Lactococcus lactis: A New Strategy for Vaccination

PDF - Export to EndNote - PubMed Central XML format - PubMed Central XML format - PubMed Central XML format
PMID: 29090064 (PubMed) - PMCID: PMC5650732 - View online: PubReader
Volume 9, Issue 4, October-December , Page 163 to 168
Wednesday, June 29, 2016 :Received , Monday, December 5, 2016 :Accepted

  • - Department of Microbiology, Arak branch, Islamic Azad University, Arak, Iran
  • Corresponding author Razi Vaccine and Serum Research Institute, Arak, Iran, Tel: +98 86 33544702, Fax: +98 86 33544704, E-mail:
    - Razi Vaccine and Serum Research Institute, Arak Branch, Arak, Iran

  • - Department of Microbiology, Islamic Azad University, Arak Branch, Arak, Iran

  • - Department of Microbiology, Islamic Azad University, Arak Branch, Arak, Iran


Needle free vaccines have a several advantages and very attractive way for vaccination. In a body, mucosal surfaces provide a universal entry portal for all the known and emerging infectious pathogenic microbes. Therefore, it seems, vaccination strategies need to be reorganized for vaccines that are hindering the entry capability of pathogenic microbes through mucosal surfaces. Lactic acid Bacteria (LAB) are widely used in the food industry and at the present, used as delivery vehicles for biological investigations. In this review, we summarized the Results of several studies which Lac-tococcus lactis (L. lactis) used as a live vector for vaccines. These bacteria are considered as promising candidates for heterologous expression of proteins and biotechnological usage. LAB are considered as promising candidates for heterologous expression of proteins and biotechnological usage. The results showed that these bacteria have an ability to deliver antigen to immune system. Therefore, developing mucosal live vaccines using lactic acid bacterium, L. lactis, as an antigen delivery vector, is an attractive alternative choice and a safer vaccination strategy against pathogens.



Introduction :

In 1980, Walter Schaffner demonstrated that the bacteria are able to transfer genetic material into mammalian cells in vitro. So, they suggested new vectors for plasmid vaccines transfer 1-3. Later, it was shown that the gram-positive bacteria like Listeria monocytogenes are capable of conveying DNA plasmid 4. Since then, attenuated or artificially engineered  invasive bacteria have been tested as a vehicle for transgene delivery 5.
For centuries, people have recognized that the consumption of fermented products can have a positive effect on human health. Over decades, it has become clear that these probiotic, Lactic Acid Bacteria (LAB) are classified as safe GRAS by the United States Food and Drug Administration (USFDA) 6. Moreover, a number of LAB can induce the immune system response like adjuvants, because of their probiotic properties and their capacity for inducing the host immune system 7. While commensal and pathogenic bacteria as a mucosal delivery vehicles have benefits and drawbacks, lactic acid bacteria are more desirable for their safety and lower side effects 8.
Lactococcus lactis (L. lactis) with a good history of safety in food fermentation and the ability to  survive in passage through the gastrointestinal tract of animals and humans 9 (until now, with a 2 to 3 days survival time) does not invade or colonize the mucosal surfaces of the host. Furthermore, L. lactis does not have lipopolysaccharides and for this rea-son, does not stimulate host immune responses powerfully 10-12.  Because of the progress in many genetic tools and sequenced complete genome, it is easier for researchers to manipulate the gene and produce proteins to the host mucosal surfaces, via the oral, genital or intranasal 12-15. Now, many studies are designed which use  recombinant L. lactis  to stimulate an  immune response against various antigens 9.
In this paper, the ability of L. lactis to transfer antigenic and therapeutic proteins was described. For this purpose, first, the interaction between L. lactis and host gastrointestinal mucosal tract was explained. So, new investigations which use the recombinant L. lactis as a mucosal vaccine were reviewed. Eventually, some early outcomes of such antigen producing bacteria were included in this study in order to pave the way for future developments.
L. lactis and host interaction
Microfold (M) cells have a significant role in inducing mucosal immune response and perpetuity of the mucosal surface barrier. M cells transfer pathogens and foreign molecules from apical lumen side to basal side via using transcytosis. M cells do not have a mucus layer on their apical side 5,16. This character allows M cells to uptake antigens efficiently from the luminal space. The basal side of M cells, which formed from invaginated membranes, has pockets and house Den-dritic Cells (DCs) (Figure 1). These DCs take up transported pathogens and molecules and help to manage the adaptive immune response 17. This close vicinity of DCs to M cells is especially remarkable because of the rapid process of the transcytosed antigens and presentation of antigenic peptides to B and T cells for inducing immune responses. Germinal center contains a net-work of follicular dendritic cells and many B cells, IgA-producing B cells 16. These B cells can migrate into the intestinal lamina propria and secrete IgA (sIgA, Figure 1). The space between neighborhood fol-licles in the Peyer’s Patches (PPs) is called Intrafollicu-lar Region (IFR). The IFR  is full of T cells and DCs and helps to ad-minister the adaptive immune response in the PPs 18. L. lactis enters through Intestinal Epithe-lial Cells (IECs) or M cells, so internalizes and reproduces within phagocytic cells, and causes cellular death mechanism used to spread to a deeper layer. In a usual manner, inflammatory response induced and infiltration of polymorphonuclear cells occurred cause the activation of inflammatory cascades and produce pro-inflammatory cytokines and severe tissue damages. So, the microbes from infected lesions were cleared and the production of antimicrobial neutralizing antibodies occurred. Thus, a dynamic immune network with native and acquired mucosal responses was created 19-21.
L. lactis as a live vehicle for mucosal vaccine delivery
Developing the molecular ways and genetic manipulating to effectively produce antigens and curative molecules in various cells to deliver protein and DNA to host cells was important to present LAB as a live vehicle. A remarkable property of genetically-engine-ered LAB is that mucosal administration elicits both systemic and mucosal immunity 12. In LAB, a hopeful candidate for vaccines development is L. lactis because (1) various genetic ways have been devel-oped for it, (2) its genome is completely sequenced, (3) and its safety property has been revealed. Iwaki et al in 1990 attempted to use L. lactis as a live vaccine 22. Many investigations with recombinant L. lactis  strains have been performed and protection or incomplete protection was observed 23. Lately, LAB as a live vehicle has been inves-tigated in different studies 24-26. In this study, some recent studies for using LAB as a vaccine are included.


