Microsoft Word 罗爱红-二校

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1 6 12 Vol. 6 No Journal of Food Safety and Quality Dec., 2015 Q67 luxab luxcdabe 罗爱红 1, 冯炘 1, 宁保安 2, 白家磊 2, 彭媛 2, 高志贤 (1., ; 2.,, ) 2* 摘要 : 目的 Q67 luxab luxcdabe, 方法 Q67 luxab luxcdabe, pet-22b, DH5α, BL21(DE3) 结果 (RLU) OD600 : luxab luxcdabe IPTG 1 µl/ml 1 RLU, Cd 2+ Hg 2+ Cr 6+, EC 50 ( ) : luxab : mg/l; luxcdabe : mg/l 结论, luxab luxcdabe RLU, luxab, luxab EC 50, 关键词 : ; ; ; Comparison of the expression and luminescence of luxab and luxcdabe genes from Vibrio qinghaiensis sp. Q67 in Escherichia coli LUO Ai-Hong 1, FENG Xin 1, NING Bao-An 2, BAI Jia-Lei 2, PENG Yuan 2, GAO Zhi-Xian 2* (1. College of Environmental Science and Safety Engineering, Tianjin University of Technology, Tianjin , China; 2. Key Laboratory of Risk Assessment and Control Technology for Environment and Food Safety, Institute of Health and Environmental Medicine, Academy of Military Medical Sciences, Tianjin , China) ABSTRACT: Objective To compare the expression of luxab and luxcdabe genes from freshwater luminescent bacteria Vibrio qinghaiensis sp. Q67 in Escherichia coli, and to make preparations for the construction of recombinant bacteria which could especially detect heavy metals. Methods Primers were designed in order to obtain the homologous fragments of luxab and luxcdabe, then got the product through polymerase chain reaction (PCR). As the next process, the product was cloned into vector pet-22b and then transferred into Escherichia coli DH5α for genetic sequencing. The positive clones were propagated and the plasmids extracted were transferred into Escherichia coli BL21 (DE3). Performance of bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Vibrio qinghaiensis was detected. Results 基金项目 : (BWS11J019) Fund: Supported by Key Project of PLA (BWS11J019) * 通讯作者 :,,, gaozhx@163.com *Corresponding author: GAO Zhi-Xian, Professor, Institute of Health and Environmental Medicine, No.1, Dali Road, Tianjin , China. gaozhx@163.com

2 12, : Q67 luxab luxcdabe 4817 After about 1 h culture with 1 µl/ml decanal and 1 µl/ml IPTG, the luxab and luxcdabe recombinant bacteria got the highest RLU: and respectively. EC 50 of luxab and luxcdabe recombinant bacteria of Cd 2+, Hg 2+, and Cr 6+ are 5.160,5.129,7.097 mg/l and 6.137, 7.643,7.736 mg/l respectively. Conclusion The expression of luxab is more effective than luxcdabe in E. Coli. LuxAB recombinant bacteria have lower EC 50 values, and the luxab recombinant bacterias are more sensitive towards heavy metals. KEY WORDS: Vibrio qinghaiensis; luciferase genes; recombinant bacterias; performance 1 引言 [1], [2] [3] [4], ( ) [5],, NaCl, ph,, PFOA( ) PFOS( ) [6] [7] [8], (20 ~25 ), [9],, luxab [10] luxcdabe [11],,,, [12,13], [14] luxab luxcdabe, 2 材料与方法 2.1 实验材料 Q67, ; DH5α, BL21(DE3), ( ) ; pet-22b, T4 DNA, Thermo ; Plasmid Mini Kit(200) Gel Extract Kit(200), Omega ; TIANamp Bacteria DNA Kit, ( ) ; Yeast Extract( ) Tryptone( ), Oxoid ; Agar Powder LA Taq,, 2.2 实验仪器 BHP9511, ; Mastercycler personal PCR, Eppendorf ; DYY-11, ; WD-9403F, ; TGL-16C, ; TU-1901, ; HH B11, ;, ESCO ; ZWY-240, ;, ; DZKW-S-4, ; MS3, IKA 2.3 试验方法配制培养基 Q67 [15] : 5 g, 5 g, MgCl 2 3.2g, KBr 0.2 g, CaSO g, NaCl 4 g, KCl 4 g, 3 ml, 2%, 1000 ml, min LB : Yeast Extract 5 g, Tryptone 10 g, NaCl 10 g, 15 g Agar, 1000 ml, min 青海弧菌 Q67 平板培养, 5 ml

