CN117844791A - Oxidation squalene cyclase gene NiOSC5 and its coded product heterocycle triterpene - Google Patents
Oxidation squalene cyclase gene NiOSC5 and its coded product heterocycle triterpene Download PDFInfo
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- CN117844791A CN117844791A CN202410031796.0A CN202410031796A CN117844791A CN 117844791 A CN117844791 A CN 117844791A CN 202410031796 A CN202410031796 A CN 202410031796A CN 117844791 A CN117844791 A CN 117844791A
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- 229960000723 ampicillin Drugs 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 108010031959 cognin Proteins 0.000 description 1
- 238000002856 computational phylogenetic analysis Methods 0.000 description 1
- 239000006184 cosolvent Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- OORMXZNMRWBSTK-LGFJJATJSA-N dammarane Chemical compound C1CCC(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@H](C)CCCC(C)C)[C@H]4CC[C@@H]3[C@]21C OORMXZNMRWBSTK-LGFJJATJSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 1
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
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- 239000007903 gelatin capsule Substances 0.000 description 1
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- 125000003147 glycosyl group Chemical group 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
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- 238000000338 in vitro Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 102000008554 lanosterol synthase activity proteins Human genes 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- JURZHOVRCOWZFN-UHFFFAOYSA-N notoginsenoside R1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5C(CC34C)OC6OC(COC7OCC(O)C(O)C7O)C(O)C(O)C6O)C JURZHOVRCOWZFN-UHFFFAOYSA-N 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
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- 239000002243 precursor Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- NWMIYTWHUDFRPL-UHFFFAOYSA-N sapogenin Natural products COC(=O)C1(CO)C(O)CCC2(C)C1CCC3(C)C2CC=C4C5C(C)(O)C(C)CCC5(CCC34C)C(=O)O NWMIYTWHUDFRPL-UHFFFAOYSA-N 0.000 description 1
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- 229940031439 squalene Drugs 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 150000003521 tetracyclic triterpenoids Chemical class 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- LEIMLDGFXIOXMT-UHFFFAOYSA-N trimethylsilyl cyanide Chemical compound C[Si](C)(C)C#N LEIMLDGFXIOXMT-UHFFFAOYSA-N 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention discloses an oxidation squalene cyclase NiOSC5 and its coding product (20)S,24S) -20, 24-epoxydammarenediol, (20)S,24R) The application of 20, 24-epoxy dammarenediol belongs to the field of synthetic biology and natural medicine technology. The invention is prepared from cucurbitaceae plant cucurbita pepoNeoalsomitra integrifoliola) The method comprises the steps of starting, cloning and functionally identifying a functional hybrid triterpene synthase NiOSC5 coding gene, cloning the gene, connecting the gene with an expression vector pYES2 after the nucleotide sequence is shown as a Seq ID No.2, constructing a recombinant plasmid of pYES2-NiOSC5, and converting the recombinant plasmid into saccharomyces cerevisiae to construct engineering saccharomycetes, thereby realizing the heterogenous efficient cyclization of the saccharomyces cerevisiae to produce heterocyclic triterpene by 2,3;22, 23-bisepoxysqualene. The NiOSC5 product heterocyclic triterpene provided by the invention can be used for preparing medicines for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation and cancers and protecting nerves.
Description
Technical Field
The invention belongs to the technical fields of synthetic biology and natural medicines. In particular to an oxidation squalene cyclase NiOSC5 and a coded product (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol thereof, and application of the product in preparing medicines for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation and cancer and protecting nerves.
Background
The octocrocetin type ginsenoside (Ocotillol type ginsenosides) is tetracyclic triterpenoid type saponin with tetrahydrofuran ring at side chain, and can be divided into (20S, 24S) according to different configurations of C-20 and C-24; (20S, 24R); (20R, 24S); four types (20R, 24R). Modern pharmacological studies show that the octopus type ginsenoside and aglycone thereof have various pharmacological effects, mainly including antiarrhythmic, antibacterial, anti-inflammatory, anticancer, nerve protecting and the like. In recent years, in vivo and in vitro metabolism studies of secondary ginsenoside (ginsenoside Rg3, rh2, rg2 and Rh 1) and aglycones (PPD and PPT) of C-20 non-linked glycosyl indicate that the octopus type metabolites of C-20 and C-24 epoxy can be truly effective components for in vivo functions.