Results :

The first investigation for L. lactis based mucosal vaccine was against the Streptococcus mutans surface protein (Pac). When cytoplasm expressed this gene in  L. lactis and supplied orally the killed bacteria, the valuable responses of IgA and IgG were seen 22. In addition, next studies on Clostridium tetani toxin, fragment C (TTFC-Tetanus Toxin Frag-ment C) with L. lactis strain showed the highly immunogenic property 6,27. Studies showed that the nasal route of sur-face which displayed recombinant TTFC was preferred 28. The intracellularly expressed T3SS (type III secretory system protein) vaccines against EspB which were orally used, after ten days, have no particular serum and faucal antibodies. Besides, in BALB/c mice, intra-peritoneal vaccination of the EspB protein increases serum IgG and faucal IgA levels 29. The comparative efficacy was explored when given orally and intramuscularly in piglets 30. The intramuscular inocula-tion with recombinant L. lactis producing FaeG (fimbria adhesion) can stimulate a specific systemic response. In another study, nasal inoculation with recombinant L. lactis expressing a conserved stretch peptide of the avian influenza M2 antigen in birds can increase survival times against high pathogenic avian influenza virus A subtype H5N2 31.
In another challenge on mice, nasal and Broncho-alveolar Lavages (BAL) inoculation with recombinant L. lactis ex-pressing Brucella abortus (B. abortus) Cu-Zn Superoxide Dismutase (SOD), showed SOD-speci-fic IgM and SOD-specific sIgA antibodies which protected the mice against virulent B. abortus strain 9. Oral and intra-nasal vaccination with L. lactis strain expressing Rhodococcus equi (R. equi) VapA (virulence-associated protein A) in mice led to a spe-cific mucosal immune response against VapA in a challenge with a virulent strain of R. equi 32. In another investigation, intragastric route vaccination with recombinant L. lactis  producing VP7 could induce systemic IgG antibody response against rotavirus 33. So, mice orally administered with recombinant L. lactis  producing intra-cellular rotavirus spike-protein subunit VP8, showed the significant levels of intestinal IgA antibodies, while the secreted cytoplasm expressed protein or as a surface-anchored antigen induced anti-VP8 antibodies at both mucosal and systemic levels 34. Oral administration of recombinant L. lactis producing enterotoxin B of Staphylococcus aureus (S. aureus) in mice elicited cellular or systemic immune responses and increased survival rate in vaccinated mice against S. aureus 14. Moreover, vaccination of animal with L. lactis expressed papillomavirus type16 (HPV16) E7 protein, persuasion of humoral and cellular immune responses and protected the animals against HPV‐16 induced tumors 34. In mice, intranasal administration of recombinant L. lactis strain expressing Yersinia pseudotuber-culosis Low-calcium response V (LcrV) antigen was able to elicit specific systemic and mucosal antibody and cellular immune responses against Yersinia infection. This investigation revealed that the type of antigen and administration place of  vaccine are very important which can have an effect on antigen-specific immune responses 35,36. These studies are very valuable for the probability in applying vaccination or therapy with recombinant L. lactis because of their capacity for inducing mucosal and systemic immune responses 37,38.
Few general strains of L. lactis and plasmids
NZ9000 is the usual standard host strain for nisin regulated gene expression (NICE®). Moreover, in this bacteria, nisK and nisR genes were cloned into the pepN gene of MG1363 39. In the strain NZ9100, nisin genes were inserted into a neutral locus. All used strains were obtained from L. lactis subsp. cremoris MG1363.
