WO2017215174A1 - 一种海洋细菌基因LfliZ及应用 - Google Patents

一种海洋细菌基因LfliZ及应用 Download PDF

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WO2017215174A1
WO2017215174A1 PCT/CN2016/103059 CN2016103059W WO2017215174A1 WO 2017215174 A1 WO2017215174 A1 WO 2017215174A1 CN 2016103059 W CN2016103059 W CN 2016103059W WO 2017215174 A1 WO2017215174 A1 WO 2017215174A1
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solution
lfliz
recombinant
liquid
monophosphate
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陈秀兰
张玉忠
刘昂
宋晓妍
于洋
李平一
张熙颖
石梅
解彬彬
苏海楠
秦启龙
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山东大学
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C07—ORGANIC CHEMISTRY
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
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    • C12P19/305—Pyrimidine nucleotides
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
    • C12Y301/04016—2',3'-Cyclic-nucleotide 2'-phosphodiesterase (3.1.4.16)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the invention relates to a marine bacterial gene LfliZ and an application thereof, and belongs to the technical field of biotechnology.
  • Cyclic nucleotides are important second messengers in the cell and are involved in the regulation of multiple signal transduction pathways in cells.
  • the second messengers known to be found include cyclic adenosine monophosphate (3', 5'-cAMP), cyclic guanosine monophosphate (3', 5'-cGMP), and cyclic cytidine monophosphate (3', 5' -cCMP), cyclic uridine monophosphate (3', 5'-cUMP), cyclic diguanosine monophosphate (c-di-GMP), cyclic diadenylate (c-di-AMP) and cyclic guanosine adenosine Acid (cGAMP).
  • cyclic adenosine monophosphate 3', 5'-cAMP
  • cyclic guanosine monophosphate 3', 5'-cGMP
  • cyclic cytidine monophosphate 3', 5'-cCMP
  • the researchers also detected adenosine-2', 3'-cyclic monophosphate and guanosine-2', 3'-cyclic monophosphate (2', 3'-cGMP) from plant cells, from a fluorescent fake
  • adenosine-2', 3'-cyclic monophosphate and guanosine-2', 3'-cyclic monophosphate (2', 3'-cGMP) from plant cells, from a fluorescent fake
  • cytidine-2', 3'-cyclic monophosphate (2', 3'-cCMP) and uridine-2', 3'-cyclic monophosphate (2', 3'-cUMP) was detected in the bacterium .
  • Existing studies have shown that these four 2',3'-cyclic nucleoside monophosphates are likely to represent another new class of biological cell second messengers.
  • the invention aims at the deficiency of the prior art, provides a marine bacterial gene LfliZ and rapidly prepares four 2',3'-cyclic nucleoside monophosphates (adenosine-2', 3'-cyclic monophosphate by using the recombinant LfliZ protein thereof. , guanosine-2', 3'-cyclic monophosphate, cytidine-2', 3'-cyclic monophosphate and uridine-2', 3'-cyclic monophosphate).
  • the recombinant protein LfliZ expressed by the above marine bacterial gene LfliZ has an amino acid sequence as shown in SEQ ID No. 2.
  • a recombinant vector in which a marine bacterial gene LfliZ is inserted into a plasmid is inserted into a plasmid.
  • the plasmid is a pET-22b (+) plasmid.
  • a recombinant cell obtained by transforming the above recombinant vector into a host cell.
  • the host cell is Escherichia coli; further preferably, the host cell is E. coli BL21 (DE3).
  • the above marine bacterial gene LfliZ is used in the preparation of 2',3'-cyclic nucleoside monophosphate.
  • the recombinant vector used in the step (2) is a plasmid of pET-22b(+); and the host cell used in the recombinant cell in the step (3) is Escherichia coli; further preferably, The host cell described is Escherichia coli BL21 (DE3).
  • the fermentation culturing step is as follows:
  • the culture was expanded at 35-38 ° C and 150-200 rpm until the absorbance of the bacterial solution at a wavelength of 600 nm was 0.8; then the culture was continued at 18-22 ° C, 100-140 rpm for 25-35 min; then IPTG (isopropyl) was added.
  • the thiogalactoside is added to a concentration of 0.1 mM, and the induction culture is continued for 22 to 25 hours;
  • the medium used for the fermentation culture is LB liquid medium, and the composition per liter is as follows:
  • the extraction and purification in the step (4) are as follows:
  • the target protein LfliZ solution prepared in the step (i) is allowed to stand at -1 to 2 ° C for 2.5 to 3.5 days, centrifuged, and the supernatant is taken, and ultrafiltration is performed to obtain a 2', 3'-cyclic nucleoside. Crude extract of monophosphate;
  • step (iii) Purifying the crude 2',3'-cyclic nucleoside monophosphate obtained in step (ii) by high performance liquid chromatography on a C18 reversed-phase column to obtain four kinds of 2', 3'- Cyclic nucleoside monophosphate.
