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WO2017148163A1 - 一种提取2',3'-环形核苷单磷酸的方法 - Google Patents

一种提取2',3'-环形核苷单磷酸的方法 Download PDF

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WO2017148163A1
WO2017148163A1 PCT/CN2016/103058 CN2016103058W WO2017148163A1 WO 2017148163 A1 WO2017148163 A1 WO 2017148163A1 CN 2016103058 W CN2016103058 W CN 2016103058W WO 2017148163 A1 WO2017148163 A1 WO 2017148163A1
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陈秀兰
张玉忠
刘昂
于洋
解彬彬
秦启龙
宋晓妍
石梅
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Shandong University
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Definitions

  • the invention relates to a method for extracting 2',3'-cyclic nucleoside monophosphate, in particular to a method for simultaneously extracting adenosine-2', 3'-cyclic monophosphate and guanosine from Escherichia coli engineering bacteria by using recombinant protein.
  • the method belongs to the 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 messenger theory was proposed by EW Sutherland in 1965. He believes that various nitrogen-containing hormones (proteins, peptides, and amino acid derivatives) in the human body pass through the intracellular cyclic adenosine monophosphate (3', 5'-cAMP). To play a role, and to call 3',5'-cAMP as the second messenger, this is the first discovered ring nucleotide second messenger.
  • cyclic guanylate (3',5'-cGMP), cyclic cytidine (3',5'-cCMP), and cyclic uridine (3',5'- in various biological cells.
  • cUMP cyclic diguanosine monophosphate
  • c-di-AMP cyclic diadenylate
  • cGAMP cyclic guanosine adenosine monophosphate
  • 2',3'-cyclic nucleoside monophosphates attract more and more attention, there will be more demand for scientific research and medical applications.
  • the preparation of 2',3'-cyclic nucleoside monophosphates is carried out by chemical synthesis, which has low yield and high price, which greatly limits its development and application.
  • the present invention addresses the deficiencies of the prior art and provides a method for rapidly preparing four 2',3'-cyclic nucleotides using the interaction of recombinant IF3 protein with four 2',3'-cyclic monophosphates.
  • This method eliminates the need for specific precursors, synthesizes four 2',3'-cyclic nucleoside monophosphates by the growth of microorganisms, and rapidly synthesizes four 2',3'-rings using a recombinant protein. Purification of nucleoside monophosphate.
  • a method for extracting 2',3'-cyclic nucleoside monophosphate comprising the following steps:
  • PCR specific primer sequences are as follows:
  • the gene if3 obtained in the step (1) is digested, ligated with the same digested expression plasmid PET-22b, and then transformed into E. coli BL21 (DE3) strain, and subjected to fermentation culture to obtain a recombinant strain. Fermentation broth
  • the PCR amplification system (50 ⁇ L) in step (1) is as follows:
  • the PCR reaction conditions are as follows:
  • the restriction enzyme digestion in the step (2) is carried out by restriction enzymes NdeI and XhoI, and the enzyme digestion reaction system is as follows:
  • the reaction system connected in the step (2) is as follows:
  • the fermentation culturing step is as follows:
  • the culture was expanded at 35-38 ° C and 150-200 rpm until the absorbance at a wavelength of 600 nm was 0.8; then the incubation was continued at 18-22 ° C, 100-140 rpm for 30 min; then IPTG was added at a final concentration of 0.1 mM. Inducing culture for 22 to 25 hours;
  • the fermentation medium is fresh water LB medium, and the components are as follows:
  • the steps of disrupting the cells are as follows:
  • the isolated bacteria are suspended in a lysis buffer, and the cells are disrupted under a pressure of 950 to 1050 bar. Centrifuge at 12000 rpm for 50 min, and take the supernatant;