In pNZ8008, pNZ8148, pNZ8149, and pNZ8150 vectors, replicon was the same and arose from pSH71 plasmid of L. lactis. These plasmids can be multiplied in various gram-positive bacteria, for example, Streptococcus thermophilus and Lactobacillus plantarum (L. plantarum) and they replicate in Escherichia coli (E. coli), but need a recA+strain like MC1061. The pNZ8149 vector contains the lacF gene as a food grade selection marker. In such vectors for transformation process, a host strain, such a L. lactis NZ3900, which has lactose operon and lacks lacF gene, was necessary 40,41. In pNZ9530, the replication genes came from Enterococcus faecalis pAMß1 plasmid which replicate only in gram-positive bacteria, like, L. lactis and L. plantarum 42,43. In table 1, common host strains and plasmids are summarized.
Safety concerns
The potential risk of using lactic acid bacteria based mucosal vaccines is the entry of the genetically manipulated crea-tures to the environment. The manipulated bacteria which produce antigens and antibiotic markers may lead to the horizontal transfer of plasmid to other bacteria. Therefore, the auxotrophic mutants which are unable to multiply in the environment were designed. For this reason, in L. lactis, scientists substituted the thyA gene (thymidylate synthase) with the human IL–10 and made an auxotrophic strain which could not survive in an environment without thymidine 44. So, a recombinant L. lactis was made which contained LLO (Listeriolysin O of Listeria monocytogenes) gene. Therefore, such bacteria not only need a vector with antibiotic markers but also minimize the probability of gene transfer to another bacteria in the environment 45. Also, a novel vaccination method was the external linkage of ARV (avian retro virus) sigma C to LAB cell wall. When this antigen was cloned in E. coli and conjugated on the surface of Enterococcus faecium, it induced mucosal and systemic immunity in mouse 46.


Conclusion :

A big concern about the use of live LAB mucosal vaccines was the risk of transmission of genetically manipulated creatures to nature. So, the use of auxotrophic mutants can prevent the reproduction of such organisms in the environment. Also, food grade plasmids and auxotrophic strains can be used for solving the problem about the horizontal transfer of plasmids which carry antibiotic resistance markers to the environmental and host microflora.
In this paper, some LAB mucosal vaccines were reviewed which had some advantages in comparison to injected vaccines: (a) their ability to induce the systemic and mucosal immune responses in the host cell, (b) their easy manipulation (c) not requiring expert personnel. Moreover, its safety concerns about releasing recombinant plasmids and chromosomally modified bacterial strains in the environment can be controlled. So, lactic acid bacteria are very good mucosal delivery vectors for heterologous antigens and can be used in clinical trials. The studies revealed that recombinant L. lactis can stimulate mucosal immunity response. So, vaccination or therapy strategy with these bacteria is valuable.


Acknowledgement :

The authors thank Dr. Hosseini, the head of Razi vaccine and serum Research institute, Arak Branch, Dr. Jafari, De-partment of Microbiology, Islamic Azad Uni-versity, Arak Branch, Arak, Iran, Dr. Akbary, Department of Microbiolo-gy, Islamic Azad University, Arak Branch, Arak, Iran for their constant support, guidance and inspiration.


<p>Figure 1. Schematic representation of Peyer&rsquo;s patches, M cells, and the different immune cell populations. M cells have no mucus. IFR: intra-follicular region, B: B cells, IEL: intraepithelial lymphocyte, T: T cells, FoDC: follicular dendritic cell, DC: dendritic cells.</p>

Figure 1. Schematic representation of Peyer’s patches, M cells, and the different immune cell populations. M cells have no mucus. IFR: intra-follicular region, B: B cells, IEL: intraepithelial lymphocyte, T: T cells, FoDC: follicular dendritic cell, DC: dendritic cells.

