Transgenic Research

, Volume 24, Issue 2, pp 365–373 | Cite as

Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk

  • Dan Cui
  • Jia Li
  • Linlin Zhang
  • Shen Liu
  • Xiao Wen
  • Qiuyan Li
  • Yaofeng Zhao
  • Xiaoxiang Hu
  • Ran Zhang
  • Ning Li
Brief Communication

Abstract

Intensive swine production industry uses antibiotics to treat diseases and improve pig growth. This can not only cause antibiotic resistance, but can also pollute the environment or eventually affect human public health. To date, human lactoferrin (hLF) and human lysozyme (hLZ) have been known as non-adaptive but interactive antimicrobial members and could act in concert against bacteria, which contribute to host defense. Therefore, their expression in pigs might be an alternative strategy for replacing antibiotics in the pig production industry. In our study, we produced hLF and hLZ bi-transgenic pigs and assessed the milk’s antibacterial ability. Integration of both transgenes was confirmed by PCR and southern blot. Both the hLF and hLZ were expressed in the mammary gland of bi-transgenic pigs, as detected by western blotting. The expression amounts were 6.5 g/L for hLF and 1.1 mg/L for hLZ using ELISA. Interestingly, pig milk containing hLF and hLZ had synergistic antimicrobial activity. Our results suggest an alternative approach for avoiding the use of antibiotics in the pig industry, which would be of great benefit to the commercial swine production.

Keywords

Human lactoferrin Human lysozyme Bi-transgenic Pig 

Notes

Acknowledgments

We thanked Dr. Jin He, Sergio Ardanza, Amanda Wiinamaki, Priya Rao, and Ashok Rao for critical discussions and reading of the manuscript. This work was supported by National Program of Transgenical Breeding Project of China (Project No. 2013ZX08006).