The octopus type ginsenoside is mainly present in Panax ginseng (Panax vietnamensis), panax quinquefolium (P.quinquefoil), panax quinquefolium (P.japonica. Major), panax japonicus (P.japonica), and Gynostemma pentaphyllum (Gynostemma pentaphyllum) belonging to Cucurbitaceae. Since naturally occurring octopus type ginsenosides are less in variety and mainly have (20S, 24S) configuration and are low in content, researchers convert the ginsenosides into (20R, 24S), (20S, 24R) and (20R, 24R) configuration octopus type compounds through semi-synthetic means. The dammarane type ginsenoside with anticancer activity, which is unique to heterologous synthesized ginseng (Panax l.), is one of the hot spots in research on synthetic biology. Therefore, the method utilizes means of synthetic biology and metabolic engineering to excavate key genes in the biological synthesis path of the okadayl alcohol type saponin, and realizes the efficient heterologous biosynthesis of the okadayl alcohol type saponin, in particular to the key precursor skeleton epoxy dammarenediol.
The cucumber (Neoalsomitra integrifoliola (cognin.) Hutch) is a plant of genus Coptis (Neoalsonmitra) of family Cucurbitaceae, and is grown in rainforest or secondary forest at 550-840 m altitude. The genus plant is about 22 species, distributed in india to brinesian and australia, and there are the n.integrifoliola (cogni.) Hutch and the n.clavigera (wall.) hutch.2 species in our country, the former species distributed in the Yunnan and the like, and the latter species produced in the southeast part of the tibetan (ink drop). The clavulanate saponins are rich in dammarane saponins, and are a new resource plant which contains high ginsenoside content in cucurbitaceae plants, wherein the content of the triterpenoid A of the octopus loltype saponins reaches more than 3%, so that the key gene research of the synthesis of the octopus loltype saponins in the clavulanate is very necessary, particularly the key gene involved in the synthesis of the rare (20R) and (24R) configuration epoxy dammarenediol is found, and the rare compounds are synthesized in a heterogeneous manner.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides an oxidation squalene cyclase gene NiOSC5 and a coding product thereof, wherein the gene is a key enzyme gene participating in the synthesis of triterpenoid sapogenin of the clavulanate, can be used as a biosynthesis regulatory gene of (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol and applied to the preparation of (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol. Thus, a novel method for biosynthesis of (20S, 24S) -20, 24-epoxydammarenediol and (20S, 24R) -20, 24-epoxydammarenediol is provided.
In order to achieve the above object of the present invention, the present invention adopts the following technical scheme:
an oxidosqualene cyclase nioc 5, which is:
(1) A protein comprising the amino acid sequence shown in Seq ID No. 1;
(2) The amino acid sequence shown in the Seq ID No.1 is a derivative protein with the same function by substituting and/or deleting and/or adding one or more amino acid residues.
The coding gene of the oxidation squalene cyclase NiOSC5 is as follows:
(a) A nucleotide sequence shown as Seq ID No.2;
(b) The nucleotide sequence shown in Seq ID No.2 is a nucleotide sequence which is substituted and/or deleted and/or added with one or several nucleotides and expresses the same functional protein.
A recombinant vector containing the gene encoding the oxidation squalene cyclase NiOSC 5.
Recombinant bacteria containing the gene encoding the oxidation squalene cyclase NiOSC 5.
