WO2021120566A1 - 一种基因工程菌及其应用，生产前列腺素e2的方法 - Google Patents

一种基因工程菌及其应用，生产前列腺素e2的方法 Download PDF

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WO2021120566A1
WO2021120566A1 PCT/CN2020/097211 CN2020097211W WO2021120566A1 WO 2021120566 A1 WO2021120566 A1 WO 2021120566A1 CN 2020097211 W CN2020097211 W CN 2020097211W WO 2021120566 A1 WO2021120566 A1 WO 2021120566A1
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prostaglandin
escherichia coli
pghs
recombinant escherichia
pet28a
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王伟
王康林
袁哲诚
金永红
王颀彤
贾和平
胡志浩
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合肥康诺生物制药有限公司
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    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12Y114/99—Miscellaneous (1.14.99)
    • C12Y114/99001—Prostaglandin-endoperoxide synthase (1.14.99.1), i.e. cyclooxygenase
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    • C12Y503/99—Other intramolecular oxidoreductases (5.3.99)
    • C12Y503/99003—Prostaglandin-E synthase (5.3.99.3)

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  • the invention relates to the field of biotechnology, in particular to a recombinant Escherichia coli and its application, and a method for producing prostaglandin E2.
  • Prostaglandin E2 is a metabolite of arachidonic acid cyclooxygenase. It is a twenty-carbon unsaturated fatty acid and a type of prostaglandin (PG). Its molecular formula is C 20 H 32 O 5 . It has a molecular weight of 352, white crystals, and a melting point of 68-69°C. It is soluble in organic solvents such as ethyl acetate, acetone, ethyl ether, methanol, and ethanol, but insoluble in water. PGE2 is an important cell growth and regulatory factor.
  • the main production method of PGE2 is to incubate the suspension of related enzymes obtained from sheep seminal vesicles with arachidonic acid.
  • This process material is inconvenient to obtain, high cost, complicated operation, low conversion rate, many impurities and by-products, and subsequent purification is complicated.
  • the present invention proposes a recombinant Escherichia coli and its application, and a method for producing prostaglandin E2.
  • the present invention constructs an Escherichia coli strain capable of expressing prostaglandin H synthase and prostaglandin E2 synthase.
  • the expressed enzyme can directly catalyze the conversion of arachidonic acid into prostaglandin E2.
  • the present invention provides a recombinant Escherichia coli (Escherichia coli) that overexpresses the coding gene of prostaglandin H synthase and the coding gene of prostaglandin E synthase.
  • nucleotide sequence of the gene encoding prostaglandin H synthase is shown in SEQ ID No. 1.
  • nucleotide sequence of the gene encoding prostaglandin E synthase is shown in SEQ ID No. 2 or shown in SEQ ID No. 3.
  • the recombinant Escherichia coli BL21(DE3)/pET28a-PGHS-mPGES1 is deposited in the General Microbiology Center CGMCC of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No.19049.
  • the recombinant Escherichia coli BL21(DE3)/pET28a-PGHS-mPGES2 is deposited in the General Microbiology Center CGMCC of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No.19050.
  • the present invention also proposes the application of the above-mentioned recombinant Escherichia coli in the production of prostaglandin E2.
  • the present invention also proposes a method for producing prostaglandin E2, which uses arachidonic acid as a substrate to produce prostaglandin E2 using the above-mentioned recombinant Escherichia coli (Escherichia coli).
  • the present invention constructs an Escherichia coli strain capable of expressing prostaglandin H synthase and prostaglandin E2 synthetase, and the expressed enzyme can directly catalyze the conversion of arachidonic acid into prostaglandin E2.
  • the preparation method of the present invention is simple and efficient.
  • the present invention uses both prostaglandin H synthase and prostaglandin E2 synthetase to directly catalyze the production of PGE2 from arachidonic acid.
  • Figure 1 is a synthetic route diagram of arachidonic acid to produce prostaglandin E2, where arachidonic acid is arachidonic acid, prostaglandin H2 is prostaglandin H2, prostaglandin E2 is prostaglandin E2, PGHS is prostaglandin H synthase, and PGES is Prostaglandin E synthetase.