4818 第6卷食品安全质量检测学报液体培养基中, 22, 180 r/min 震荡过夜培养 0.01 0.05 0.1 1 5 10 mg/l 用水做对照, 设置 2.3.

3 4818 第6卷食品安全质量检测学报液体培养基中, 22, 180 r/min 震荡过夜培养 mg/l 用水做对照, 设置个平行样测试组每个浓度取 2 ml 被检测溶液, 加青海弧菌基因组 DNA 提取取培养过夜菌液, 使用 TIANamp Bacteria DNA 入诱导培养 1 h 后的菌液 100 μl 菌液混合均匀, 15 Kit 制剂盒提取, 方法参照说明书 min 后检测相对发光强度, 计算相对发光强度(测试组发光强度/对照组发光强度)和 EC50 值(半有效浓度, luxab 和 luxcdabe 基因的克隆分别设计 luxab 引物 Fab:CGCCATATGAAATT 发光强度抑制率为 50%对应的被测物浓度) TGGAAATTTCTT Rab:CTTCTCGAGTGTGGTATC CATTGTTTATTC; luxcdabe 引物 Fce:CTTGGATC CGATGAAAAAACACATGCCATT Rce:GCGCTCG AGCATTTTCTTATTCCTATTCTTA, 华大基因合成用青海弧菌基因组 DNA 作为模板, 进行 PCR 扩增扩增产物电泳验证并进行胶回收纯化, 纯化产物连 p MD18-T, 转化入 DH5α 感受态细胞转化后菌株平板培养, 挑单菌落进行菌落 PCR, 阳性菌株送华大基因测序, 得到各自的拼接结果, 进行序列比对结果与分析青海弧菌基因组 DNA 提取用 TIANamp Bacteria DNA Kit 制剂盒提取培养 15 h 的菌液的基因组 DNA, 提取结果琼脂糖凝胶电泳验证表明, 青海弧菌 Q67 基因组 DNA 大小介于 1008~11849 bp 之间 3.2 青海弧菌 LuxAB luxcdabe 基因的克隆 luxab 和 luxcdabe 基因 PCR 产物琼脂糖凝胶电荧光素酶基因表达菌的构建取测序正确的菌株过夜培养活化, 用 Plasmid 泳验证结果如图 1 和图 2 由图 1 和图 2 可知, luxab PCR Mini Kit(200)试剂盒提取质粒, 提取结果电泳验证, 产物大小约为 2000 bp, luxcdabe PCR 产物大小约为并进行胶回收, 取胶回收产物与表达载体 pet-22b 分 6000 bp, 与预期大小相符 PCR 产物连接 pmd18-t, 转别进行双酶切, 酶切位点为 luxab: NdeⅠ, XhoⅠ; 化入 DH5α 感受态细胞, 测序得到完整序列 luxcdabe: BamHⅠ, XhoⅠ 酶切产物电泳验证并胶 3.3 回收, 回收产物通过 T4 DNA 连接酶连接, 连接产物转化入大肠杆菌 BL21(DE3)感受态细胞在含有氨苄重组菌性质测定随着培养时间的延长, 重组菌在 600 nm 波长处的吸光度及对应发光量如图 3 和图 4 所示由图 3 西林(Amp)的固体培养基上 37 过夜培养, 挑单菌和图 4 可知, 重组菌随着菌密度的增大, 在诱导培养落进行菌落 PCR, 后的第一个小时左右相对发光强度就已经达到了最阳性菌株 37 液体培养基培养, 测定 OD600 值和 RLU 大值, 分别是和 (单位为 1), luxab 重组菌性质测定选癸醛作为发光底物, 用无水乙醇配成体积分重组菌的最大相对发光强度是 luxcdabe 重组菌的 5 数为 5%的癸醛溶液取阳性重组菌株平板培养, 挑能是发光底物或者诱导剂的浓度影响了发光此外, 单菌落于加入了 5 μl 癸醛溶液的 5 ml LB 液体培养次级代谢产物的积累也会造成一定影响倍多约 1 h 后迅速下降到一个非常低的水平这可基中 r/min 培养 2 h, 然后按 2%接种量接种至含 5 μl 癸醛溶液的 5 ml LB 液体培养基中 r/mim 培养, 2 h 后加入 5 μl 异丙基硫代 -β-d-半乳糖苷(iptg)诱导, 继续培养用 BHP9511 水质毒性检测仪和 TU-1901 双光束紫外可见分光光度计检测 RLU 和 OD600 值重金属离子对重组菌毒性分析用 CdCl2 2. 5H2O K2Cr2O7 HgCl2(均为分析纯) 配制不同浓度的溶液依照水质检测国标检出限设置重金属浓度 Cd2+浓度设置为: M: 500 bp DNA Ladder Marker mg/l, Cr6+浓度设置为: mg/l, Hg2+浓度设置为: Fig. 1 图 1 luxab PCR 扩增产物电泳结果 Electrophoretic results of PCR products of luxab