  • nickel column affinity chromatography is used in the step (i), and the steps are as follows:
  • each nickel column was filled with 2 mL of nickel glue. After the supernatant was passed through the nickel gel, each nickel column was equilibrated with 20 mL of lysis buffer and rinsed with 20 mL of washing buffer. Finally, eluted with 10 mL of elution buffer to prepare a recombinant protein LfliZ solution;
  • the lysis buffer component is: 50 mM Tris-HCl, 150 mM NaCl, pH 8.0;
  • the washing buffer component is: 50 mM Tris-HCl, 150 mM NaCl, 50 mM imidazole, pH 8.0;
  • the elution buffer component was: 50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0.
  • the ultrafiltration is ultrafiltration using an ultrafiltration tube having a molecular weight cut off of 3K.
  • the separation procedure for the high-performance liquid phase separation of the C 18 reverse chromatography column in the step (iii) is: 0 min, B solution 0%; 2.5 min, B solution 0%; 5 min, B solution 30 %; 10min, B solution 60%; 14min, B solution 100%; 21min, B solution 100%; 22min, B solution 50%; 23min, B solution 0%; 30min, B solution 0%; flow rate is 10ml / min,
  • the detection wavelength is 254 nm;
  • the mobile phase was: liquid A, 10 mM ammonium acetate solution, pH 5.0; liquid B, and liquid A was mixed with methanol in a ratio of 3:1 by volume.
  • the gene LfliZ encoded by the cloned gene LfliZ of the present invention is capable of binding four 2',3'-cyclic nucleoside monophosphates in the cell of Escherichia coli, namely adenosine-2', 3'-cyclic monophosphate, guanosine-2' , 3'-cyclic monophosphate, cytidine-2', 3'-cyclic monophosphate and uridine-2', 3'-cyclic monophosphate.
  • Simultaneous direct extraction of four 2',3'-cyclic nucleoside monophosphates from E. coli can be achieved by purifying the recombinant LfliZ protein. It can be calculated that about 2.5 mg of four pure 2',3'-cyclic nucleoside monophosphates can be extracted per liter of culture solution, which has good application potential.
  • M nucleic acid molecular weight marker (marker); LfliZ, LfliZ gene;
  • M protein molecular weight marker (marker);
  • LfliZ nickel column affinity chromatography purification of recombinant LfliZ protein;
  • E. coli DH5 ⁇ , E. coli BL21 (DE3) strain was purchased from TransGen Biotech;
  • the pET-22b(+) expression vector was purchased from Novagen;
  • PCR amplification reagents were purchased from TransGen Biotech;
  • Restriction enzyme and ligase Solution I were purchased from Takara;
  • the bacterial genome extraction kit and the plasmid extraction kit were purchased from Beijing Baitek Biotechnology Co., Ltd.;
  • DNA gel recovery kit was purchased from Omega;
  • 2',3'-cUMP, 2',3'-cGMP, 3',5'-cCMP, 3',5'-cUMP, 3',5'-cGMP and 3',5'-cAMP were purchased from Biolog the company;
  • Penicillium ampicillin and inducer IPTG was purchased from Merck;
  • Methanol was purchased from Tianjin Komi Chemical Reagent Co., Ltd.;
  • the medium preparation materials are all commonly used raw materials in the field, and are commercially available;
  • Pseudoalteromonas sp. SM9913 is purchased from China National Culture Collection, deposit number CCTCC NO: M2010223.
  • PCR amplification was carried out using genomic DNA as a template; PCR reaction conditions were: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, extension at 72 °C for 30 sec, 30 cycles The final extension was 72 min at 72 °C.
  • the PCR amplification system (50 ⁇ L) is as follows:
  • the obtained gene LfliZ and the vector pET-22b(+) were digested with NdeI and XhoI, and the digestion reaction system was as follows:
  • the reaction system is as follows:
  • the ligated recombinant pET-22b-LfliZ vector was transferred to E. coli BL21 (DE3) competent state according to the heat shock transformation method in the Guide to Molecular Cloning.
  • the specific steps are as follows: adding the ligation solution to 50 ⁇ L of E. coli BL21 (DE3) competent cells, mixing and ice bath for 30 min; 42 ° C water bath for 90 s; rapidly transferring to ice bath, cooling for 1-2 min; adding 600 ⁇ L of liquid LB medium The mixture was incubated at 37 ° C for 1 h; after centrifugation, about 100 ⁇ L of the resuspended cells were applied to a MacConkey plate containing ampicillin, and cultured overnight at 37 °C.