  • Figure 2 is an HPLC detection spectrum of the reaction solution prepared in Example 5.
  • Figure 3 is a PCR detection gel map of the co-expression plasmid pET28a-PGHS-mPGES1 and the co-expression plasmid pET28a-PGHS-mPGES2, where 1 is the co-expression plasmid pET28a-PGHS-mPGES1, and 2 is the co-expression plasmid pET28a-PGHS-mPGES2.
  • M is Marker.
  • Plasmids and strains Escherichia coli TG1 strain (Wuhan Miaoling Biotechnology Co., Ltd., item number P1468); Escherichia coli BL21 (DE3) strain (Wuhan Miaoling Biotechnology Co., Ltd., item number P1472); expression vector pET28a (Wuhan Miaoling Biotechnology Co., Ltd., item number P1472) Limited company, article number P0023).
  • restriction endonucleases such as NcoI, BamHI, HindIII and EcoRI (Thermo Fisher Technology (China) Co., Ltd.), gel recovery purification kit (Shanghai Jierui Biological Engineering Co., Ltd., catalog number GK2043-200) , GBclonart seamless cloning kit (Suzhou Shenzhou Gene Co., Ltd., article number GB2001-48), JY92-IIN ultrasonic cell crusher (Ningbo Xinyi Ultrasonic Equipment Co., Ltd.).
  • the inventors published the prostaglandin H synthase, human prostaglandin E synthetase, cynomolgus monkey's prostaglandin H synthase, human prostaglandin E synthetase, and cynomolgus prostaglandin H synthase of the Gracilaria genus according to the NCBI website (https://www.ncbi.nlm.nih.gov/) published by the present inventors.
  • the nucleotide sequence of prostaglandin E synthetase was codon optimized for the E. coli expression system to obtain the corresponding optimized nucleotide sequence, and Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to carry out gene synthesis; the optimized nucleotide sequences were respectively for:
  • the optimized nucleotide sequence of the prostaglandin H synthase encoding gene (referred to as the PGHS encoding gene) of Gracilaria esculenta is shown in SEQ ID No. 1, and its amino acid sequence is:
  • mPGES1 encoding gene The optimized nucleotide sequence of the human prostaglandin E synthetase encoding gene (referred to as mPGES1 encoding gene) is shown in SEQ ID No. 2, and its amino acid sequence is:
  • mPGES2 encoding gene The nucleotide sequence of the optimized gene encoding prostaglandin E synthetase of cynomolgus monkey (referred to as mPGES2 encoding gene) is shown in SEQ ID No. 3, and its amino acid sequence is:
  • Example 1 Using the PGHS encoding gene synthesized in Example 1 as a template, amplify using the PGHS-F/PGHS-R primer pair to obtain a PGHS fragment with a size of 1738bp;
  • the mPGES1-F/mPGES1-R primer pair was used for amplification to obtain a mPGES1 fragment with a size of 525 bp;
  • Example 2 Using the mPGES2 encoding gene synthesized in Example 1 as a template, amplify using mPGES2-F/mPGES2-R primer pair to obtain mPGES2 fragment with a size of 960 bp;
  • plasmid pET28a-PGHS use restriction enzymes EcoRI and HindIII to digest to obtain vector fragment 2. After gel recovery and purification of vector fragment 2, mPGES1 fragment and mPGES2 fragment with gel recovery and purification kit, they are cloned seamlessly Technically constructed the co-expression plasmid pET28a-PGHS-mPGES1 and the co-expression plasmid pET28a-PGHS-mPGES2;
  • FIG. 3 is the co-expression plasmid pET28a- The PCR detection gel map of PGHS-mPGES1 and co-expression plasmid pET28a-PGHS-mPGES2, where 1 is the co-expression plasmid pET28a-PGHS-mPGES1, 2 is the co-expression plasmid pET28a-PGHS-mPGES2, and M is the Marker; It can be seen that the PCR verification band size of the co-expression plasmid pET28a-PGHS-mPGES1 is 2543 bp and the band size is correct; the PCR verification band size of the co-expression plasmid pET28a-
  • Example 3 Take the co-expression plasmid pET28a-PGHS-mPGES2, and prepare the recombinant Escherichia coli strain BL21(DE3)/pET28a-PGHS-mPGES2 according to the two-step operation in Example 3, which is deposited in the Chinese Microbial Culture Collection Management Committee. Microbiology Center CGMCC, the deposit number is CGMCC No.19050.