  • the lysis buffer component was: 50 mM Tris-HCl, 150 mM NaCl, pH 8.0.
  • the step of purifying the recombinant protein IF3 by nickel column affinity chromatography is 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 IF3 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, 20 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 of the C18 reverse chromatography column for high-performance liquid phase separation is: 0 min, B solution 0%; 2.5 min, B solution 0%; 5 min, B Liquid 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 method provided by the present invention is the first to directly extract four kinds of 2',3'-cyclic nucleoside monophosphates from Escherichia coli.
  • four kinds of 2',3'-cyclic nucleoside monophosphates are synthesized de novo, no specific precursors are needed, no chemical synthesis is used for pollution, simple and convenient, and 5 mg of four pures can be extracted per liter of culture solution.
  • the invention adopts Escherichia coli for biological fermentation, and because of its clear genetic background and fast growth rate, it is beneficial to further adopt bioengineering methods for improvement and increase yield.
  • Figure 1 Electropherogram of recombinant protein IF3 purified by nickel column affinity chromatography.
  • M protein molecular weight marker
  • IF3 recombinant IF3 protein purified by nickel column affinity chromatography
  • the Escherichia coli K12 strain in the examples was purchased from the China Industrial Microbial Culture Collection Management Center, Address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, China, Culture Collection No. CICC 10424;
  • the E. coli BL21 (DE3) receptor was purchased from Beijing Quanjin Biotechnology Co., Ltd., Address: 4th Floor, Building B-3, Zhongsheng Village, Zhongguancun, No. 66 Xixiaokou Road, Haidian District, Beijing.
  • a method for constructing a recombinant protein expression strain the steps are as follows:
  • 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 if3 and the vector pET-22b were digested with NdeI and XhoI, and the digestion reaction system was as follows:
  • DNA fragment is linked to an expression vector
  • the reaction system is as follows:
  • the expression strain transferred to the recombinant pET-22b-if3 vector on the plate was picked up to 100 mL of liquid LB medium supplemented with a final concentration of 100 ⁇ g/mL ampicillin, and cultured overnight at 37 ° C, 180 rpm.
  • the cultured seed liquid 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 was 0.8, the culture conditions were changed to 20 ° C, 120 rpm, and the culture was continued for 30 min, and then IPTG was added at a final concentration of 0.1 mM. Continue to culture for 24hr.
  • the buffer used to purify the protein is
  • Lysis buffer 50 mM Tris-HCl, 150 mM NaCl, pH 8.0.
  • Wash buffer 50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8.0.
  • Elution buffer 50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH 8.0.
  • the cells were collected by centrifugation at 10,000 rpm for 5 min, and the cells collected by the 1 L bacterial solution were resuspended in the lysis buffer by a ratio of 50 mL of the lysis buffer, and the cells were disrupted under pressure (1000 bar).
  • the target protein eluted is allowed to stand at 0 ° C for 3 days, at which time the protein solution will show significant precipitation.
  • the protein solution was centrifuged at 12,000 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.
  • the obtained 2',3'-cyclic nucleoside monophosphate solution was separated by high performance liquid chromatography using a C18 reversed-phase column to obtain four pure 2',3'-cyclic nucleoside monophosphates.
  • the mobile phase used was: solution A, 10 mM ammonium acetate solution, pH 5.0; solution B, 75% solution A was 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.