  • the transformants were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing verification, and the expression strains transformed into the recombinant pET-22b-LfliZ vector were obtained.
  • the nucleotide sequence of the gene LfliZ was determined as shown in SEQ ID NO.
  • the expression strain transferred to the recombinant pET-22b-LfliZ vector on the plate was picked up to 100 mL of LB liquid medium supplemented with a final concentration of 100 ⁇ g/mL ampicillin, and cultured at 37 ° C, 180 rpm overnight;
  • Inoculation was carried out according to the inoculation amount of 1% by volume, and the cultured seed solution was transferred to a shake flask having a liquid volume of 1 L. Incubate at 37 ° C, 180 rpm until the absorbance at a wavelength of 600 nm is 0.8, the culture conditions are changed to 20 ° C, 120 rpm, continue to culture for 30 min, then add IPTG (isopropyl thiogalactoside) to a concentration of 0.1 mM, Continue to train for 24 hours;
  • composition of LB liquid medium per liter is as follows:
  • the buffer used to purify the protein is
  • Lysis buffer 50 mM Tris-HCl, 150 mM NaCl, pH 8.0;
  • Rinsing buffer 50 mM Tris-HCl, 150 mM NaCl, 50 mM imidazole, pH 8.0;
  • Elution buffer 50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0;
  • the cell disrupted liquid obtained in the step (2) is centrifuged at 12,000 rpm for 50 min at a temperature of 4 ° C to remove an insoluble precipitate;
  • amino acid sequence of the protein LfliZ was determined as shown in SEQ ID No. 2.
  • the eluted target protein LfliZ was allowed to stand at 0 ° C for 3 days, at which time the protein solution would show significant precipitation.
  • the protein solution was centrifuged at 12000 rpm for 20 min to obtain a supernatant;
  • the supernatant obtained in the step (1) is ultrafiltered with an ultrafiltration tube having a molecular weight cut off of 3K to remove residual protein, and the ultrafiltration membrane filtrate contains four kinds of 2', 3'-rings.
  • Nucleoside monophosphate solution Nucleoside monophosphate solution
  • the mobile phase used was: liquid A, 10 mM ammonium acetate solution, pH 5.0; liquid B, 75% liquid added to 25% methanol;
  • the separation procedure used in the above steps is: 0 min, B solution 0%; 2.5 min, B solution 0%; 5 min, B solution 30%; 10 min, B solution 60%; 14 min, B solution 100%; 21 min, B solution 100%; 22min, B solution 50%; 23min, B solution 0%; 30min, B solution 0%.
  • the flow rate was 10 mL/min and the detection wavelength was 254 nm.
  • the four 2',3'-cyclic nucleoside monophosphate standards purchased were diluted with distilled water to the following concentration gradients: 0 ⁇ M, 20 ⁇ M, 40 ⁇ M, 60 ⁇ M, 80 ⁇ M, and 100 ⁇ M, using a C 18 reverse column for high efficiency.
  • the peak area corresponding to each concentration of the cyclic nucleoside monophosphate standard was determined by liquid chromatography, and then the standard curve was drawn with the peak area as the abscissa and the concentration as the ordinate.
  • the above molar amount can be calculated by mass conversion, and about 2.5 mg of four pure 2', 3'-ring cores can be extracted per liter of the culture solution.
  • Monophosphate monophosphate Monophosphate monophosphate.