  • IPTG isopropyl- ⁇ -D- Thiogalactoside
  • the crude enzyme solution of prostaglandin H synthase and prostaglandin E synthetase the volume ratio of the crude enzyme solution to the substrate solution is 1:1, and the reaction solution is obtained by stirring and aeration reaction at 26°C for 4 hours.
  • the reaction solution is detected by HPLC.
  • Prostaglandin E2 The yield is 0.85g/L.
  • concentration of phosphate buffer can be replaced with any value in 50-100mM; the pH of phosphate buffer can be replaced with any value in pH 7.8-8.1; the reaction temperature of 26°C can be replaced with 25-28°C Any value of; reaction time 4h can be replaced with any value in 2-6h.
  • the flow rate will gradually change from 1.0ml/min to 1.2ml/min; at 12.01-19.00min, the flow rate will be 1.2ml/min.
  • the specific operation is: take 0.8ml of the reaction solution obtained in Example 5 into a 2ml centrifuge tube, add 0.8ml of acetonitrile, centrifuge, pass through a 0.22 ⁇ m filter membrane, take the filtrate for sample injection, record the chromatogram, the typical chromatogram is shown in Figure 2.
  • 2 is the HPLC detection spectrum of the reaction solution prepared in Example 5. It can be seen from Figure 2 that the retention time of prostaglandin E2 is 5.24 min, and the retention time of arachidonic acid is 14.46 min.
  • Example 7 Whole cells catalyze arachidonic acid to produce prostaglandin E2
  • IPTG isopropyl- ⁇ -D -Thiogalactoside
  • the final concentration of IPTG is 0.1mM
  • the final concentration of arachidonic acid is 100 ⁇ M
  • the induction culture is continued for 20h at 20°C, centrifuged at 7000rpm for 30min, collect the supernatant, and use ethyl acetate Ester extraction takes the organic phase, rotary steams until the ethyl acetate is completely volatilized, then add 1.5ml of acetonitrile to fully dissolve it, filter with an organic phase filter, and perform HPLC detection (the chromatographic conditions of HPLC are the same as in Example 6), the purity of prostaglandin E2 It is 2.547%.