12, : Q67 luxab luxcdabe 4819 3.4 重金属离子对重组菌毒性分析 5 6 7, EC 50 1 表 1 各金属离子对重组菌的毒性效应 Table 1 Toxic effects of heavy metals on recombinant bacteria EC 50 (mg/l) Fig.

4 12, : Q67 luxab luxcdabe 重金属离子对重组菌毒性分析 5 6 7, EC 50 1 表 1 各金属离子对重组菌的毒性效应 Table 1 Toxic effects of heavy metals on recombinant bacteria EC 50 (mg/l) Fig. 2 M: 1 kb DNA Ladder 2 luxcdabe PCR Electrophoretic results of pcr products of luxcdabe Cd 2+ luxab luxcdabe Hg 2+ luxab luxcdabe Cr 6+ luxab luxcdabe Fig. 3 3 luxab Characteristics of luxab Recombinant bacteria 5 Hg 2+ Fig.5 Toxic effects of Hg 2+ on recombinant bacteria Fig. 4 4 luxcdabe Characteristics of LuxCDABE Recombinant bacteria 6 Cr 6+ Fig.6 Toxic effects of Cr 6+ on recombinant bacteria

5 contaminants [J]. Yangtze River, 2014, 45(18): [2],,,. [J]., 2013, 23(5): Zhang JJ, Jia JM, Ma YH, et al The application of luminescent bacteria in heavy terrorist attacks of chemical poisons emergency monitoring system [J]. Chin J Health Lab Technol, 2013, 23(5): [3],,,. [J]., 2014, 24(6): Zou YP, Zhang L, Zhang P, et al, Determination of sodium benzoate level in sodas by luminescent bacteria Virbrio 7 Cd 2+ Fig.7 Toxic effects of Cd 2+ on recombinant bacteria 1, luxab EC 50 luxcdabe, luxab luxcdabe luxab, 4 结论 luxab luxcdabe, luxab, EC 50, luxab, luxab, luxab luxcdabe,, 参考文献 [1],,,. [J]., 2014, 45(18): Chen J, Yuan L, Ye D. Test and study of toxicity of two photogenic bacteria affected by different heavy metal qinghaiensis sp Q67 [J]. Chin J Health Lab Tec, 2014, 24(6): [4],,. [J]., 2015, 7(3): 4 7. Huang CK, Liu TT, Tang XW. Application of the photobacteria toxicity detection method in water quality assessment and early warning of drinking water [J]. Environ Monit Forewarn, 2015, 7(3): 4 7. [5],,,. _ [J]., 1994, 25(3): Zhu WJ, Wang J, Chen XY, et al. A new species of luminous bacteria Vibrio qinghaiensis sp.nov. [J]. Oceanologia Et Limnologi Sinica, 1994, 25(3): [6],,,. PFOA PFOS Q67 [J]., 2015, 35(1): Liu HY, Qin GJ, Mo LY, et al. Evaluation of aquatic toxcity of PFOA and PFOS to Vibrio qinghaiensis sp.