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Abstract

本发明提供了一种提取2',3'-环形核苷单磷酸的方法，包括如下步骤：(1)提取基因组DNA，PCR扩增，制得基因if3；(2)将基因if3与表达质粒连接，转化后，经发酵培养，制得重组菌发酵液；(3)分离重组菌发酵液中的菌体，破碎细胞，镍柱亲和层析提纯，静置，固液分离，取上清液；(4)将上清液经超滤去除蛋白杂质，经C18反向色谱柱进行高效液相分离，分别制得含四种2',3'-环形核苷单磷酸溶液。

Description

一种提取2′,3′-环形核苷单磷酸的方法 技术领域
本发明涉及一种提取2',3'-环形核苷单磷酸的方法，特别涉及一种利用重组蛋白从大肠杆菌工程菌中同时提取腺苷-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'-环形单磷酸四种2',3'-环形核苷单磷酸的方法，属于生物技术技术领域。
背景技术
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。第二信使学说是E.W.萨瑟兰1965年提出，他认为人体内各种含氮激素(蛋白质、多肽和氨基酸衍生物)都是通过细胞内的环形腺苷酸(3′,5′-cAMP)而发挥作用的，并把3′,5′-cAMP称为第二信使，这是最早发现的环形核苷酸类第二信使。随后，研究人员在多种生物细胞内发现了环形鸟苷酸(3′,5′-cGMP)，环形胞苷(3′,5′-cCMP)，环形尿苷酸(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)等均可作为第二信使参与胞内信号通路的调控。除了3′,5′-环形核苷单磷酸之外，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。有关2′,3′-环形核苷单磷酸生理功能的研究较少，现有的研究结果认为，2′,3′-环形核苷单磷酸与组织细胞的损伤有一定的关系，在受损的组织细胞内，2′,3′-环形核苷单磷酸的含量会明显升高；腺苷-2',3'-环形单磷酸具有激活细胞线粒体膜上通透性孔洞的作用，引起细胞凋亡；研究还发现与正常的脑部细胞相比，感染了HIV病毒的脑部细胞内2′,3′-环形核苷单磷酸降解酶的磷酸化水平更高。2',3'-环形核苷单磷酸(2′,3′-cNMPs)与细胞损伤的密切相关性激发了科研人员越来越大的兴趣。
随着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应用上将有更多的需求。目前2′,3′-环形核苷单磷酸的制备均采用化学合成的方法，产量低，价格高，从而极大限制了其开发应用。
发明内容：
本发明针对现有技术的不足，提供了一种利用重组IF3蛋白与四种2′,3′-环形单磷酸的相互作用快速制备四种2′,3′-环形核苷酸的方法。该方法无需特定的前体物质，利用微生物的生长从头合成四种2′,3′-环形核苷单磷酸，并利用一种重组蛋白快速地将微生物合成的四种2′,3′-环形核苷单磷酸提纯。
本发明技术方案如下：
一种提取2',3'-环形核苷单磷酸的方法，包括如下步骤：
(1)提取大肠埃希氏菌(Escherichia coli)K12菌株的基因组DNA，利用PCR扩增编码IF3蛋白的基因if3，制得基因if3；
所述PCR特异引物序列如下：
F:GGAATTCCATATGATTAAAGGCGGAAAACG，
R:CCGCTCGAGCTGTTTCTTCTTAGG；
(2)将步骤(1)制得的基因if3酶切后与同样经酶切的表达质粒PET-22b连接，然后转化到E.coli BL21(DE3)菌株中，进行发酵培养，制得重组菌发酵液；
(3)分离步骤(2)制得的重组菌发酵液中的菌体，破碎细胞，镍柱亲和层析提纯重组蛋白IF3，然后0℃静置2.5～3.5d，固液分离，取上清液；
(4)将步骤(3)制得的上清液经超滤去除蛋白杂质，滤液经C18反向色谱柱进行高效液相分离，分别制得含四种2',3'-环形核苷单磷酸溶液。
根据本发明优选的，所述步骤(1)中的PCR扩增体系(50μL)如下：
Figure PCTCN2016103058-appb-000001
PCR反应条件如下：
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。
根据本发明优选的，所述步骤(2)中酶切采用限制性内切酶NdeI和XhoI进行双酶切，酶切反应体系如下：
Figure PCTCN2016103058-appb-000002
反应条件：37℃温浴30min。
根据本发明优选的，所述步骤(2)中连接的反应体系如下：
酶切后的载体pET-22b   1μL
酶切后的基因if3       4μL
Solution I            5μL
反应条件：16℃连接过夜。
根据本发明优选的，所述步骤(2)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吸光度为0.8；然后在18～22℃、100～140rpm继续培养30min；再加入终浓度为0.1mM的IPTG，继续诱导培养22～25小时；
所述发酵培养基为淡水LB培养基，组分如下：
10g NaCl，10g蛋白胨，5g酵母粉，pH 8.0，蒸馏水定容至1L。
根据本发明优选的，所述步骤(3)中，破碎细胞步骤如下：
将分离得到的菌体重悬于裂解缓冲液中，在压力为950～1050bar的条件下破碎细胞， 12000rpm离心50min，取上清液；
所述的裂解缓冲液组分为：50mM Tris-HCl、150mM NaCl，pH 8.0。
根据本发明优选的，所述步骤(3)中，镍柱亲和层析提纯重组蛋白IF3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IF3溶液；
所述裂解缓冲液组分为：50mM Tris-HCl、150mM NaCl，pH 8.0；
所述冲洗缓冲液组分为：50mM Tris-HCl、150mM NaCl、20mM咪唑，pH 8.0；
所述洗脱缓冲液组分为：50mM Tris-HCl、150mM NaCl、250mM咪唑，pH 8.0。
根据本发明优选的，所述步骤(4)中，超滤为采用截留分子量为3K的超滤管超滤。