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Abstract

提供一种重组大肠杆菌及其在生产前列腺素E2中的应用，所述重组大肠杆菌过表达前列腺素H合成酶的编码基因和前列腺素E合成酶的编码基因。还提供了一种生产前列腺素E2的方法，以花生四烯酸作为底物利用上述重组大肠杆菌生产前列腺素E2。

Description

[根据细则91更正 17.07.2020]　一种重组大肠杆菌及其应用，生产前列腺素E2的方法 技术领域
[根据细则91更正 17.07.2020]　
本发明涉及生物技术领域，尤其涉及一种重组大肠杆菌及其应用，生产前列腺素E2的方法。
背景技术
前列腺素E2(prostaglandin E2，PGE2)是花生四烯酸环氧合酶代谢产物，为二十碳不饱和脂肪酸，是前列腺素(prostaglandin，PG)的一种，分子式为C 20H 32O 5，分子量为352，呈白色晶体，熔点68-69℃，溶于乙酸乙酯、丙酮、乙醚甲醇及乙醇等有机溶剂，不溶于水。PGE2是一种重要的细胞生长和调节因子，其生理功能主要表现在：扩张血管，增加器官血流量，降低血管外周阻力，降低血压；使支气管平滑肌舒张，降低通气阻力；抑制胃酸分泌，促进胃肠平滑肌蠕动；具有免疫抑制和抗炎作用。
目前PGE2的主要生产方法为利用羊精囊获得的相关酶的混悬液与花生四烯酸混合孵育。此工艺材料获得不便，成本高，操作繁琐，转化率低，杂质和副产物较多，后续纯化复杂。
另外，已有报道在大肠杆菌中表达PGHS并催化花生四烯酸形成前列腺素H2，然后进一步利用SnCl 2对其进行化学还原，最终得到由前列腺素F2α、前列腺素D2和前列腺素E2组成的混合产物(Jan Christopher Guder,et al.BiotechnolLetters,2014,36:2193–2198)。因此有必要开发一种新的PGE2制备方法。
发明内容
[根据细则91更正 17.07.2020]　
基于背景技术存在的技术问题，本发明提出了一种重组大肠杆菌及其应用， 生产前列腺素E2的方法，本发明构建得到能够表达前列腺素H合成酶和前列腺素E2合成酶的大肠杆菌菌株，利用所表达的酶能够直接催化花生四烯酸转化为前列腺素E2。
本发明提出的一种重组大肠杆菌(Escherichia coli)，所述重组大肠杆菌(Escherichia coli)过表达前列腺素H合成酶的编码基因和前列腺素E合成酶的编码基因。
优选地，前列腺素H合成酶的编码基因的核苷酸序列如SEQ ID No.1所示。
优选地，前列腺素E合成酶的编码基因的核苷酸序列如SEQ ID No.2所示或如SEQ ID No.3所示。
优选地，所述重组大肠杆菌(Escherichia coli)BL21(DE3)/pET28a-PGHS-mPGES1，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19049。
优选地，所述重组大肠杆菌(Escherichia coli)BL21(DE3)/pET28a-PGHS-mPGES2，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19050。
本发明还提出了上述重组大肠杆菌(Escherichia coli)在生产前列腺素E2中的应用。
本发明还提出了一种生产前列腺素E2的方法，以花生四烯酸作为底物利用上述重组大肠杆菌(Escherichia coli)生产前列腺素E2。
有益效果：
本发明构建得到能够表达前列腺素H合成酶和前列腺素E2合成酶的大肠杆菌菌株，利用所表达的酶能够直接催化花生四烯酸转化为前列腺素E2。相较于 传统的前列腺素E2制备方法，本发明的制备方法简单、效率高。与文献中报道的利用一种酶制备前列腺素相比，本发明同时利用前列腺素H合成酶和前列腺素E2合成酶，能够直接的催化花生四烯酸产生PGE2。
生物保藏说明
重组大肠杆菌(Escherichia coli)BL21(DE3)/pET28a-PGHS-mPGES1，于2019年11月28日，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，地址为中国，北京，保藏编号为CGMCC No.19049。
重组大肠杆菌(Escherichia coli)BL21(DE3)/pET28a-PGHS-mPGES2，于2019年11月28日，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，地址为中国，北京，保藏编号为CGMCC No.19050。
附图说明
图1为花生四烯酸生成前列腺素E2的合成路线图，其中，arachidonic acid为花生四烯酸，prostaglandin H2为前列腺素H2，prostaglandin E2为前列腺素E2，PGHS为前列腺素H合成酶，PGES为前列腺素E合成酶。