<p>Table 1. <em>L. </em><em>lactis</em> strains and plasmids for expression</p>
<p>Cm<sup>R</sup>: Chloramphenicol resistance.</p>

Table 1. L. lactis strains and plasmids for expression

CmR: Chloramphenicol resistance.

References :
  1. Grillot-Courvalin C, Goussard S, Huetz F, Ojcius DM, Courvalin P. Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 1998;16:862-866.   [PubMed]
  2. Sizemore DR, Branstrom AA, Sadoff JC. Attenuated shi-gella as a DNA delivery vehicle for DNA-mediated im-munization. Science 1995;270(5234):299-302.   [PubMed]
  3. Vassaux G, Nitcheu J, Jezzard S, Lemoine NR. Bacterial gene therapy strategies. J Pathol 2006;208(2):290-298.   [PubMed]
  4. Becker PD, Noerder M, Guzmán CA. Genetic immuniz-ation: bacteria as DNA vaccine delivery vehicles. Hum Vaccin 2008;4(3):189‐202.   [PubMed]
  5. Seow Y, Wood MJ. Biological gene delivery vehicles: beyond viral vectors. Mol Ther 2009;17(5):767-777.   [PubMed]
  6. Pellissery AJ, Nair UR. Lactic acid bacteria as mucosal delivery vaccine. Adv Anim Vet Sci 2013;1(6):183-187.
  7. Seegers JF. Lactobacilli as live vaccine delivery vectors: progress and prospects. Trends Biotechnol 2002;20(12): 508-515.   [PubMed]
  8. del Rio B, Dattwyler RJ, Aroso M, Neves V, Meirelles L, Seegers JF, et al. Oral immunization with recombinant Lactobacillus plantarum induces a protective immune re-sponse in mice with Lyme disease. Clin Vaccine Immu-nol 2008;15(9):1429-1435.   [PubMed]
  9. Sáez D, Fernández P, Rivera A, Andrews E, Oñate A. Oral immunization of mice with recombinant Lactococ-cus lactis expressing Cu,Zn superoxide dismutase of Bru-cella abortus triggers protective immunity. Vaccine 2012; 30(7):1283-1290.   [PubMed]
  10. Siezen RJ, Kok J, Abee T, Schasfsma G. Lactic acid bac-teria: genetics, metabolism and applications. Antonie Van Leeuwenhoek 2002;82(1-4):1.   [PubMed]
  11. Gui-hua W, Xi-lin H, Li-yun Y, Jian-kui L, Chun-hua W. Studies on mucosal Immunity Induced by transmissible gastroenteritis virus nucleocapsid protein recombinant Lactobacillus casei in mice and sow. Agric Sci China 2009;8(2):231-237.
  12. D’Souza R, Pandeya DR, Hong ST. Lactococcus lactis: an efficient gram positive cell factory for the production and secretion of recombinant protein. Biomed Res 2012; 23(1):1-7.
  13. Wyszyńska A, Kobierecka P, Bardowski J, Jagusztyn-Krynicka EK. Lactic acid bacteria--20 years exploring their potential as live vectors for mucosal vaccination. Appl Microbiol Biotechnol 2015;99(7):2967-2977.   [PubMed]
  14. Asensi GF, de Sales NF, Dutra FF, Feijó DF, Bozza MT, Ulrich RG, et al. Oral immunization with lactococcus lactis secreting attenuated recombinant staphylococcal enterotoxin B induces a protective immune response in a murine model. Microb Cell Fact 2013;12:32.   [PubMed]
  15. Bermúdez-Humarán LG, Kharrat P, Chatel JM, langella P. Lactococci and lactobacilli as mucosal delivery vec-tors for therapeutic proteins and DNA vaccines. Microb Cell Fact 2011;10 Suppl 1:S4.   [PubMed]
  16. Mestecky J, Strober W, Russell M, Cheroutre H, Lam-brecht BN, Kelsall B. Mucosal immunology. 4th ed. USA: Academic Press; 2015. 2064 p.
  17. Yamamoto M, Pascual DW, Kiyono H. M cell-targeted mucosal vaccine strategies. Curr Top Microbiol Immunol 2012;354:39-52.   [PubMed]
  18. Shakya AK, Chowdhury MY, Tao W, Gill Hs. Mucosal vaccine delivery: current state and a pediatric perspec-tive. J Control Release 2016;240:394-413.   [PubMed]
  19. Adachi K, Kawana K, Yokoyama T, Fujii T, Tomio A, Miura S, et al. Oral immunization with a Lactobacillus casei vaccine expressing human papillomavirus (HPV) type 16 E7 is an effective strategy to induce mucosal cy-totoxic lymphocytes against HPV16 E7. Vaccine 2010; 28(16):2810-2817.   [PubMed]
