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1 5 4 Vol. 5 No Journal of Food Safety and Quality Apr., 2014 LC2W 任婧 *, 李楠, 陈臣, 董懿樱, 刘振民 (, ) 摘要 : 目的 LC2W 方法, 结果, : 1 %, 10 kv/cm, 3 h, 200 ng/μl, CFU/μg DNA 结论,,, LC2W 关键词 : ; ; ; ; Establishment of genetic transformation system for Lactobacillus casei LC2W REN Jing *, LI Nan, CHEN Chen, DONG Yi-Ying, LIU Zhen-Min (Dairy Research Institute, Bright Dairy & Food Co., Ltd., State Key Laboratory of Dairy Biotechnology, Shanghai , China) ABSTRACT: Objective To establish a genetic transformation system of Lactobacillus casei LC2W. Methods On the basis of single factor experiments, the effects of operating conditions, such as different plasmids, concentration of cell wall weakening agent, strength of electric field, recovery time, and plasmid concentration were investigated. Results The optimized parameters for electransformation were as follows: concentration of glycine used as cell wall weakening agent 1%, strength of electric field 10 kv/cm, recovery time 3 h, plasmid concentration 200 ng/μl. The high-efficient electransformation was reached at CFU/μg DNA. Conclusions A high-efficient electransformation system was estabolished, which could be used as a protocol for other Lactobacilli and the basis for genetic modification of L. casei LC2W. KEY WORDS: Lactobacillus casei; electransformation; genetic transformation; cell wall weakening agent; strength of electric field 1 引言,, [1-4], 基金项目 : (2013BAD18B01) 863 (2011AA100901) Fund: Supported by the National "Twelfth Five-Year" Plan for Science & Technology Support Program of China (2013BAD18B01) and the National High Technology Research and Development Program of China (2011AA100901). * 通讯作者 :,,,, renjing@brightdairy.com *Corresponding author: REN Jing, Ph.D, Senior Engineer, Senior Manager of Dairy Safety Research Department, Dairy Research Institute, Bright Dairy & Food Co., Ltd., renjing@brightdairy.com

2 4, : LC2W 1029, [5,6], (generally recognized as safe, GRAS),, [7-9], [10,11], LC2W, [12],, 1987, Chassy [13], [14-17], DNA,,,, DNA,,,, DNA,, LC2W 2 材料与方法 2.1 菌株和质粒 LC2W(CGMCC NO. 0828) DH5α pib184 psip403 Dr. Lars Axelsson, DH5α 200 μg/ml, LC2W 10 μg/ml 2.2 主要仪器 Eporator ( Eppendorf ); Labconco ( Labconco ); A35 ( Don Whitley Scientific ); Bio- Photometer plus ( Eppendorf ); NanoDrop 2000C ( Thermo Fisher Scientific ); Eppendorf 5424R ( Eppendorf ) 2.3 培养基和缓冲液 MRS ( Merck ) LC2W ; MRS ( 10 μg/ml) ; 30% (PEG, 1500); : MRS+500 mmol/l +20 mmol/l MgCl 2 +2 mmol/l CaCl 2 LB ( 10 g 5 g 10 g NaCl) 2.4 实验方法质粒提取 ( ), NanoDrop 2000C 干酪乳杆菌 LC2W 感受态制备 MRS 5 ml MRS, 37 ( 16 h); 2% 100 ml 1% MRS, 37 OD ( 4.5 h); 15 min, g 10 min ; 100 ml 30 % PEG 3 ; 1 ml 30 % PEG, 100 μl, 电转化 5 μl 100 μl, 0.1 cm ( BioRad ), 5 min, 1 kv 1 ml, 37 3 h; 100 μl, h,, 3, 3 结果与讨论 3.1 不同质粒转化 LC2W pib184 psip403

1030 5, P 23,, P spp, sakacin P,, LC2W, 1, pib184 LC2W, pib184,, 表 1 不同质粒转化 LC2W 的比较 Table 1 Comparison of different plasmids on the transformation of LC2W, ;,,, 10 kv/cm, 2 3.

3 1030 5, P 23,, P spp, sakacin P,, LC2W, 1, pib184 LC2W, pib184,, 表 1 不同质粒转化 LC2W 的比较 Table 1 Comparison of different plasmids on the transformation of LC2W, ;,,, 10 kv/cm, 不同复苏时间对电转效率的影响,,,,, [14], h, LC2W, 3 h, 3 (CFU/μg DNA) pib psip 不同甘氨酸浓度对电转效率的影响,, [18],,,,,, (0 0.3% 1.0% 3.0%),, LC2W,,,,,,, 1%, 不同电场强度对电转效率的影响,,,,,,, [14] ( kv/cm), Fig. 1 Fig. 2 1 Effect of glycine concentration on the transformation efficiency 2 Effect of the strength of electric field on the transformation efficiency