图2为实施例5制得的反应液的HPLC检测图谱。
图3为共表达质粒pET28a-PGHS-mPGES1和共表达质粒pET28a-PGHS-mPGES2的PCR检测胶图，其中，1为共表达质粒pET28a-PGHS-mPGES1，2为共表达质粒pET28a-PGHS-mPGES2，M为Marker。
具体实施方式
下面，通过具体实施例对本发明的技术方案进行详细说明。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
质粒和菌株：大肠杆菌TG1菌株(武汉淼灵生物科技有限公司，货号P1468)；大肠杆菌BL21(DE3)菌株(武汉淼灵生物科技有限公司，货号P1472)；表达载体pET28a(武汉淼灵生物科技有限公司，货号P0023)。
试剂及仪器：NcoI、BamHI、HindIII和EcoRI等限制性内切酶(赛默飞世尔科技(中国)有限公司)，胶回收纯化试剂盒(上海捷瑞生物工程有限公司，货号GK2043-200)，GBclonart无缝克隆试剂盒(苏州神洲基因有限公司，货号GB2001-48)，JY92-IIN型超声波细胞粉碎仪(宁波新艺超声设备有限公司)。
实施例1 前列腺素H合成酶基因和前列腺素E合成酶基因合成
本发明人根据NCBI网站(https://www.ncbi.nlm.nih.gov/)公布的江蓠属植物真江蓠的前列腺素H合成酶、人的前列腺素E合成酶、食蟹猴的前列腺素E合成酶的核苷酸序列，针对大肠杆菌表达系统进行密码子优化获得相应的优化后核苷酸序列，并委托苏州金唯智生物科技有限公司进行基因合成；优化后核苷酸序列分别为：
优化后的真江蓠的前列腺素H合成酶的编码基因(简称为PGHS编码基因)的核苷酸序列如SEQ ID No.1所示，其氨基酸序列为：
Figure PCTCN2020097211-appb-000001
优化后的人的前列腺素E合成酶的编码基因(简称为mPGES1编码基因)的核苷酸序列如SEQ ID No.2所示，其氨基酸序列为：
Figure PCTCN2020097211-appb-000002
优化后的食蟹猴的前列腺素E合成酶的编码基因(简称为mPGES2编码基因)的核苷酸序列如SEQ ID No.3所示，其氨基酸序列为：
Figure PCTCN2020097211-appb-000003
实施例2 前列腺素H合成酶PGHS和前列腺素E合成酶PGES表达质粒的构建
1质粒pET28a-PGHS的构建
1.1以实施例1中合成的PGHS编码基因为模板，利用PGHS-F/PGHS-R引物对进行扩增，得到PGHS片段，大小1738bp；
1.2取表达载体pET28a，使用限制性内切酶NcoI和BamHI进行酶切得到载体片段1；用胶回收纯化试剂盒对载体片段1、PGHS片段进行胶回收纯化后，用GBclonart无缝克隆试剂盒无缝克隆重组，转化至大肠杆菌TG1菌株感受态细胞，最终构建得到质粒pET28a-PGHS；
2共表达质粒pET28a-PGHS-mPGES1、共表达质粒pET28a-PGHS-mPGES2的构建
2.1以实施例1中合成的mPGES1编码基因为模板，利用 mPGES1-F/mPGES1-R引物对进行扩增，得到mPGES1片段，大小525bp；
2.2以实施例1中合成的mPGES2编码基因为模板，利用mPGES2-F/mPGES2-R引物对进行扩增，得到mPGES2片段，大小960bp；
2.3取质粒pET28a-PGHS，使用限制性内切酶EcoRI和HindIII进行酶切得到载体片段2；用胶回收纯化试剂盒对载体片段2、mPGES1片段、mPGES2片段进行胶回收纯化后，通过无缝克隆技术构建得到共表达质粒pET28a-PGHS-mPGES1和共表达质粒pET28a-PGHS-mPGES2；
2.4利用引物对pET28a-YZ-F/pET28a-YZ-R对共表达质粒pET28a-PGHS-mPGES1和共表达质粒pET28a-PGHS-mPGES2进行PCR验证，结果参见图3，图3为共表达质粒pET28a-PGHS-mPGES1和共表达质粒pET28a-PGHS-mPGES2的PCR检测胶图，其中，1为共表达质粒pET28a-PGHS-mPGES1，2为共表达质粒pET28a-PGHS-mPGES2，M为Marker；由图3可以看出共表达质粒pET28a-PGHS-mPGES1的PCR验证条带大小为2543bp，条带大小正确；共表达质粒pET28a-PGHS-mPGES2的PCR验证条带大小为2967bp，条带大小正确；
2.5将共表达质粒pET28a-PGHS-mPGES1和共表达质粒pET28a-PGHS-mPGES2送苏州金唯智生物科技有限公司进行测序检测，测序正确后备用；
表1 实施例2中引物对的序列