References

  1. Addison JB (1984) Antibiotics in sediments and run-off waters from feedlots. Residue rev 92:1–28CrossRefPubMedGoogle Scholar
  2. Barton MD (2000) Antibiotic use in animal feed and its impact on human healt. Nutr Res Rev 13(2):279–299. doi: 10.1079/095442200108729106 CrossRefPubMedGoogle Scholar
  3. Britigan BE, Serody JS, Cohen MS (1994) The role of lactoferrin as an anti-inflammatory molecule. Adv Exp Med Biol 357:143–156CrossRefPubMedGoogle Scholar
  4. Brock J (1995) Lactoferrin: a multifunctional immunoregulatory protein? Immunol Today 16(9):417–419CrossRefPubMedGoogle Scholar
  5. Brock JH (2002) The physiology of lactoferrin. Biochem Cell Biol 80(1):1–6CrossRefPubMedGoogle Scholar
  6. Brock JH (2012) Lactoferrin–50 years on. Biochem Cell Biol 90(3):245–251. doi: 10.1139/o2012-018 CrossRefPubMedGoogle Scholar
  7. Brundige DR, Maga EA, Klasing KC, Murray JD (2008) Lysozyme transgenic goats’ milk influences gastrointestinal morphology in young pigs. J nutr 138(5):921–926PubMedGoogle Scholar
  8. Callewaert L, Michiels CW (2010) Lysozymes in the animal kingdom. J Biosci 35(1):127–160CrossRefPubMedGoogle Scholar
  9. Carroll MA (1980) The right to treatment and involuntary commitment. J Med Philos 5(4):278–291CrossRefPubMedGoogle Scholar
  10. Coker RJ, Hunter BM, Rudge JW, Liverani M, Hanvoravongchai P (2011) Emerging infectious diseases in southeast Asia: regional challenges to control. Lancet 377(9765):599–609. doi: 10.1016/S0140-6736(10)62004-1 CrossRefPubMedGoogle Scholar
  11. Cooper CA, Brundige DR, Reh WA, Maga EA, Murray JD (2011) Lysozyme transgenic goats’ milk positively impacts intestinal cytokine expression and morphology. Transgenic Res 20(6):1235–1243. doi: 10.1007/s11248-011-9489-7 CrossRefPubMedCentralPubMedGoogle Scholar
  12. Cooper CA, Nelson KM, Maga EA, Murray JD (2013) Consumption of transgenic cows’ milk containing human lactoferrin results in beneficial changes in the gastrointestinal tract and systemic health of young pigs. Transgenic Res 22(3):571–578. doi: 10.1007/s11248-012-9662-7 CrossRefPubMedGoogle Scholar
  13. Ellison RT 3rd, Giehl TJ (1991) Killing of gram-negative bacteria by lactoferrin and lysozyme. J Clin Invest 88(4):1080–1091. doi: 10.1172/JCI115407 CrossRefPubMedCentralPubMedGoogle Scholar
  14. Ellison RT 3rd, Giehl TJ, LaForce FM (1988) Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin. Infect Immun 56(11):2774–2781PubMedCentralPubMedGoogle Scholar
  15. Fairbrother JM, Nadeau E, Gyles CL (2005) Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention strategies. Anim health res rev 6(1):17–39CrossRefPubMedGoogle Scholar
  16. Giraldo P, Montoliu L (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10(2):83–103CrossRefPubMedGoogle Scholar
  17. Groves ML (1960) The isolation of a red protein from milk. J Am Chem Soc 82(13):3345–3350CrossRefGoogle Scholar
  18. Hennart PF, Brasseur DJ, Delogne-Desnoeck JB, Dramaix MM, Robyn CE (1991) Lysozyme, lactoferrin, and secretory immunoglobulin A content in breast milk: influence of duration of lactation, nutrition status, prolactin status, and parity of mother. Am J Clin Nutr 53(1):32–39PubMedGoogle Scholar
  19. Humphrey BD, Huang N, Klasing KC (2002) Rice expressing lactoferrin and lysozyme has antibiotic-like properties when fed to chicks. J nutr 132(6):1214–1218PubMedGoogle Scholar
  20. Johanson B (1960) Isolation of an iron-containing red protein from human milk. Acta Chem Scand 14(2):510–512CrossRefGoogle Scholar
  21. Lee-Huang S, Huang PL, Sun Y, Huang PL, Kung HF, Blithe DL, Chen HC (1999) Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin. Proc Natl Acad Sci USA 96(6):2678–2681CrossRefPubMedCentralPubMedGoogle Scholar