The oxidation squalene cyclase NiOSC5 or the coding gene thereof is applied to the preparation of recombinant vectors, expression cassettes, transgenic cell lines and recombinant bacteria containing (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The application of the oxidation squalene cyclase NiOSC5 or the coding gene thereof in preparing fermentation liquor containing the compound (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol is characterized in that the application is as follows: constructing an expression vector containing the coding gene, transforming the recombinant vector into saccharomyces cerevisiae cells, and fermenting and culturing the obtained genetically engineered saccharomycetes to obtain fermentation liquor containing (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The application of the oxidation squalene cyclase NiOSC5 or the coding gene thereof in synthesizing or preparing the compound (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol,
the oxidation squalene cyclase NiOSC5 or the coding gene thereof is applied to the preparation of antiarrhythmic, antiarrhythmic injury, antibacterial, anti-inflammatory, anticancer and nerve-protecting drugs.
A process for the preparation of the compound (20 s,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol, comprising the steps of:
constructing an expression vector containing a coding gene for coding an oxidation squalene cyclase NiOSC5, converting the recombinant vector into saccharomyces cerevisiae, fermenting and culturing the obtained genetically engineered saccharomyces cerevisiae to obtain a fermentation liquor containing (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol, extracting the fermentation liquor by petroleum ether or ethyl acetate or dichloromethane or chloroform to obtain an extract containing (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol, separating and purifying the extract by a silica gel column chromatography method and a high performance liquid chromatography method to finally obtain the compound (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol shown in the following structural formula,
the compound (20S, 24S) -20, 24-epoxy dammarenediol and the application of the (20S, 24R) -20, 24-epoxy dammarenediol in preparing medicaments for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation, cancer and nerve.
The Open Reading Frame (ORF) of the gene encoding the oxidation squalene cyclase gene NiOSC5 provided by the invention is 2295bp (Seq ID No. 2), 764 amino acids (Seq ID No. 1) are encoded, and the gene is placed in NCBI for BLASTN analysis and comparison, so that the homology with SgBAS1 of Momordica grosvenori (Siraitia grosvenorii) of Cucurbitaceae is 76%.
The coding gene of the oxidation squalene cyclase NiOSC5 provided by the invention is a gene cloned from the cucumis metuliferus (Neoalsomitra integrifoliola), and can cyclize 2,3;22, 23-bis-epoxy squalene forming compound (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol. The NiOSC5 is cloned from the plant for the first time, and the discovery of the oxidation squalene cyclase NiOSC5 and the coding gene thereof enriches the diversity of enzymes.
The invention clones and functionally identifies the oxidation squalene cyclase NiOSC5 from cucurbitaceae plant Alternaria verrucosa, and uses saccharomyces cerevisiae to generate (20S, 24S) -20, 24-epoxy dammarenediol and the application of (20S, 24R) -20, 24-epoxy dammarenediol.
The invention discloses an oxidation squalene cyclase NiOSC5 and an application of a coded product (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol thereof, belonging to the technical fields of synthetic biology and natural medicines. According to the invention, starting from cucurbitaceae plant mallow (Neoalsomitra integrifoliola), a functional hybrid triterpene synthase NiOSC5 coding gene is cloned and functionally identified, and can cyclize 2,3; the 22, 23-double epoxy squalene generates heterocyclic triterpene (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol, the nucleotide sequence of which is shown as SEQ ID No.2, and the gene engineering cell is connected with an expression vector pYES2 after gene cloning, so as to construct a recombinant plasmid of pYES2-NiOSC5, and then the recombinant plasmid is transformed into saccharomyces cerevisiae to construct engineering saccharomycetes, thereby realizing the heterologous efficient synthesis of the heterocyclic triterpene (20S, 24S) -20, 24-epoxy dammarenediol by the saccharomyces cerevisiae, and the gene engineering cell constructed by the invention is safe and stable, has short production period and shows great value in application and development. The NiOSC5 product (20S, 24S) -20, 24-epoxy dammarenediol provided by the invention is applied to the preparation of medicines for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation and cancers and protecting nerves.
Drawings
FIG. 1 is a three-dimensional structure prediction diagram of the oxidosqualene cyclase NiOSC5 in example 1.