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Abstract

提供了一种海洋细菌基因LfliZ及其应用。所述海洋细菌基因LfliZ的核苷酸序列如SEQ ID NO.1所示；还提供了该基因LfliZ表达的重组蛋白LfliZ及其在制备2',3'-环形核苷单磷酸中的应用。

Description

一种海洋细菌基因LfliZ及应用 技术领域
本发明涉及一种海洋细菌基因LfliZ及其应用，属于生物技术技术领域。
背景技术
海洋微生物数量巨大，新种资源极其丰富，也存在大量新基因资源。随着技术的进步，海洋微生物资源的开发成为各国研究的焦点。但是，由于采样和培养等困难，目前对海洋微生物资源及其基因资源的开发利用还很有限。发现海洋中新基因并研究其功能，对开发和利用海洋微生物基因资源具有重要意义。
环形核苷酸是细胞内重要的第二信使，参与细胞内多种信号转导途径的调控。现已知发现的第二信使包括环形腺苷单磷酸(3′,5′-cAMP)、环形鸟苷单磷酸(3′,5′-cGMP)、环形胞苷单磷酸(3′,5′-cCMP)、环形尿苷单磷酸(3′,5′-cUMP)、环形二鸟苷酸(c-di-GMP)，环形二腺苷酸(c-di-AMP)及环形鸟苷腺苷酸(cGAMP)。除了上述已发现的环形核苷酸类第二信使外，在2009年埃德温(Edwin)等人还首次在人的肾脏细胞中发现了腺苷-2',3'-环形单磷酸(2′,3′-cAMP)，随后人们又在哺乳动物的大脑等组织细胞中发现了腺苷-2',3'-环形单磷酸的存在。科研人员还从植物细胞中检测到腺苷-2',3'-环形单磷酸和鸟苷-2',3'-环形单磷酸(2′,3′-cGMP)，从一株荧光假单胞菌中检测到胞苷-2',3'-环形单磷酸(2′,3′-cCMP)和尿苷-2',3'-环形单磷酸(2′,3′-cUMP)的存在。现有的研究结果表明，这四种2′,3′-环形核苷单磷酸很可能代表了另外一类新型的生物细胞第二信使。
由于2′,3′-环形核苷单磷酸吸引了越来越多的关注，其在科学研究和医学上的需求也日益增多。目前2′,3′-环形核苷单磷酸的制备均采用化学合成的方法，尚未有生物制备方法。化学合成法生产2′,3′-环形核苷单磷酸，产量低，价格高，在一定程度上限制了其开发应用。
发明内容
本发明针对现有技术的不足，提供一种海洋细菌基因LfliZ及利用其重组LfliZ蛋白快速制备四种2′,3′-环形核苷单磷酸(腺苷-2',3'-环形单磷酸、鸟苷-2',3'-环形单磷酸、胞苷-2',3'-环形单磷酸和尿苷-2',3'-环形单磷酸)的方法。
本发明技术方案如下：
一种海洋细菌基因LfliZ，核苷酸序列如SEQ ID NO.1所示。
上述海洋细菌基因LfliZ表达的重组蛋白LfliZ，氨基酸序列如SEQ ID No.2所示。
一种重组载体，在质粒中插入含有上述海洋细菌基因LfliZ。
根据本发明优选的，所述的质粒为pET-22b(+)质粒。
一种重组细胞，将上述重组载体转化入宿主细胞中获得。
根据本发明优选的，所述的宿主细胞为大肠杆菌；进一步优选的，所述的宿主细胞为 大肠杆菌BL21(DE3)。
上述海洋细菌基因LfliZ在制备2′,3′-环形核苷单磷酸中的应用。
上述应用，步骤如下：
(1)制备核酸序列如SEQ ID No.1所示的海洋细菌基因LfliZ；
(2)构建含有SEQ ID No.1所示核苷酸序列的重组载体；
(3)构建含有步骤(2)所示重组载体的重组细胞；
(4)发酵培养上述重组细胞，经提取纯化，制得2',3'-环形核苷单磷酸。
根据本发明优选的，所述步骤(2)的重组载体采用的质粒为pET-22b(+)质粒；所述步骤(3)中的重组细胞采用的宿主细胞为大肠杆菌；进一步优选的，所述的宿主细胞为大肠杆菌BL21(DE3)。
根据本发明优选的，所述步骤(4)中，发酵培养步骤如下：
先在35～38℃、150～200rpm条件下扩大培养至菌液在波长为600nm时的吸光度为0.8；然后在18～22℃、100～140rpm继续培养25～35min；然后加入IPTG(异丙基硫代半乳糖苷)至浓度为0.1mM，继续诱导培养22～25小时；
所述发酵培养采用的培养基为LB液体培养基，每升组分如下：
10g NaCl，10g蛋白胨，5g酵母粉，蒸馏水定容至1L，pH 8.0。
根据本发明优选的，所述步骤(4)中的提取纯化，步骤如下：
(i)将发酵液经固液分离，取菌体，破碎菌体，固液分离，取上清液，经镍柱亲和层析，洗脱后，制得目的蛋白LfliZ溶液；
(ii)将步骤(i)制得的目的蛋白LfliZ溶液在-1～2℃静置2.5～3.5天，离心，取上清液，经超滤，制得2',3'-环形核苷单磷酸粗提液；