  22. Leitch EC, Willcox MD (1998) Synergic antistaphylococcal properties of lactoferrin and lysozyme. J Med Microbiol 47(9):837–842CrossRefPubMedGoogle Scholar
  23. Leitch EC, Willcox MD (1999) Elucidation of the antistaphylococcal action of lactoferrin and lysozyme. J Med Microbiol 48(9):867–871CrossRefPubMedGoogle Scholar
  24. Levay PF, Viljoen M (1995) Lactoferrin: a general review. Haematologica 80(3):252–267PubMedGoogle Scholar
  25. Liu Z, Zhao C, Fan B, Dai Y, Zhao Z, Wang L, Zheng M, Feng J, Chen Y, Duan Y, Li N (2004) Variable expression of human lactoferrin gene in mice milk driven by its 90 KB upstream flanking sequences. Anim biotechnol 15(1):21–31. doi: 10.1081/ABIO-120029810 CrossRefPubMedGoogle Scholar
  26. Liu S, Li X, Lu D, Shang S, Wang M, Zheng M, Zhang R, Tang B, Li Q, Dai Y, Li N (2012) High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC. Transgenic Res 21(2):407–414. doi: 10.1007/s11248-011-9536-4 CrossRefPubMedGoogle Scholar
  27. Lonnerdal B (2003) Nutritional and physiologic significance of human milk proteins. Am J Clin Nutr 77(6):1537S–1543SPubMedGoogle Scholar
  28. Lonnerdal B, Iyer S (1995) Lactoferrin: molecular structure and biological function. Annu Rev Nutr 15:93–110. doi: 10.1146/annurev.nu.15.070195.000521 CrossRefPubMedGoogle Scholar
  29. Maga EA, Anderson GB, Cullor JS, Smith W, Murray JD (1998) Antimicrobial properties of human lysozyme transgenic mouse milk. J Food Prot 61(1):52–56PubMedGoogle Scholar
  30. Masson PL, Heremans JF (1971) Lactoferrin in milk from different species. Comp Biochem Physiol B Comp Biochem 39(1):119–129CrossRefGoogle Scholar
  31. Mathur NB, Dwarkadas AM, Sharma VK, Saha K, Jain N (1990) Anti-infective factors in preterm human colostrum. Acta Paediatr Scand 79(11):1039–1044CrossRefPubMedGoogle Scholar
  32. Matsuda Y, Saoo K, Hosokawa K, Yamakawa K, Yokohira M, Zeng Y, Takeuchi H, Imaida K (2007) Post-initiation chemopreventive effects of dietary bovine lactoferrin on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in female A/J mice. Cancer Lett 246(1–2):41–46. doi: 10.1016/j.canlet.2006.01.034 CrossRefPubMedGoogle Scholar
  33. McEwen SA, Fedorka-Cray PJ (2002) Antimicrobial use and resistance in animals. Clin infect dis 34(Suppl 3):S93–S106. doi: 10.1086/340246 CrossRefPubMedGoogle Scholar
  34. Mir MA (1977) Lysozyme: a brief review. Postgrad Med J 53(619):257–259CrossRefPubMedCentralPubMedGoogle Scholar
  35. Montagne P, Cuilliere ML, Mole C, Bene MC, Faure G (1998) Microparticle-enhanced nephelometric immunoassay of lysozyme in milk and other human body fluids. Clin Chem 44(8 Pt 1):1610–1615PubMedGoogle Scholar
  36. Montagne P, Cuilliere ML, Mole C, Bene MC, Faure G (2001) Changes in lactoferrin and lysozyme levels in human milk during the first twelve weeks of lactation. Adv Exp Med Biol 501:241–247CrossRefPubMedGoogle Scholar
  37. Morse SI (1965) Biological attributes of staphylococcal cell walls. Ann N Y Acad Sci 128(1):191–213CrossRefPubMedGoogle Scholar
  38. Muller M, Brenig B, Winnacker EL, Brem G (1992) Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection. Gene 121(2):263–270CrossRefPubMedGoogle Scholar
  39. Pluske JR, Pethick DW, Hopwood DE, Hampson DJ (2002) Nutritional influences on some major enteric bacterial diseases of pig. Nutr Res Rev 15(2):333–371. doi: 10.1079/NRR200242 CrossRefPubMedGoogle Scholar
  40. Salton MR (1957) The properties of lysozyme and its action on microorganisms. Bacteriol Rev 21(2):82–100PubMedCentralPubMedGoogle Scholar
  41. Samaranayake YH, Samaranayake LP, Pow EH, Beena VT, Yeung KW (2001) Antifungal effects of lysozyme and lactoferrin against genetically similar, sequential Candida albicans isolates from a human immunodeficiency virus-infected southern Chinese cohort. J Clin Microbiol 39(9):3296–3302CrossRefPubMedCentralPubMedGoogle Scholar