Figure PCTCN2020097211-appb-000004
Figure PCTCN2020097211-appb-000005
实施例3 重组菌株的获得
1制备BL21(DE3)感受态细胞：
从甘油管挑取一环大肠杆菌BL21(DE3)在LB平板划线纯化，置于37℃培养箱，培养16h，挑取单菌落接种到装有4mL LB培养基(胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L)的试管中，于37℃、250rpm条件下培养16h，然后按照2％转接量转接到装有50mL LB培养基的摇瓶中，37℃培养至OD 600＝0.8，收集菌体置于冰上预冷，离心去上清后用冰浴的0.1M CaCl 2溶液洗涤菌体两次，最后用2ml预冷的含15％甘油的0.1M CaCl 2溶液重悬菌体，分装后放置冰上即得大肠杆菌BL21(DE3)感受态细胞；
2制备重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES1：
取100ng共表达质粒pET28a-PGHS-mPGES1，加入至100μL大肠杆菌BL21(DE3)感受态细胞中，冰上放置片刻，42℃水浴2min，迅速置于冰上冷却3-5min，加入1ml LB液体培养基混匀，然后于37℃，250rpm振荡复苏培养1h得到培养液，取100-300μL培养液涂布于含50μg/ml卡那霉素的LB平板上，37℃培养16h，最终得到重组大肠杆菌(Escherichia coli)菌株BL21(DE3) /pET28a-PGHS-mPGES1，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19049；
3制备重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES2：
取共表达质粒pET28a-PGHS-mPGES2，按照实施例3中2步骤的操作制得重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES2，保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19050。
实施例4 摇瓶制备前列腺素H合成酶和前列腺素E合成酶
1培养重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES1：
将实施例3得到的重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES1接种于含有50μg/ml卡那霉素的LB培养基中，置37℃摇床中，250rpm培养20h得到活化菌种；取活化菌种按照0.8％接种量接种至50mL含50μg/ml卡那霉素的LB培养基，37℃生长到OD 600＝0.6-0.8，添加异丙基-β-D-硫代半乳糖苷(IPTG)，使IPTG的终浓度为0.2mM，于30℃诱导培养20h后，离心收集菌体，用50mM、pH8.0的磷酸缓冲液重悬菌体至5ml得到培养菌液；
2培养重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES2：
取实施例3得到的重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES2，按照实施例4中1步骤的操作制得培养菌液；
3制备含前列腺素H合成酶和前列腺素E合成酶的粗酶液：
使用JY92-IIN型超声波细胞粉碎仪分别对实施例4中1步骤和2步骤制得 的培养菌液进行超声破碎得到两种破胞液；两种破胞液分别离心取上清得到两种含前列腺素H合成酶和前列腺素E合成酶的粗酶液，其中，超声破碎条件为：以每超声2s后间隙4s的模式循环超声，超声总时长为10min，超声温度为0℃，超声功率为40％。
实施例5 酶催化法制备前列腺素E2
取花生四烯酸(终浓度为1g/L)溶解到50mM，pH 8.0的磷酸缓冲液(称量0.2067g NaH 2PO 4·2H 2O和8.4757g Na 2HPO 4·12H 2O加去离子水溶解并定容至500ml容量瓶中)中，然后添加终浓度为0.75mg/ml的谷胱甘肽(简称GSH)得到底物溶液，再向底物溶液中加入实施例4制得的含前列腺素H合成酶和前列腺素E合成酶的粗酶液，粗酶液与底物溶液的体积比为1：1，于26℃搅拌通气反应4h得到反应液，HPLC检测反应液，前列腺素E2产量为0.85g/L。
需要说明的是，磷酸缓冲液的浓度可以替换为50-100mM中的任意值；磷酸缓冲液的pH可替换为pH 7.8-8.1中的任意值；反应温度26℃可替换为25-28℃中的任意值；反应时间4h可替换为2-6h中的任意值。
实施例6 HPLC检测花生四烯酸和前列腺素E2