  20. Bermúdez-Humarán LG. Lactococcus lactis as a live vector for mucosal delivery of therapeutic proteins. Hum Vaccin 2009;5(4):264-267.   [PubMed]
  21. Lebeer S, Vanderleyden J, Keersmaecker SC. Host inter-actions of probiotic bacterial surface molecules: com-parison with commensals and pathogens. Nat Rev Micro-biol 2010;8(3):171-184.   [PubMed]
  22. Iwaki M, Okahashi N, Takahashi I, Kanamoto T, Sugita-Konishi Y, Aibara K, et al. Oral immunization with re-combinant Streptococcus lactis carrying the Streptococ-cus mutans surface protein antigen gene. Infect Immun 1990;58(9):2929-2934.   [PubMed]
  23. Pontes DS, de Azevedo MS, Chatel JM, Langella P, Aze-vedo V, Miyoshi A. Lactococcus lactis as a live vector: heterologous protein production and DNA delivery sys-tems. Protein Expr Purif 2011;79(2):165-175.   [PubMed]
  24. Guimarães VD, Innocentin S, Lefèvre F, Azevedo V, Wal JM, Langella P, et al. Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells. Appl Environ Microbiol 2006;72(11):7091-7097.   [PubMed]
  25. Chatel JM, Pothelune L, Ah-Leung S, Corthier G, Wal JM, Langella P. In vivo transfer of plasmid from food-grade transiting lactococci to murine epithelial cells. Gene Ther 2008;15(16):1184-1190.   [PubMed]
  26. Tao L, Pavlova SI, Ji X, Jin L, Spear G. A novel plasmid for delivering genes into mammalian cells with noninvas-ive food and commensal lactic acid bacteria. Plasmid 2011;65(1):8-14.   [PubMed]
  27. Wells JM, Wilson PW, Norton PM, Gasson MJ, Le Page RW. Lactococcus lactis: high–level expression of tetanus toxin fragment C and protection against lethal challenge. Mol Microbiol 1993;8(6):1155-1162.   [PubMed]
  28. Nouaille S, Ribeiro LA, Miyoshi A, Pontes D, Le Loir Y, Oliveira SC, et al. Heterologous protein production and delivery systems for lactococcus lactis. Genet Mol Res 2003;2(1):102-111.   [PubMed]
  29. Ahmed B, Loos M, Vanrompay D, Cox E. Mucosal priming of the murine immune system against entero-hemorrhagic Escherichia coli O157:H7 using Lactococ-cus lactis expressing the type III secretion system protein EspB. Vet Immunol Immunopathol 2013;152(1-2):141-145.   [PubMed]
  30. Liu S, Li Y, Xu Z, Wang Y. Subcutaneous or oral Im-munization of mice with lactococcus lactis expressing F4 fimbrial adhesin FaeG. J Vet Med Sci 2013;75(6):779-784.   [PubMed]
  31. Ferbas J, Belouski SS, Horner M, Kaliyaperumal A, Chen L, Boyce M, et al. A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus ery-thematosus. Br J Clin Pharmacol 2013;76(2):188-202.   [PubMed]
  32. Cauchard S, Bermúdez-Humarán LG, Blugeon S, Lau-gier C, Langella P, Cauchard J. Mucosal co-immuniz-ation of mice with recombinant lactococci secreting VapA antigen and leptin elicits a protective immune re-sponse against Rhodococcus equi infection. Vaccine 2011;30(1):95-102.   [PubMed]
  33. Marelli B, Perez AR, Banchio C, de Mendoza D, Magni C. Oral immunization with live Lactococcus lactis ex-pressing rotavirus VP8 subunit induces specific immune response in mice. J Virol Methods 2011;175(1):28-37.   [PubMed]
  34. Bermúdez-Humarán LG, Cortes-Perez NG, Lefèvre F, Guimarães V, Rabot S, Alcocer-Gonzalez JM, et al. A novel mucosal vaccine based on live Lactococci express-ing E7 antigen and IL-12 induces systemic and mucosal immune responses and protects mice against human pa-pillomavirus type 16-induced tumors. J Immunol 2005; 175(11):7297-7302.   [PubMed]
  35. Cortes-Perez NG, Lefèvre F, Corthier G, Adel-Patient K, Langella P, Bermúdez-Humarán LG. Influence of the route of immunization and the nature of the bacterial vector on immunogenicity of mucosal vaccines based on lactic acid bacteria. Vaccine 2007;25(36):6581-6588.   [PubMed]