(iii)将步骤(ii)制得的2',3'-环形核苷单磷酸粗提液经C18反向色谱柱进行高效液相色谱纯化，分别制得四种2',3'-环形核苷单磷酸。
根据本发明进一步优选的，所述步骤(i)中采用镍柱亲和层析，步骤如下：
将破碎细胞后的上清液上样镍柱，每根镍柱装2mL镍胶，待上清液流过镍胶后，每根镍柱用20mL裂解缓冲液平衡，用20mL冲洗缓冲液冲洗，最后用10mL的洗脱缓冲液洗脱，制得重组蛋白LfliZ溶液；
所述裂解缓冲液组分为：50mM Tris-HCl、150mM NaCl，pH8.0；
所述冲洗缓冲液组分为：50mM Tris-HCl、150mM NaCl、50mM咪唑，pH8.0；
所述洗脱缓冲液组分为：50mM Tris-HCl、150mM NaCl、250mM咪唑，pH8.0。
根据本发明进一步优选的，所述步骤(ii)中，超滤为采用截留分子量为3K的超滤管超滤。
根据本发明进一步优选的，所述步骤(iii)中C18反向色谱柱进行高效液相分离的分离程序为：0min，B液0％；2.5min，B液0％；5min，B液30％；10min，B液60％；14min，B液100％；21min，B液100％；22min，B液50％；23min，B液0％；30min，B液0％； 流速为10ml/min，检测波长为254nm；
流动相为：A液，10mM醋酸铵溶液，pH 5.0；B液，将A液与甲醇按体积比3:1的比例混合。
有益效果
本发明克隆的基因LfliZ编码的蛋白LfliZ能够在大肠杆菌胞内结合四种2′,3′-环形核苷单磷酸，即腺苷-2',3'-环形单磷酸、鸟苷-2',3'-环形单磷酸、胞苷-2',3'-环形单磷酸和尿苷-2',3'-环形单磷酸。通过提纯重组LfliZ蛋白，可以实现从大肠杆菌中同时直接提取四种2',3'-环形核苷单磷酸。经计算每升培养液可提取约2.5mg的四种纯品2',3'-环形核苷单磷酸，有较好的应用潜力。
附图说明
图1、通过PCR克隆的LfliZ基因的琼脂糖凝胶电泳图；
图中：M、核酸分子量标记(marker)；LfliZ、LfliZ基因；
图2、镍柱亲和层析纯化的重组蛋白LfliZ的SDS-PAGE电泳图；
图中：M、蛋白质分子量标记(marker)；LfliZ、镍柱亲和层析纯化后的重组LfliZ蛋白；
图3、提取含4种2′,3′-环形核苷单磷酸粗提液的HPLC分析；
图4、纯化得到的2′,3′-cCMP液相色谱和质谱分析；
(a)，液相色谱分析纯化得到的2′,3′-cCMP；
(b)，一级质谱分析纯化得到的2′,3′-cCMP，图谱显示提纯的2′,3′-cCMP的质荷比为306.0491(z＝1),与理论值306.0486极为相符；
(c)，2′,3′-cCMP结构示意图；
图5、液相色谱，质谱分析纯化得到的2′,3′-cUMP；
(a)，液相色谱分析纯化得到的2′,3′-cUMP；
(b)，一级质谱分析纯化得到的2′,3′-cUMP，图谱显示提纯的2′,3′-cCMP的质荷比为307.0331(z＝1),与理论值307.0326极为相符；
(c)，2′,3′-cUMP结构示意图；
图6、液相色谱，质谱分析纯化得到的2′,3′-cGMP；
(a)，液相色谱分析纯化得到的2′,3′-cGMP；
(b)，一级质谱分析纯化得到的2′,3′-cGMP，图谱显示提纯的2′,3′-cCMP的质荷比为346.0552(z＝1),与理论值346.0547极为相符；
(c)，2′,3′-cGMP结构示意图；
图7、液相色谱，质谱分析纯化得到的2′,3′-cAMP；
(a)，液相色谱分析纯化得到的2′,3′-cAMP；
(b)，一级质谱分析纯化得到的2′,3′-cAMP，图谱显示提纯的2′,3′-cCMP的质荷比为330.06(z＝1)，与理论值330.0598极为相符；
(c)，2′,3′-cAMP结构示意图。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明，但本发明所保护范围不限于此。
生物材料来源：
E.coli DH5α、E.coli BL21(DE3)菌株购自TransGen Biotech公司；
pET-22b(+)表达载体购自Novagen公司；
PCR扩增试剂均购自TransGen Biotech公司；
限制性内切酶及连接酶Solution I购自Takara公司；
细菌基因组提取试剂盒、质粒小提试剂盒购自北京百泰克生物技术有限公司；
DNA胶回收试剂盒购自Omega公司；
2′,3′-cCMP和2′,3′-cAMP购买自Sigma-Aldrich公司；
2′,3′-cUMP、2′,3′-cGMP、3′,5′-cCMP、3′,5′-cUMP、3′,5′-cGMP和3′,5′-cAMP购买自Biolog公司；
所用抗生素氨苄青霉及诱导剂IPTG购自Merck公司；