FIG. 2 shows the analysis of the expression level of the oxidosqualene cyclase NiOSC5 in roots, stems, leaves and flowers in example 2.
FIG. 3 is a total ion figure of GC-MS analysis of an engineered yeast extract expressing the oxidosqualene cyclase NiOSC5 in example 2.
FIG. 4 is a mass spectrum of the oxidation squalene cyclase NiOSC5 catalysis product (20S, 24S) -20, 24-epoxydammarenediol and (20S, 24R) -20, 24-epoxydammarenediol in example 2.
FIG. 5 is a chromatogram of the (20S, 24S) -20, 24-epoxydammarenediol standard of example 2 (20S, 24R) -20, 24-epoxydammarenediol.
FIG. 6 is a standard quality spectrum of (20S, 24S) -20, 24-epoxydammarenediol and (20S, 24R) -20, 24-epoxydammarenediol in example 2.
FIG. 7 is a schematic diagram showing the construction of recombinant expression plasmid pYES2-NiOSC5 in example 2.
Detailed description of the preferred embodiments
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise indicated, the examples were conducted under conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or under conditions recommended by the manufacturer's instructions.
Example 1
Cloning of the Oxidation squalene cyclase Gene in Calophyllum Instrongylus.
1. According to the sequenced data of the gloriope clavatus transcriptome, the candidate oxidation squalene cyclase gene cDNA sequence in the gloriope clavatus saponin synthesis pathway is obtained through operations such as splicing, annotation, screening and the like.
2. Designing a primer of the candidate oxidation squalene cyclase gene, wherein the primer sequence is as follows:
forward primer (NiOSC 5-F):
gggaatattaagcttggtaccATGTGGCGGCTTAAGATTGCAG，ID NO.3；
reverse primer (NiOSC 5-R):
ccctctagatgcatgctcgagTTAAAATACAGAGGAAGTAGTTGGCAATTTGAC，IDNO.4；
primers were synthesized by Kunming division, inc. of Biotech, beijing.
3. Individual tissues (root, stem, leaf and flower) of the plant of the Hamamelis mollis were taken, total RNA was extracted using TRIzol kit (Invitrogen, carlsbad, calif., USA) using PrimeScript TM Reverse transcription was performed using RT kit (Takara, china) to obtain cDNA, and the gene sequence of NiOSC5 was amplified using the cDNA as a template.
4. The amplified product agarose gel electrophoresis shows a specific band at about 2.3kb, the target band is subjected to gel cutting recovery, the gel recovery product is connected to a vector pYES2, and E.coli DH5 alpha is transformed, positive clones are selected for sequencing (Kunming division of Beijing qing biological science and technology Co., ltd.), and NiOSC5 gene clones with correct sequences are selected and saved for the construction of subsequent expression vectors.
Sequencing to obtain the triterpenoid saponin anabolism pathway oxidation squalene cyclase gene NiOSC5 with the length of 2295bp and nucleotide sequence as ID No.2; encodes 764 amino acids, and the amino acid sequence is ID NO.1.
By multiple sequence alignment and phylogenetic tree analysis of NiOSC5 with the identified OSCs, it was shown that NiOSC5 has the highly conserved domains QW and DCTAE of the OSCs gene family.
Example 2
Eukaryotic expression and functional analysis of NiOSC5 gene.
NiOSC5 functional preliminary analysis.
Extracting RNA of root, stem, leaf and flower of Coptis chinensis respectively, referring to PrimeScript TM RT kit (Takara, china) was reverse transcribed into cDNA using 2X ChamQ Universal SYBR qPCR Master Mix (Vazyme) at Applied Biosystems QuantStudio TM Real-time fluorescent quantitative PCR amplification was performed on platform 5 (Life Technologies).
Forward primer (NiOSC 5-qRT-F): GCTGGTTTGCTATTGGTGGT, ID NO.5;
reverse primer (NiOSC 5-qRT-R): ACCGGTTTCCTTCAAGAGGT, ID NO.6.