  42. Sanchez L, Calvo M, Brock JH (1992) Biological role of lactoferrin. Arch Dis Child 67(5):657–661CrossRefPubMedCentralPubMedGoogle Scholar
  43. Sexton C, Buss D, Powell B, O’Connor M, Rainer R, Woodruff R, Cruz J, Pettenati M, Rao PN, Case LD (1996) Usefulness and limitations of serum and urine lysozyme levels in the classification of acute myeloid leukemia: an analysis of 208 cases. Leuk Res 20(6):467–472CrossRefPubMedGoogle Scholar
  44. Shau H, Kim A, Golub SH (1992) Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin. J Leukoc Biol 51(4):343–349PubMedGoogle Scholar
  45. Soukka T, Lumikari M, Tenovuo J (1991) Combined inhibitory effect of lactoferrin and lactoperoxidase system on the viability of Streptococcus mutans, serotype c. Scand J Dent Res 99(5):390–396PubMedGoogle Scholar
  46. Spadaro M, Caorsi C, Ceruti P, Varadhachary A, Forni G, Pericle F, Giovarelli M (2008) Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells. FASEB J 22(8):2747–2757. doi: 10.1096/fj.07-098038 CrossRefPubMedGoogle Scholar
  47. Steijns JM, van Hooijdonk AC (2000) Occurrence, structure, biochemical properties and technological characteristics of lactoferrin. Br J Nutr 84(Suppl 1):S11–S17PubMedGoogle Scholar
  48. Suzuki T, Yamauchi K, Kawase K, Tomita M, Kiyosawa I, Okonogi S (1989) Collaborative bacteriostatic activity of bovine lactoferrin with lysozyme against Escherichia coli 0111. Agric Biol Chem 53(6):1705–1706CrossRefGoogle Scholar
  49. Tang L, Wu JJ, Ma Q, Cui T, Andreopoulos FM, Gil J, Valdes J, Davis SC, Li J (2010) Human lactoferrin stimulates skin keratinocyte function and wound re-epithelialization. Br J Dermatol 163(1):38–47. doi: 10.1111/j.1365-2133.2010.09748.x PubMedGoogle Scholar
  50. Tong J, Wei H, Liu X, Hu W, Bi M, Wang Y, Li Q, Li N (2011) Production of recombinant human lysozyme in the milk of transgenic pigs. Transgenic Res 20(2):417–419. doi: 10.1007/s11248-010-9409-2 CrossRefPubMedGoogle Scholar
  51. Ward PP, Uribe-Luna S, Conneely OM (2002) Lactoferrin and host defense. Biochem cell biol 80(1):95–102CrossRefPubMedGoogle Scholar
  52. Wei HX, Zhang K, Ma YF, Li Y, Li QY, Dai YP, Li N (2009) Stage-dependent effect of leptin on development of porcine embryos derived from parthenogenetic activation and transgenic somatic cell nuclear transfer. J Reprod Dev 55(2):99–104CrossRefPubMedGoogle Scholar
  53. Weidle UH, Lenz H, Brem G (1991) Genes encoding a mouse monoclonal antibody are expressed in transgenic mice, rabbits and pigs. Gene 98(2):185–191CrossRefPubMedGoogle Scholar
  54. Yang P, Wang J, Gong G, Sun X, Zhang R, Du Z, Liu Y, Li R, Ding F, Tang B, Dai Y, Li N (2008) Cattle mammary bioreactor generated by a novel procedure of transgenic cloning for large-scale production of functional human lactoferrin. PLoS One 3(10):e3453. doi: 10.1371/journal.pone.0003453 CrossRefPubMedCentralPubMedGoogle Scholar
  55. Zhang Y, Pan D, Sun X, Sun G, Wang X, Liu X, Li Y, Dai Y, Li N (2006) Production of porcine cloned transgenic embryos expressing green fluorescent protein by somatic cell nuclear transfer. Sci China Ser C 49(2):164–171CrossRefGoogle Scholar
  56. Zimecki M, Mazurier J, Spik G, Kapp JA (1995) Human lactoferrin induces phenotypic and functional changes in murine splenic B cells. Immunology 86(1):122–127PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Dan Cui
    • 1
  • Jia Li
    • 1
  • Linlin Zhang
    • 1
  • Shen Liu
    • 2
  • Xiao Wen
    • 2
  • Qiuyan Li
    • 2
  • Yaofeng Zhao
    • 1
  • Xiaoxiang Hu
    • 1
  • Ran Zhang
    • 1
  • Ning Li
    • 1
  1. 1.State Key Laboratory for AgrobiotechnologyChina Agricultural UniversityBeijingPeople’s Republic of China
  2. 2.GenProtein Biotech Ltd.BeijingPeople’s Republic of China

Personalised recommendations