-q67 [J]. J Guilin Univ Technol, 2015, 35(1): [7],,,. Q67 [J]., 2015, 10(2): Li X, Li N, Rao KF, et al. Toxicities of benzothiazole and benzothiazole derivatives (BTs) to Vibrio qinghaiensis sp. -Q67 [J]. Asian J Ecotoxicol, 2015, 10(2): [8],,,. Q67 [J]., 2013, 8(6): Zhang J, Liu S S, Deng HP, et al. Evaluation on the combined toxicity of pyridium-based Ionic liquids to Vibrio qinghaiensis sp.-q67 [J]. Asian J Ecotoxicol, 2013, 8(6): [9] Nora Kováts MR, Varanka B. Comparison of conventional and vibrio fischeri bioassays for the assessment of municipal wastewater toxicity [J]. Environ Eng Manag J, 2012, 11(11): [10],, 璠,, LuxAB [J]., 2015, 15(1):

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6 12, : Q67 luxab luxcdabe 4821 Wang YW, Ding W, Li F, et al, Prokaryotic Expression of Luciferase Gene LuxAB of Vibrio qinghaiensis [J]. J Chin Inst Food Sci Tecnol, 2015, 15(1): [11],,. lux [J]., 2014, 20(6): Zhang LL, Ding W, Wang YW, et al. Heterologous expression of lux gene from Vibrio qinghaiensis in E. coli [J]. Chin J Appl Environ Biol, 2014, 20(6): [12] Meighen EA. Bacterial bioluminescence: organisation, regulation, and application of the lux genes [J]. FASEB, 1993, 7(11): [13],. Lux [J]., 2008, 21(18): 1 5. Xia TJ, Chang HP. Investigation and application of Lux genes from luminous bacteria [J]. J Puyang Vocat Tech Coll, 2008, 21(4): 1 5. [14],,,. lux [J]., 2003, 3(3): Jiang AM, Yang GM, Li YR, et al, Studies on the construetion method of luminescent Lactoccus [J]. J Chin Inst Food Sci Tecnol, 2003, 3(3): [15],,,. [J]. ( ),1999, 1: Zheng JM, Zhao SX, Zhu WJ, et al. The Influence of Environmental Factors on the Growth and Luminescence of Vibrio qinghaiensis sp. nov [J]. J East China Norm Univ (Nat Sci), 1999, 1: 作者简介 ( 责任编辑 : 白洪健 ) 罗爱红, 硕士研究生, 主要研究方向为水与食品中有毒物质的快速检测 aihongluo1201@163.com 高志贤, 教授, 博士生导师, 主要研究方向为食品安全风险监控技术研究 gaozhx@163.com

Microsoft Word 聂雪梅.doc

Microsoft Word 聂雪梅.doc 4 4 Vol. 4 No. 4 2013 8 Journal of Food Safety and Quality Aug., 2013 聂雪梅 *, 李立, 孙利, 高飞 (, 100123) 摘要 : 2011 12,, 2007~2011,,,, 关键词 : ; ; ; ; Trade and notification situation on food import and export