From the results of the real-time fluorescent quantitative PCR analysis (FIG. 2), it was estimated that NiOSC5 was expressed in the highest amount in leaves and flowers, and that NiOSC5 was probably involved in biosynthesis of clavulanic saponin in the leaves.
2. Construction of a Yeast expression vector.
ERG 7-deficient Saccharomyces cerevisiae mutant GIL77, which lacks lanosterol synthase gene, can endogenously accumulate 2,3;22, 23-bisoxy squalene, 2,3;22, 23-bisoxasqualene can be used as a substrate for functional verification of NiOSC 5.
By analyzing the coding sequence and the cleavage site of the gene NiOSC5, primers with KpnI and XhoI cleavage sites are designed as follows, and the full-length ORF of NiOSC5 is amplified.
After sequencing and verifying the amplification product, connecting a target gene NiOSC5 to a yeast expression vector pYES2 by a homologous recombination method, screening a transformed colony by using an LB solid plate containing ampicillin (100 mu g/mL), selecting a monoclonal for verification, sequencing and verifying correctness to obtain a pYES2-NiOSC5 vector, inoculating the verified correct monoclonal into 5mL of LB liquid culture with the same resistance, fermenting and culturing, and extracting pYES2-NiOSC5 plasmid.
3. Yeast transformation.
The pYES2-NiOSC5 plasmid was transferred into Saccharomyces cerevisiae strain GIL77 by lithium acetate transformation, and an empty pYES2 transformation control group was set. Positive clones GIL77-pYES2-NiOSC5 and GIL77-pYES2 were selected by colony PCR.
4. Induction of expression and incubation.
Positive yeast monoclonal GIL77-pYES2-NiOSC5 and GIL77-pYES2 were picked separately and inoculated in 50ml of uracil-free synthetic complete medium [ SC-U; comprises ergosterol (20. Mu.g/ml), tween 80 (5 mg/ml) and hemin (13. Mu.g/ml), and is then cultured at 30℃for 2 days with shaking at 200 rpm.
Yeast cells were harvested, resuspended in 50mL of SC-U medium containing 2% galactose and induced by shaking at 200rpm for 2 days at 30 ℃.
After induction for 2 days, the cells were harvested and resuspended in the same volume of 0.1M potassium phosphate buffer (pH 7.0; 2% glucose and hemin (13. Mu.g/ml) were added and incubated at 30℃for 12 hours with shaking at 200 rpm.
5. And (5) extracting and identifying a catalytic product.
After 12 hours of incubation, the cells were refluxed with the same volume of saponification reagent (20% KOH/50% EtOH) for 10 minutes at 92℃and then extracted twice with the same volume of petroleum ether, the extracts were combined, concentrated to dryness under reduced pressure and the residue was derivatised with 200. Mu.l of cyano trimethylsilane at 65℃for 30 minutes.
The derived products are subjected to GC-MS analysis, and a catalytic group containing the NiOSC5 gene recombinant expression vector pYES2-NiOSC5 shows a specific peak compared with a control group containing no-load pYES2, namely new substances are generated, and the specific products are identified as (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The experiment proves that the NiOSC5 gene participates in the biosynthesis of triterpenoid saponins of the clava malleata, and the NiOSC5 gene can be used for regulating the biosynthesis of (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol in the clava malleata, so that the heterologous synthesis of (20S, 24S) -20, 24-epoxy dammarenediol is realized.
Example 3
Preparation of the compound (20S, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol.
