Construction and identification of exogenous gene expression system in Lactococcus lactis

Abstract

Abstract: Objective To construct and characterize an exogenous gene expression system in Lactococcus lactis. Methods PCR-based gene assembly was used to synthesize the gene sequence containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning site. Plasmid pBS-pu was obtained after ligation of the assembled sequence with pBluescriptⅡ SK (+), and the terminator of USP45 protein was added to construct the recombinant plasmid pNBC1000. The gene coding for Streptococcus pyogenes M6 protein was amplified and cloned into pNBC1000 to obtain the plasmid pNBC2000. To characterize the expression system, enhanced green fluorescent protein (EGFP) gene was amplified from the plasmid pEGFP-N1 and cloned into pNBC2000. Laser scanning confocal microscope was used to observe the bacteria containing pNBC2000, pNBC2000-EGFP or pBS-EGFP. Results Green fluorescence wad wisualized in the bacteria containing pNBC2000-EGFP, but not in the bacteria containing pNBC2000 or pBS-EGFP. Conclusion The plasmids pNBC1000 and pNBC2000 containing P59 promoter, USP45 signal peptide, ribosome binding site and multiple cloning sites are successfully constructed, which are capable of expressing exogenous gene in Lactococcus lactis.

CLC Number:

Q939.11

YANG Xiao-qiang¹, PENG Chun-xiu², JIANG Bo¹, XING Rui¹, ZHANG Shao-rong¹, CHEN Xue-qing¹. Construction and identification of exogenous gene expression system in Lactococcus lactis[J]. Journal of Southern Medical University, 2005, 25(10): 1232-1235,1239.

References

[1] Chatel JM, Nouaille S, Adel-Patient K, et al. Characterization of a Lactococcus lactis strain that secretes a major epitope of bovine beta-lactoglobulin and evaluation of its immunogenicity in mice [J].Appl Environ Microbiol, 2003, 69( 11): 6620-7.
[2] Bolotin A, Wincker P, Mauger S, et al. The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403[J]. Genome Res, 2001, 11(5): 731-53.
[3] Steidler L, Hans W, Schotte L, et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10[J]. Science, 2000, 289:1352-5.
[4] Steidler L, Neirynck S, Huyghebaert N, et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10[J]. Nat Biotechnol, 2003, 21 (7):785-9.
[5] Vandenbroucke K, Hans W, van Huysse J, et al. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevants and heals actue colitis in mice[J]. Gastroenterology, 2004, 127(2):502-13.
[6] van der Vossen JM, van der Lelie D, Venema G, et al. Isolation and characterization of Streptococcus cremoris Wg2-specific promoters [J]. Gene, 1987, 53(10): 2452-7.
[7] van Asseldonk M, Rutten G, Oteman M, et al. Cloning ofusp45, a gene encoding a secreted protein from Lactococcus lactis subsp.lactis MG1363[J]. Gene, 1990, 95(1): 155-60.
[8] Simon D, Chopin A. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis [J]. Biochimie,1988, 70(4): 559-66.
[9] Samal BB, Arakawa T, Boone TC, et al. High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein[J]. Biochim Biophys Acta, 1995, 1260(1): 27-33.
[10] Barnes WM. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates [J]. Proc Natl Acad Sci USA, 1994, 91(6): 2216-20.
[11] Ribeiro LA, Azevedo V, Le LY, et al. Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grad live vaccines against Brucellosis [J]. Appl Environ Microbiol, 2002, 68(2): 910-6
[12] Mannam P, Jones KF, Geller BL. Mucosal vaccine made from live,recombinant Lactococcus lactis protects mice against pharyngeal infection with Streptococcus pyogenes [J]. Infect Immun, 2004, 72(6): 3444-50.
[13] Pei H, Liu J, Cheng Y, et al. Expression of SARS-coronavirus nucleocapsid protein in Escherichia coli and Lactococcus lactis for serodiagnosis and mucosal vaccination [J]. Appl Microbiol Biotechnol, 2005, 68(2): 220-7.
[14] 凌均棨,汪喻忠,蒋少云,等.基因重组乳链菌防龋疫苗免疫性的实验研究[J].中山医科大学学报,2000,2l(4):241-4.Ling JQ, Wang YZ, Jiang SY. Experimental study on the immunity of recombinant Streptococcus lactis anticaries vaccine [J]. Acad J SUMS, 2000, 21(4): 241-4.
[15] Dieye Y, Usai S, Clier F, et al. Design of a protein-targeting system for lactic acid bacteria[J]. J Bacteriol, 2001, 183(14): 4157-66.
[16] Stemmer WP, Crammeri A, Ha KD, et al. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides[J]. Gene, 1995, 164(1): 49-53.