根据本发明优选的，所述步骤(4)中，所述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
1、本发明提供的方法首次实现了从大肠杆菌中同时直接提取四种2',3'-环形核苷单磷酸。通过微生物生物发酵，从头合成四种2′,3′-环形核苷单磷酸，不需要特定的前体物，无化学合成法的污染，简单方便，每升培养液可提取5mg的四种纯品2',3'-环形核苷单磷酸；
2、本发明采用大肠杆菌进行生物发酵，由于其遗传背景清楚、生长速度快，有利于进一步采用生物工程方法进行改进，提高产量。
附图说明
图1、镍柱亲和层析纯化的重组蛋白IF3的电泳图。
图中：M、蛋白质分子量标记(marker)；IF3、镍柱亲和层析纯化后的重组IF3蛋白；
图2、提取含4种2′,3′-环形核苷单磷酸溶液的HPLC分析；
图3、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306.0491完全相符；
(c)，二级质谱分析一级质谱中得到的质荷比为306.0491(z＝1)的离子，片段化方式如(d)所示；
(d)，2′,3′-cCMP结构示意图；
图4、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307.0331完全相符；
(c)，二级质谱分析一级质谱中得到的质荷比为307.0331(z＝1)的离子，片段化方式如(d)所示；
(d)，2′,3′-cUMP结构示意图；
图5、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346.0552完全相符；
(c)，二级质谱分析一级质谱中得到的质荷比为346.0552(z＝1)的离子，片段化方式如(d)所示；
(d)，2′,3′-cGMP结构示意图；
图6、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330.0603极为相符；
(c)，二级质谱分析一级质谱中得到的质荷比为330.06(z＝1)的离子，片段化方式如(d)所示；
(d)，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。
实施例中所述大肠杆菌K12菌株购自中国工业微生物菌种保藏管理中心，地址：北京市朝阳区酒仙桥中路24号院6号楼，菌种保藏号CICC 10424；
大肠杆菌BL21(DE3)感受态购自北京全式金生物技术有限公司，地址：北京市海淀区西小口路66号中关村东升科技园B-3号楼四层。
实施例1
一种重组蛋白表达菌株构建方法，步骤如下：
1.大肠杆菌转录起始因子IF3编码基因的克隆
1.1大肠杆菌K12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。
1.2引物设计与合成
根据IF3编码基因序列设计两条引物：
F:GGAATTCCATATGATTAAAGGCGGAAAACG，
R:CCGCTCGAGCTGTTTCTTCTTAGG，
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：
Figure PCTCN2016103058-appb-000003
Figure PCTCN2016103058-appb-000004
(2)对PCR扩增产物进行1％琼脂糖凝胶电泳，结果获得一条约500bp的DNA片段。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if3。
2.表达载体及表达菌株的构建
(i)DNA片段与表达载体酶切
将制得的基因if3和载体pET-22b用NdeI和XhoI双酶切，酶切反应体系如下：
Figure PCTCN2016103058-appb-000005
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。
(ii)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。
(iii)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if3       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。
(iv)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-if3载体转至大肠杆菌BL21(DE3)感受态。
(v)转化的大肠杆菌BL21(DE3)涂于含100μg/L氨苄青霉素LB培养基，37℃过夜培养。
(vi)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-if3载体的表达菌株。
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-if3载体的表达菌株到100mL加入了终浓度为100μg/mL氨苄青霉素的液体LB培养基中，37℃，180rpm过夜培养。
(2)按照1％接种量，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，加入终浓度为0.1mM的IPTG。继续培养24hr。
2.提纯IF3蛋白
(1)提纯蛋白所用的缓冲液为
裂解缓冲液：50mM Tris-HCl，150mM NaCl，pH 8.0。
冲洗缓冲液：50mM Tris-HCl，150mM NaCl，20mM咪唑，pH 8.0。
洗脱缓冲液：50mM Tris-HCl，150mM NaCl，250mM咪唑，pH 8.0。
(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IF3。
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，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2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3-6所示。