  36. Daniel C, Sebbane F, Poiret S, Goudercourt D, Dewulf J, Mullet C, et al. Protection against Yersinia pseudotuber-culosis infection conferred by a Lactococcus lactis mu-cosal delivery vector secreting LcrV. Vaccine 2009;27(8):1141-1144.   [PubMed]
  37. Xin KQ, Hoshino Y, Toda Y, Igimi S, Kojima Y, Jounai N, et al. Immunogenicity and protective efficacy of oral-ly administered recombinant Lactococcus lactis express-ing surface-bound HIV Env. Blood 2003;102(1):223-228.   [PubMed]
  38. Robinson K, Chamberlain LM, Lopez MC, Rush CM, Marcotte H, Le Page RW, et al. Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C. Infect Immun 2004;72(5):2753-2761.   [PubMed]
  39. Mierau I, Kleerebezem M. 10 years of the nisin-controll-ed gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 2005;68(6):705-717.   [PubMed]
  40. Vos WMD. Gene cloning and expression in lactic strep-tococci. FEMS Microbiol Rev 1987;46(3):281-295.
  41. de Ruyter PG, Kuipers OP, de Vos WM. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 1996;62(10):3662-3667.   [PubMed]
  42. Simon D, Chopin A. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lacti. Biochimie 1988;70(4):559-566.   [PubMed]
  43. Kleerebezem M, Beerthuyzen MM, Vaughan EE, de Vos WM, Kuipers OP. Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible ex-pression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol 1997;63(11):4581-4584.   [PubMed]
  44. Steidler L, Neirynck S, Huyghebaert N, Snoeck V, Ver-meire A, Goddeeris B, et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 2003; 21(7):785-789.   [PubMed]
  45. Bahey-El-Din M, Casey PG, Griffi BT, Gahan CG. Ef-ficacy of a Lactococcus lactis ΔpyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes. Bioeng Bugs 2010;1(1):66-74.   [PubMed]
  46. Lin KH, Hsu AP, Shien JH, Chang TJ, Liao JW, Chen JR, et al. Avian reovirus sigma C enhances the mucosal and systemic immune responses elicited by antigen-conjugated lactic acid bacteria. Vaccine 2012;30(33): 5019-5029.   [PubMed]
  47. de Ruyter PG, Kuipers OP, Beerthuyzen MM, van Alen-Boerrigter I, de Vos WM. Functional analysis of pro-moters in the nisin gene cluster of Lactococcus lactis. J Bacteriol 1996;178(12):3434-3439.   [PubMed]
  48. Mierau I, Leij P, van Swam I, Blommestein B, Floris E, Mond J, et al. Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: the case of lysostaphin. Microb Cell Fact 2005;4:15.   [PubMed]
  49. Bron PA, Benchimol MG, Lambert J, Palumbo E, Deg-horain M, Delcour J, et al. Use of the alr gene as a food-grade selection marker in lactic acid bacteria. Appl En-viron Microbiol 2002;68(1):5663-5670.   [PubMed]
  50. Vos P, Simons G, Siezen RJ, de Vos WM. Primary struc-ture and organization of the gene for a procaryotic, cell envelope-located serine proteinase. J Biol Chem 1989; 264(23):13579-13585.   [PubMed]
  51. Novotny R, Scheberl A, Giry-Laterriere M, Messner P, Schäffer C. Gene cloning, functional expression and sec-retion of the S-layer protein SgsE from Geobacillus stea-rothermophilus NRS 2004/3a in Lactococcus lactis. FEMS Microbiol Lett 2005;242(1):27-35.   [PubMed]
  52. van Asseldonk M, Rutten G, Oteman M, Siezen RJ, de Vos WM, Simons G. Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 1990;95(1):155-160.   [PubMed]

Home | About us | Contact us | Search | Site Map | Feeds | Rights & Permissions

"Avicenna Journal of Medical Biotechnology - A.J.M.B." is owned, published, and copyrighted by © 2022 Avicenna Research Institute. (pISSN: 2008-2835 .::. eISSN: 2008-4625)

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.

Creative Commons License