HPLC的色谱条件为：色谱柱为Agilent XDB C18(4.6×150mm，5μm)，紫外检测器检测，波长＝210nm，进样量＝10μL，柱温＝30℃，初始流速＝1.0ml/min；流动相A为乙腈，流动相B为pH＝4.0，浓度为10mM的乙酸铵水溶液，梯度洗脱，洗脱程序为：0-0.01min时，流动相A与流动相B的体积比为30：70；0.01-12.00min时，流动相A和流动相B的体积比由30：70匀速渐变为95：5；12.00-14.00min时，流动相A和流动相B的体积比为95：5；14.00-15.00min时，流动相A和流动相B的体积比由95：5匀速渐变为30：70；15.00-19.00min时， 流动相A与流动相B的体积比为30：70；
12.00-12.01min时，流速由1.0ml/min匀速渐变为1.2ml/min；12.01-19.00min时，流速为1.2ml/min。
具体操作为：取实施例5得到的反应液0.8ml于2ml离心管中，加入0.8ml乙腈，离心，过0.22μm滤膜，取滤液进样，记录色谱图，典型色谱图参见图2，图2为实施例5制得的反应液的HPLC检测图谱，由图2可以看出前列腺素E2的保留时间为5.24min，花生四烯酸的保留时间为14.46min。
实施例7 全细胞催化花生四烯酸生成前列腺素E2
1用重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES1全细胞催化生产前列腺素E2：
将实施例3得到的重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES1接种于含有50μg/ml卡那霉素的液态LB培养基中，置37℃摇床中，250rpm培养20h得到活化菌种；取活化菌种按照0.8％接种量接种至400mL含50μg/ml卡那霉素的LB培养基，37℃生长到OD 600＝0.6-0.8，添加异丙基-β-D-硫代半乳糖苷(IPTG)和花生四烯酸，IPTG终浓度为0.1mM，花生四烯酸终浓度为100μM，20℃继续诱导培养20h后，7000rpm离心30min，收集上清，用乙酸乙酯萃取取有机相，旋蒸至乙酸乙酯完全挥发，再加入1.5ml乙腈充分溶解，用有机相滤膜过滤后，进行HPLC检测(HPLC的色谱条件同实施例6)，前列腺素E2的纯度为2.547％。
2用重组大肠杆菌(Escherichia coli)菌株BL21(DE3)/pET28a-PGHS-mPGES2全细胞催化生产前列腺素E2：
取实施例3得到的重组大肠杆菌(Escherichia coli)菌株 BL21(DE3)/pET28a-PGHS-mPGES2，按照实施例7中1步骤的操作制得前列腺素E2，其纯度为1.945％。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。

Claims (7)

  1. 一种重组大肠杆菌(Escherichia coli)，其特征在于，所述重组大肠杆菌(Escherichia coli)过表达前列腺素H合成酶的编码基因和前列腺素E合成酶的编码基因。
  2. 根据权利要求1所述重组大肠杆菌(Escherichia coli)，其特征在于，前列腺素H合成酶的编码基因的核苷酸序列如SEQ ID No.1所示。
  3. 根据权利要求1或2所述重组大肠杆菌(Escherichia coli)，其特征在于，前列腺素E合成酶的编码基因的核苷酸序列如SEQ ID No.2所示或如SEQ ID No.3所示。
  4. 根据权利要求1-3任一项所述重组大肠杆菌(Escherichia coli)，其特征在于，所述重组大肠杆菌(Escherichia coli)BL21(DE3)/pET28a-PGHS-mPGES1保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19049。
  5. 根据权利要求1-3任一项所述重组大肠杆菌(Escherichia coli)，其特征在于，所述重组大肠杆菌(Escherichia coli)BL21(DE3)/pET28a-PGHS-mPGES2保藏在中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏编号为CGMCC No.19050。
  6. 一种如权利要求1-5任一项所述重组大肠杆菌(Escherichia coli)在生产前列腺素E2中的应用。
  7. 一种生产前列腺素E2的方法，其特征在于，以花生四烯酸作为底物利用权利要求1-5任一项所述重组大肠杆菌(Escherichia coli)生产前列腺素E2。
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