin. Int J Biochem. 24, 275-80.

Hutchens, T.W., Henry, J.F., Yip, T.T., Hachey, D.L., Schanler, R.J., Motil, K.J. and Garza, C. (1991) Origin of intact lactoferrin and its DNA-binding fragments found in the urine of human milk-fed preterm infants. Evaluation by stable isotopic enrichment. Pediatr Res. 29, 243-50.

Ikeda, M., Nozaki, A., Sugiyama, K., Tanaka, T., Naganuma, A., Tanaka, K., Sekihara, H., Shimotohno, K., Saito, M. and Kato, N. (2000) Characterization of antiviral activity of lactoferrin against hepatitis C virus infection in human cultured cells. Virus Res. 66, 51-63.

Ikeda, M., Sugiyama, K., Tanaka, T., Tanaka, K., Sekihara, H., Shimotohno, K. and Kato, N. (1998) Lactoferrin markedly inhibits hepatitis C virus infection in cultured human hepatocytes. Biochem Biophys Res Commun. 245, 549-53.

Iyer, S., Yip, T.T., Hutchens, T.W. and Lonnerdal, B. (1994) Lactoferrin-receptor interaction. Effect of surface exposed histidine residues. Adv Exp Med Biol. 357, 245-52.

Janssen, P.T. and van Bijsterveld, O.P. (1983) Origin and biosynthesis of human tear fluid proteins. Invest Ophthalmol Vis Sci. 24, 623-30.

Kuwana T, Maeda-Suga H, Fujimoto T. (1986) Attraction of chick primordial germ cells by gonadal anlage in vitro. Anat Rec. 215, 403-6

LaForce, F.M. and Boose, D.S. (1987) Release of lactoferrin by polymorphonuclear leukocytes after aerosol challenge with Escherichia coli. Infect Immun. 55, 2293-5.

Mazurier, J. and Spik, G. (1980) Comparative study of the iron-binding properties of human transferrins. I. Complete and sequential iron saturation and desaturation of the lactotransferrin. Biochim Biophys Acta. 629, 399-408.

McMillan, J.A., Oski, F.A., Lourie, G., Tomarelli, R.M. and Landaw, S.A. (1977) Iron absorption from human milk, simulated human milk, and proprietary formulas. Pediatrics. 60, 896-900.

Metz-Boutigue, M.H., Jolles, J., Mazurier, J., Schoentgen, F., Legrand, D., Spik, G., Montreuil, J. and Jolles, P. (1984) Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins. Eur J Biochem. 145, 659-76.

Montagne, P., Cuilliere, M.L., Mole, C., Bene, M.C. and Faure, G. (2001) Changes in lactoferrin and lysozyme levels in human milk during the first twelve weeks of lactation. Adv Exp Med Bio.l 501 241-7.

Nakamura, M., Kuwana, T., Miyayama, Y. and Fujimoto, T. (1988) Extragonadal distribution of primordial germ cells in the early chick embryo. Anat Rec 222, 90-4.

Olsson, I., Lantz, M., Persson, A.M. and Arnljots, K. (1988) Biosynthesis and processing of lactoferrin in bone marrow cells, a comparison with processing of myeloperoxidase. Blood. 71, 441-7.

Orr RM. (2000) Technology evaluation: electroporation therapy, Genetronics Inc. Curr Opin Mol Ther. 2, 205-10

Pentecost, B.T. and Teng, C.T. (1987) Lactotransferrin is the major estrogen inducible protein of mouse uterine secretions. J Biol Chem. 262, 10134-9.

Powell, M.J. and Ogden, J.E. (1990) Nucleotide sequence of human lactoferrin cDNA. Nucleic Acids Res. 18, 4013.

Prentice, A., Ewing, G., Roberts, S.B., Lucas, A., MacCarthy, A., Jarjou, L.M. and Whitehead, R.G. (1987) The nutritional role of breast-milk IgA and lactoferrin. Acta Paediatr Scand. 76, 592-8.