Claims (8)

  1. 一种提取2',3'-环形核苷单磷酸的方法，包括如下步骤：
    (1)提取大肠埃希氏菌(Escherichia coli)K12菌株的基因组DNA，利用PCR扩增编码IF3蛋白的基因if3，制得基因if3；
    所述PCR特异引物序列如下：
    F:GGAATTCCATATGATTAAAGGCGGAAAACG，
    R:CCGCTCGAGCTGTTTCTTCTTAGG；
    (2)将步骤(1)制得的基因if3酶切后与同样经酶切的表达质粒PET-22b连接，然后转化到E.coli BL21(DE3)菌株中，进行发酵培养，制得重组菌发酵液；
    (3)分离步骤(2)制得的重组菌发酵液中的菌体，破碎细胞，镍柱亲和层析提纯重组蛋白IF3，然后0℃静置2.5～3.5d，固液分离，取上清液；
    (4)将步骤(3)制得的上清液经超滤去除蛋白杂质，滤液经C18反向色谱柱进行高效液相分离，分别制得含四种2',3'-环形核苷单磷酸溶液。
  2. 如权利要求1所述的方法，其特征在于，所述步骤(1)中的PCR扩增体系如下：
    Figure PCTCN2016103058-appb-100001
    PCR反应条件如下：
    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。
  3. 如权利要求1所述的方法，其特征在于，所述步骤(2)中酶切采用限制性内切酶NdeI和XhoI进行双酶切，酶切反应体系如下：
    Figure PCTCN2016103058-appb-100002
    反应条件：37℃温浴30min。
  4. 如权利要求1所述的方法，其特征在于，所述步骤(2)中连接的反应体系如下：
    酶切后的载体pET-22b       1μL
    酶切后的基因if3           4μL
    Solution I                5μL
    反应条件：16℃连接过夜。
  5. 如权利要求1所述的方法，其特征在于，所述步骤(2)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吸光度为0.8；然后在18～22℃、100～140rpm继续培养30min；再加入终浓度为0.1mM的IPTG，继续诱导培养22～25小时；
    所述发酵培养基为淡水LB培养基，组分如下：
    10g NaCl，10g蛋白胨，5g酵母粉，pH 8.0，蒸馏水定容至1L。
    根据本发明优选的，所述步骤(3)中，破碎细胞步骤如下：
    将分离得到的菌体重悬于裂解缓冲液中，在压力为950～1050bar的条件下破碎细胞，12000rpm离心50min，取上清液；
    所述的裂解缓冲液组分为：50mM Tris-HCl、150mM NaCl，pH 8.0。
  6. 如权利要求1所述的方法，其特征在于，所述步骤(3)中，镍柱亲和层析提纯重组蛋白IF3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IF3溶液；
    所述裂解缓冲液组分为：50mM Tris-HCl、150mM NaCl，pH 8.0；
    所述冲洗缓冲液组分为：50mM Tris-HCl、150mM NaCl、20mM咪唑，pH 8.0；
    所述洗脱缓冲液组分为：50mM Tris-HCl、150mM NaCl、250mM咪唑，pH 8.0。
  7. 如权利要求1所述的方法，其特征在于，所述步骤(4)中，超滤为采用截留分子量为3K的超滤管超滤。
  8. 如权利要求1所述的方法，其特征在于，所述步骤(4)中，所述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。
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