4 4, : LC2W Fig. 3 Effect of recovery time on the transformation efficiency 3.5 不同质粒浓度对电转效率的影响, DNA [16] (200 ng/μl) (1000 ng/μl), ( 2), 1 μg/μl DNA 表 2 Table 2 4 结论不同浓度质粒对电转效率的影响 Effect of plasmid concentrations on the transformation efficiency (ng/μl) (CFU/μg DNA) ,, LC2W 参考文献 [1],. [J]., 2009, S1: Cao RB, Wang JM. Research on functional property of Lactobacillus casei and its application [J]. China Food Addit, 2009, S1: [2],,,. H9 [J]., 2013, (11): Liu CC, Ji FD, Jiang LP, et al. Bio-preservative effects of Lactobacillus paracasei H9 in sodium alginate coating on grass carp [J]. Food Sci Technol, 2013, (11): [3],,,. [J]., 2009, (8): Bai WD, Zhao WH, Liang GF, et al. Characteristics of Lactobacillus bulgaricus and its application [J]. China Brew, 2009, (8): [4],,. [J]., 2009, (2): Ma PF, Chen YL, Jin NJ. Research and development on the functional properties of Lactobacillus rhamnous [J]. Bull Sci Technol, 2009, (2): [5] Kailasapathy K. Commercial sources of probiotic strains and their validated and potential health benefits - a review [J]. Int J Ferment Food, 2013, (2): [6] del Rio B, Seegers JF, Gomes-Solecki M. Immune response to Lactobacillus plantarum expressing Borrelia burgdorferi OspA is modulated by the lipid modification of the antigen [J]. PLoS One, 2010, (5): e [7] Kajikawa A, Satoh E, Leer RJ, et al. Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis [J]. Vaccine, 2007, (25): [8] Wu CM, Chung TC. Mice protected by oral immunization with Lactobacillus reuteri secreting fusion protein of Escherichia coli enterotoxin subunit protein [J]. FEMS Immunol Med Microbiol, 2007, (50): [9] del Rio B, Dattwyler RJ, Aroso M, et al. Oral immunization with recombinant Lactobacillus plantarum induces a protective immune response in mice with Lyme disease [J]. Clin Vaccine Immunol, 2008, (15): [10] Thompson K, Collins MA. Improvement in electroporation efficiency for Lactobacillus plantarum by the inclusion of high concentrations of glycine in the growth medium [J]. J Microbiol Method, 1996, (26): [11],,,. HD1.7 [J]., 2011, (23): Ge JP, Gao XJ, You T, et al. Initial Establishment of Genetic Transformation System for Lactobacillus paracasei HD1.7 [J]. Chin Agr Sci Bull, 2011, (23): [12] Ai LZ, Zhang H, Guo BH, et al. Preparation, partial characterization and bioactivity of exopolysaccharides from Lactobacillus

1032 5 casei LC2W [J]. Carbohyd Polym, 2008, (74): 353 357. [13] Chassy BM, Flickinger JL. Transformation of Lactobacillus casei by electroporation [J]. FEMS Microbiol Lett, 1987, (44): 173 177.

5 casei LC2W [J]. Carbohyd Polym, 2008, (74): [13] Chassy BM, Flickinger JL. Transformation of Lactobacillus casei by electroporation [J]. FEMS Microbiol Lett, 1987, (44): [14],,,. [J]., 2008, (6): Cao XM, Zhang HC, Li M, et al. Progress in study on electroporation efficiency for Lactobacillus [J]. Bull Acad Mil Med Sci, 2008, (6): [15],,,. [J]., 2008, (2): Xia ZF, Shi GY, Zhang L, et al. Optimization of electroporation processing of Lactobacilli [J]. Food Sci, 2008, (2): [16],,,. [J]. :, 2009, (zl): Zhang X, Cui YH, Zhang LW, et al. Development of electrotransformation in lactic acid bacteria [J]. J Lanzhou Univ: Nat Sci, 2009, (zl): [17],,,. [J]., 1998, (4): Jia SF, Wang YY, Guo XH, et al. The Factors Affected Transformation Efficiency of Lactobacillus by Electroporation [J]. Chin J Biotechnol, 1998, (4): [18] Holo H, Nes IF. High-frequency transformation, by electropration, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media [J]. Appl Environ Microbiol, 1989, (12): 作者简介 ( 责任编辑 : 张宏梁 ) 任婧, 高级工程师, 主要研究方向为食品安全及乳酸菌分子生物学 renjing@brightdairy.com

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<4D6963726F736F667420576F7264202D203631372D3632312032303133303430333030312DBACEC0F25FD0A3B6D4B8E55F2DB6FED0A32D2D2DC8A5B5F4CDBCD6D0B5C4BBD8B3B5B7FBBAC52E646F63> 第 4 卷第 2 期食品安全质量检测学报 Vol. 4 No. 2 2013 年 4 月 Journal of Food Safety and Quality Apr., 2013 何莉 *, 姜笑寒 ( 广东省食品药品职业技术学校, 广州 510663) 摘要 : 本文通过科学地运用 html5+jsp+sql 技术, 建