甲醇购自天津市科密欧化学试剂有限公司；
培养基配制原料均为本领域常用原料，可市场购得；
DNA测序在北京博尚生物技术有限公司完成；
引物合成在华大基因完成；
深海适冷菌假交替单胞菌株(Pseudoalteromonas sp.)SM9913，购自中国典型培养物保藏中心，保藏号CCTCC NO:M2010223。
实施例1
LfliZ基因的克隆及表达载体的构建，步骤如下：
1LfliZ基因的克隆
1.1 Ps.sp.SM9913基因组DNA的提取
参照百泰克公司基因组提取试剂盒说明书提取基因组DNA(常规提取步骤)。
1.2 引物设计与合成
根据LfliZ基因序列设计两条引物：
F:GGAATTCCATATGAGTAACCAATCAG
R:CCGCTCGAGTGCATTGGTTTTTTTTGC
由上海生工生物技术公司合成。
1.3 利用PCR进行基因序列扩增及产物回收
(1)以F和R为引物，以基因组DNA为模板，进行PCR扩增；PCR反应条件为：95℃预变性5min；95℃变性30sec，55℃退火30sec，72℃延伸30sec，30个循环；72℃终延伸5min。
PCR扩增体系(50μL)如下：
灭菌蒸馏水              32.2μL
5×TransStart FastPfu缓冲液10μL
dNTP混合物5μL
引物F(50μM) 0.4μL
引物R(50μM) 0.4μL
基因组DNA              1μL
TransStartFastPfuDNA聚合酶1μL
(2)对PCR扩增产物进行1％琼脂糖凝胶电泳，结果获得一条约400bp的DNA片段(如图1所示)，然后用Omega公司的DNA回收试剂盒按照其说明回收扩增出的DNA片段，制得基因LfliZ。
2. 表达载体及表达菌株的构建
(1)DNA片段与表达载体酶切
将制得的基因LfliZ和载体pET-22b(+)用NdeI和XhoI双酶切，酶切反应体系如下：
缓冲液 2μL
基因LlifZ或载体pET-22b(+)    8μL
NdeI内切酶 1μL
XhoI内切酶 1μL
灭菌蒸馏水 8μL
盖紧盖子，手指轻弹离心管，混匀样品，在离心机上瞬时离心2sec，把样品集中在管底，37℃温浴30min。
(2)对酶切产物进行1％琼脂糖凝胶电泳，将目的条带切胶，然后用Omega公司的DNA回收试剂盒按照其说明回收目的DNA片段。
(3)DNA片段与表达载体连接
连接反应体系如下：
酶切后的载体pET-22b(+)  1μL
酶切后的基因LfliZ       4μL
Solution I              5μL
盖紧盖子，手指轻弹离心管，混匀样品，在离心机上瞬时离心2sec，把样品集中在管底，16℃连接过夜。
(4)按《分子克隆实验指南》上的热激转化方法将连接好的重组pET-22b-LfliZ载体转至大肠杆菌BL21(DE3)感受态。具体的步骤为：将连接液加入50μL大肠杆菌BL21(DE3)感受态细胞中，混匀后冰浴30min；42℃水浴90s；快速转至冰浴，冷却1-2min；加入600μL液体LB培养基，37℃水浴1h；离心后用约100μL重悬菌体涂布至含有氨苄青霉素的麦康凯平板上，37℃过夜培养。
转化子送上海生工生物技术有限公司测序验证，获得转入重组pET-22b-LfliZ载体的表达菌株。
经检测，基因LfliZ核苷酸序列如SEQ ID NO.1所示。
实施例2
1. 重组菌株的发酵培养
(1)种子的培养
挑取平板上的转入重组pET-22b-LfliZ载体的表达菌株到100mL加入了终浓度为100μg/mL氨苄青霉素的LB液体培养基中，37℃，180rpm过夜培养；
(2)按照体积百分比1％的接种量进行接种，将培养好的种子液转接到装液量为1L的摇瓶中。37℃，180rpm培养至菌液在波长为600nm吸光度为0.8时，将培养条件改为20℃，120rpm，继续培养30min后，加入IPTG(异丙基硫代半乳糖苷)至浓度为0.1mM，继续培养24小时；
LB液体培养基每升组分如下：
10g NaCl，10g蛋白胨，5g酵母粉，蒸馏水定容至1L，pH 8.0。
2. 提纯LfliZ蛋白
(1)提纯蛋白所用的缓冲液为
裂解缓冲液：50mM Tris-HCl，150mM NaCl，pH8.0；
冲洗缓冲液：50mM Tris-HCl，150mM NaCl，50mM咪唑，pH8.0；
洗脱缓冲液：50mM Tris-HCl，150mM NaCl，250mM咪唑，pH8.0；
(2)10000rpm离心5min收集菌体，按照1L菌液收集的菌体用50mL裂解缓冲液的比例加入裂解缓冲液重悬菌体，压力(1000bar)破碎细胞；
(3)将步骤(2)得到的细胞破碎液，在4℃温度下，12000rpm离心50min，去除不可溶沉淀；