Rado, T.A., Wei, X.P. and Benz, E.J., Jr. (1987) Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis. Blood. 70, 989-93.

Rejman, J.J., Hegarty, H.M. and Hurley, W.L. (1989) Purification and characterization of bovine lactoferrin from secretions of the involuting mammary gland: identification of multiple molecular weight forms. Comp Biochem Physiol B. 93, 929-34.

Rey, M.W., Woloshuk, S.L., deBoer, H.A. and Pieper, F.R. (1990) Complete nucleotide sequence of human mammary gland lactoferrin. Nucleic Acids Res. 18, 5288.

Saito, K. and Nakanuma, Y. (1992) Lactoferrin and lysozyme in the intrahepatic bile duct of normal livers and hepatolithiasis. An immunohistochemical study. J Hepatol. 15, 147-53.

Son, K.N., Park, J., Chung, C.K., Chung, D.K., Yu, D.Y., Lee, K.K. and Kim, J. (2002) Human lactoferrin activates transcription of IL-1beta gene in mammalian cells. Biochem Biophys Res Commun. 290 236-41.

Spik, G., Brunet, B., Mazurier-Dehaine, C., Fontaine, G. and Montreuil, J. (1982a) Characterization and properties of the human and bovine lactotransferrins extracted from the faeces of newborn infants. Acta Paediatr Scand. 71, 979-85.

Spik, G., Strecker, G., Fournet, B., Bouquelet, S., Montreuil, J., Dorland, L., van Halbeek, H. and Vliegenthart, J.F. (1982b) Primary structure of the glycans from human lactotransferrin. Eur J Biochem. 121, 413-9.

Stuart, J., Norrell, S. and Harrington, J.P. (1984) Kinetic effect of human lactoferrin on the growth of Escherichia coli 0111. Int J Biochem. 16, 1043-7.

Tabak, L., Mandel, I.D., Herrera, M. and Baurmash, H. (1978) Changes in lactoferrin and other proteins in a case of chronic recurrent parotitis. J Oral Pathol. 7, 91-9.

Tanaka, K., Ikeda, M., Nozaki, A., Kato, N., Tsuda, H., Saito, S. and Sekihara, H. (1999) Lactoferrin inhibits hepatitis C virus viremia in patients with chronic hepatitis C: a pilot study. Jpn J Cancer Res. 90, 367-71.

Teng, C.T. (2002) Lactoferrin gene expression and regulation: an overview. Biochem Cell Biol. 80, 7-16.

Tomita, M., Takase, M., Bellamy, W. and Shimamura, S. (1994) A review: the active peptide of lactoferrin. Acta Paediatr Jpn. 36 585-91.

Urven, L.E., Abbott, U.K. and Erickson, C.A. (1989) Distribution of extracellular matrix in the migratory pathway of avian primordial germ cells. Anat Rec. 224, 14-21.

Van Berkel, P.H., Welling, M.M., Geerts, M., Van Veen, H.A., Ravensbergen, B., Salaheddine, M., Pauwels, E.K., Pieper, F., Nuijens, J.H. and Nibbering, P.H. (2002) Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol. 20 484-7.

Wada, T., Aiba, Y., Shimizu, K., Takagi, A., Miwa, T. and Koga, Y. (1999) The therapeutic effect of bovine lactoferrin in the host infected with Helicobacter pylori. Scand J Gastroenterol. 34, 238-43.

Xie TD, Tsong TY. (1993) Study of mechanisms of electric field-induced DNA transfection. V. Effects of DNA topology on surface binding, cell uptake, expression, and integration into host chromosomes of DNA in the mammalian cell. Biophys J. 65, 1684-9

Yi, M., Kaneko, S., Yu, D.Y. and Murakami, S. (1997) Hepatitis C virus envelope proteins bind lactoferrin. J Virol. 71, 5997-6002.