RNA of Hakka Swinhonis was extracted, and reference was made to PrimeScript TM RT kit (Takara, china) is reverse transcribed into cDNA, the cDNA is used as template to amplify the gene sequence of NiOSC5, the amplified product agarose gel electrophoresis shows specific band at about 2.3kb, the target band is cut and recovered, the recovered product is connected to vector pYES2, and E.coli DH5 alpha is transformed, positive clone is selected for sequencing (Kunming division Co., ltd. Of Beijing family biotechnology), the NiOSC5 gene with correct sequencing is selected, the target gene NiOSC5 is connected to yeast expression vector pYES2 by homologous recombination method, pYES2-NiOSC5 vector is obtained, and sequencing verifies correctness. Extracting pYES2-NiOSC5 plasmid, introducing the pYES2-NiOSC5 plasmid into Saccharomyces cerevisiae GIL77 strain by adopting a lithium acetate method, selecting positive clones GIL77-pYES2-NiOSC5 and GIL77-pYES2 by adopting a colony PCR method, and inoculating the positive yeast monoclonal GIL77-pYES2-NiOSC5 into 50ml of synthetic complete culture medium [ SC-U ] without uracil; comprises ergosterol (20 μg/ml), tween 80 (5 mg/ml) and hemin (13 μg/ml)]Then incubated at 30℃for 2 days. 50ml of the bacterial liquid is inoculated to 10L of synthetic complete medium [ SC-U ] without uracil; comprises ergosterol (20 μg/ml), tween 80 (5 mg/ml) and hemin (13 μg/ml)]In the following, yeast cells were harvested by shaking culture at 200rpm for 2 days at 30℃and resuspended in 10L of SC-U medium containing 2% galactose and protein expression was induced by shaking culture at 200rpm for 2 days at 30 ℃. After induction for 2 days, the yeast cells were harvested and resuspended in the same volume of 0.1M potassium phosphate buffer (pH 7.0; 2% glucose and hemin (13. Mu.g/ml) were added and incubated at 30℃for 12 hours with shaking at 200rpm, after incubation for 12 hoursThe cells were refluxed with the same volume of saponification reagent (20% KOH/50% EtOH) at 92℃for 10 minutes, then extracted 3 times with the same volume of petroleum ether, the extracts were combined, and the extracts were concentrated to dryness under reduced pressure to give an extract containing (20S, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol, weighed, stirred with 1.5 times the amount of silica gel, column chromatographed with 15 times the amount of silica gel, 6 times the column volume of petroleum ether: ethyl acetate (10:1, v/v-4:1, v/v), collecting fractions, detecting by thin layer chromatography, collecting fractions containing the mixture (20S, 24S) -20, 24-epoxydammarenediol and (20S, 24R) -20, 24-epoxydammarenediol, and concentrating under reduced pressure to dry. Then separating by preparative high performance liquid chromatography, using Agilent Zorbax SB-C18 chromatographic column (9.4X105 mm,5 μm), acetonitrile (A) -water (B) as mobile phase, 0-10min,83% A-85% A,10-18min,85% A-85% A,18-21min,85% A-100% A, running for 25min, running for 5min later, flow rate of 8ml/min, and detection wavelength of 194nm. (20S, 24S) -20, 24-epoxy dammarenediol is prepared by preparing liquid phase, and the structures of the (20S, 24R) -20, 24-epoxy dammarenediol are identified by nuclear magnetic resonance. The structural data are shown in table 1.
TABLE 1 two Compounds of the invention 1 H NMR 13 C NMR data
a Measured at 151 MHz; b measured at 600 MHz; "m" refers to overlapping or multiple repetition with other signals.
Pharmaceutical formulation examples 1-8:
in the following formulation examples, conventional reagents are selected and formulation preparation is performed according to the conventional methods, and this application example only shows that the compound of the present invention, (20 s,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol can be prepared into different formulations, and specific reagents and operations are not specifically limited:
1. mixing one or two of the compounds (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol, dissolving with absolute ethyl alcohol, adding water for injection according to a conventional method, fine filtering, packaging and sterilizing to prepare injection, wherein the concentration of the injection is 0.5-5mg/mL.
2. Mixing one or two of (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol, dissolving in water for sterile injection, stirring to dissolve, filtering with a sterile suction filter funnel, sterile fine filtering, packaging in ampoule, freeze-drying at low temperature, and sealing to obtain powder for injection.