(4)将上述离心后的上清液，上样镍柱(每根镍柱装2mL镍胶)。待上清液流过镍胶后，每根镍柱用20mL裂解缓冲液平衡，之后每根镍柱用20mL冲洗缓冲液冲洗。最后每根镍柱用10mL的洗脱缓冲液洗脱蛋白，即得到提纯的目的蛋白LfliZ。对提纯的LfliZ的SDS-PAGE电泳分析如图2所示。
经检测，蛋白LfliZ的氨基酸序列如SEQ ID No.2所示。
实施例3
1. 2′,3′-环形核苷单磷酸混合物的提取
(1)将洗脱得到的目的蛋白LfliZ，0℃静置3d，此时蛋白溶液将会出现明显的沉淀。将蛋白溶液12000rpm离心20min，得到上清液；
(2)将步骤(1)中得到的上清液，用截留分子量为3K的超滤管超滤，去除残留的蛋白，超滤膜滤过液即为含有4种2′,3′-环形核苷单磷酸溶液；
2. 四种环形核苷酸2′,3′-cAMP，2′,3′-cGMP，2′,3′-cCMP和2′,3′-cUMP纯品的提取
(1)将得到的2′,3′-环形核苷单磷酸溶液，采用C18反向色谱柱进行高效液相色谱分离得到四种2′,3′-环形核苷单磷酸纯品；
所用的流动相为：A液，10mM醋酸铵溶液，pH 5.0；B液，75％A液加入25％甲醇；
(2)上述步骤中所用的分离程序为：0min，B液0％；2.5min，B液0％；5min，B液30％；10min，B液60％；14min，B液100％；21min，B液100％；22min，B液50％；23min，B液0％；30min，B液0％。流速为10mL/min，检测波长为254nm。
对上述4种2′,3′-环形核苷单磷酸溶液进行HPLC分析，结果如图3所示，对上述4种2′,3′-环形核苷单磷酸溶液进行液相色谱和质谱分析，结果如图4-7所示。
3. 四种纯品2',3'-环形核苷单磷酸产量检测
(1)将购买的4种2′,3′-环形核苷单磷酸标准品用蒸馏水稀释到如下浓度梯度：0μM、20μM、40μM、60μM、80μM和100μM，采用C18反向色谱柱进行高效液相色谱检测各浓度的环形核苷单磷酸标准品所对应的峰面积，之后以峰面积为横坐标，浓度为纵坐标绘制标准曲线。
(2)利用做好的标准曲线，将制备好的4种2′,3′-环形核苷单磷酸样品用C18反向色谱柱进行高效液相色谱分别检测浓度可得，每升培养液可提取1.30±0.17μmol的2',3'-cCMP、1.40±0.12μmol的2',3'-cUMP、2.81±0.25μmol的2',3'-cGMP和2.61±0.19μmol的2',3'-cAMP。根据4种2′,3′-环形核苷单磷酸的分子量，将上述摩尔量换算成质量计算可得，每升培养液可提取约2.5mg的四种纯品2',3'-环形核苷单磷酸。

Claims (10)

  1. 一种海洋细菌基因LfliZ，核苷酸序列如SEQ ID NO.1所示。
  2. 权利要求1所述海洋细菌基因LfliZ表达的重组蛋白LfliZ，氨基酸序列如SEQ ID No.2所示。
  3. 一种重组载体，在质粒中插入含有权利要求1所述海洋细菌基因LfliZ。
  4. 如权利要求3所述的重组载体，其特征在于，所述的质粒为pET-22b(+)质粒。
  5. 一种重组细胞，将权利要求3所述重组载体转化入宿主细胞中获得。
  6. 如权利要求5所述的重组细胞，其特征在于，所述的宿主细胞为大肠杆菌；进一步优选的，所述的宿主细胞为大肠杆菌BL21(DE3)。
  7. 权利要求1所述海洋细菌基因LfliZ在制备2′,3′-环形核苷单磷酸中的应用。
  8. 如权利要求7所述的应用，其特征在于，步骤如下：
    (1)制备核酸序列如SEQ ID No.1所示的海洋细菌基因LfliZ；
    (2)构建含有SEQ ID No.1所示核苷酸序列的重组载体；
    (3)构建含有步骤(2)所示重组载体的重组细胞；
    (4)发酵培养上述重组细胞，经提取纯化，制得2',3'-环形核苷单磷酸。
  9. 如权利要求8所述的应用，其特征在于，所述步骤(2)的重组载体采用的质粒为pET-22b(+)质粒；所述步骤(3)中的重组细胞采用的宿主细胞为大肠杆菌；进一步优选的，所述的宿主细胞为大肠杆菌BL21(DE3)。
  10. 如权利要求8所述的应用，其特征在于，所述步骤(4)中，发酵培养步骤如下：
    先在35～38℃、150～200rpm条件下扩大培养至菌液在波长为600nm时的吸光度为0.8；然后在18～22℃、100～140rpm继续培养25～35min；然后加入IPTG至浓度为0.1mM，继续诱导培养22～25小时；
    所述发酵培养采用的培养基为LB液体培养基，每升组分如下：
    10g NaCl，10g蛋白胨，5g酵母粉，蒸馏水定容至1L，pH 8.0；