3. Mixing one or two of (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol, adding excipient according to the mass ratio of the compound to the excipient of 9:1, and preparing into powder.
4. Mixing one or two of the compounds (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol, adding excipient according to the mass ratio of the compound to the excipient of 5:1, granulating and tabletting.
5. Mixing one or two of (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol, and making into oral liquid by conventional oral liquid preparation method.
6. The compound (20S, 24S) -20, 24-epoxy dammarenediol and the compound (20S, 24R) -20, 24-epoxy dammarenediol are added with excipient according to the mass ratio of the compound to the excipient of 5:1, and are prepared into capsules.
7. Mixing one or two of (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol, adding excipient at a mass ratio of 5:1, and making into granule.
8. The capsule comprises the following components: compound (20 s,24 s) -20, 24-epoxydamageenediol, (20 s,24 r) -20, 24-epoxydamageenediol or a mixture of two of them 20mg, lactose 180mg, magnesium stearate 5mg.
The preparation method comprises the following steps: the compound or mixture was mixed with a cosolvent uniformly, sieved, and the resulting mixture was filled into gelatin capsules each weighing 205mg and having an active ingredient content of 20mg.
The generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. An oxidation squalene cyclase nioc 5, characterized in that it is:
(1) A protein comprising the amino acid sequence shown in Seq ID No. 1;
(2) The amino acid sequence shown in the Seq ID No.1 is a derivative protein with the same function by substituting and/or deleting and/or adding one or more amino acid residues.
2. The gene encoding the oxidosqualene cyclase nioc 5 according to claim 1, characterized in that it is:
(a) A nucleotide sequence shown as Seq ID No.2;
(b) The nucleotide sequence shown in Seq ID No.2 is a nucleotide sequence which is substituted and/or deleted and/or added with one or several nucleotides and expresses the same functional protein.
3. A recombinant vector comprising a gene encoding the oxidosqualene cyclase nioc 5 according to claim 2.
4. A recombinant bacterium comprising a gene encoding the oxidosqualene cyclase nioc 5 of claim 2.
5. Use of an oxidosqualene cyclase nioc 5 or a gene encoding it according to claim 1 or 2 for the preparation of recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria comprising (20 s,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol.
6. Use of an oxidosqualene cyclase nioc 5 or a gene encoding it according to claim 1 or 2 for the preparation of a fermentation broth comprising the compound (20 s,24 s) -20, 24-epoxydamagediol, (20 s,24 r) -20, 24-epoxydamagediol, characterized in that said use is by the following: constructing an expression vector containing the coding gene, transforming the recombinant vector into saccharomyces cerevisiae cells, and fermenting and culturing the obtained genetically engineered saccharomycetes to obtain fermentation liquor containing (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
7. Use of an oxidative squalene cyclase nioc 5 or a gene encoding it according to claim 1 or 2 for the preparation of an antiarrhythmic, antibacterial, antiinflammatory, anticancer, neuroprotective drug.
8. The use of the oxidosqualene cyclase NiOSC5 or its coding gene according to claim 1 or 2 for the synthesis or preparation of a compound (20S, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol) represented by the following structural formula,
9. a process for the preparation of a heterocyclic triterpene (20 s,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol, characterized in that it comprises the steps of:
constructing an expression vector containing a coding gene for coding an oxidation squalene cyclase NiOSC5, converting the recombinant vector into saccharomyces cerevisiae, fermenting and culturing the obtained genetically engineered saccharomyces cerevisiae to obtain a fermentation liquor containing (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol, extracting the fermentation liquor by petroleum ether or ethyl acetate or dichloromethane or chloroform to obtain an extract containing (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol, separating and purifying the extract by a silica gel column chromatography method and high performance liquid chromatography to finally obtain the compound (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol shown in the following structural formula,
10. the use of the compound (20S, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol obtained by the preparation method of claim 9 in the preparation of drugs for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation, cancer and nerves.
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