    根据本发明优选的，所述步骤(4)中的提取纯化，步骤如下：
    (i)将发酵液经固液分离，取菌体，破碎菌体，固液分离，取上清液，经镍柱亲和层析，洗脱后，制得目的蛋白LfliZ溶液；
    (ii)将步骤(i)制得的目的蛋白LfliZ溶液在-1～2℃静置2.5～3.5天，离心，取上清液，经超滤，制得2',3'-环形核苷单磷酸粗提液；
    (iii)将步骤(ii)制得的2',3'-环形核苷单磷酸粗提液经C18反向色谱柱进行高效液相色谱纯化，分别制得四种2',3'-环形核苷单磷酸；
    根据本发明进一步优选的，所述步骤(i)中采用镍柱亲和层析，步骤如下：
    将破碎细胞后的上清液上样镍柱，每根镍柱装2mL镍胶，待上清液流过镍胶后，每根镍柱用20mL裂解缓冲液平衡，用20mL冲洗缓冲液冲洗，最后用10mL的洗脱缓冲液洗 脱，制得重组蛋白LfliZ溶液；
    所述裂解缓冲液组分为：50mM Tris-HCl、150mM NaCl，pH8.0；
    所述冲洗缓冲液组分为：50mM Tris-HCl、150mM NaCl、50mM咪唑，pH8.0；
    所述洗脱缓冲液组分为：50mM Tris-HCl、150mM NaCl、250mM咪唑，pH8.0；
    根据本发明进一步优选的，所述步骤(ii)中，超滤为采用截留分子量为3K的超滤管超滤；
    根据本发明进一步优选的，所述步骤(iii)中C18反向色谱柱进行高效液相分离的分离程序为：0min，B液0％；2.5min，B液0％；5min，B液30％；10min，B液60％；14min，B液100％；21min，B液100％；22min，B液50％；23min，B液0％；30min，B液0％；流速为10ml/min，检测波长为254nm；
    流动相为：A液，10mM醋酸铵溶液，pH 5.0；B液，将A液与甲醇按体积比3:1的比例混合。
PCT/CN2016/103059 2016-06-15 2016-10-24 一种海洋细菌基因LfliZ及应用 WO2017215174A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4720458A (en) * 1983-10-19 1988-01-19 Sullivan Cornelius W Heat sensitive bacterial alkaline phosphatase
CN103215238A (zh) * 2013-04-12 2013-07-24 国家海洋局第二海洋研究所 一种海洋细菌新型酯酶及其制备方法与应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647995B (zh) * 2016-02-29 2019-03-08 山东大学 一种提取2′，3′-环形核苷单磷酸的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4720458A (en) * 1983-10-19 1988-01-19 Sullivan Cornelius W Heat sensitive bacterial alkaline phosphatase
CN103215238A (zh) * 2013-04-12 2013-07-24 国家海洋局第二海洋研究所 一种海洋细菌新型酯酶及其制备方法与应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN [O] 18 May 2013 (2013-05-18), XP055451877, Database accession no. WP_013464393 *
EDWIN,K. ET AL.: "In Vivo Cardiovascular Pharmacology of 2', 3'-Camp, 2,-AMP, and 3'-AMP in the Rat", THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL *
YUAN, RONGRONG ET AL.: "Research Progress in Signal Transduction System of Escherichia Coli", JOURNAL OF GREEN SCIENCE AND TECHNOLOGY, 30 June 2013 (2013-06-30), pages 310 - 314, ISSN: 1674-9944 *

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