CN113186183A - Difunctional sesterterpene/diterpene synthase LcTPS2, coding gene and product and application thereof - Google Patents

Difunctional sesterterpene/diterpene synthase LcTPS2, coding gene and product and application thereof Download PDF

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CN113186183A
CN113186183A CN202110479022.0A CN202110479022A CN113186183A CN 113186183 A CN113186183 A CN 113186183A CN 202110479022 A CN202110479022 A CN 202110479022A CN 113186183 A CN113186183 A CN 113186183A
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lctps2
sesterterpene
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黎胜红
刘燕
陈月桂
凌伊
刘艳春
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Kunming Institute of Botany of CAS
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Abstract

The invention provides a bifunctional sesterterpene/diterpene synthase LcTPS2, a coding gene and application thereof, and relates to the technical field of synthetic biology and natural medicinal chemistry. The invention starts from a Labiatae plant of the Leucosceptrum micranthum, clones and functionally identifies a terpene synthase LcTPS2 coding gene for synthesizing 14-/18-membered ring terpenoid, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. The gene engineering cell constructed by the invention is safe and stable, the production period is short, the synthesized macrocyclic sesterterpene product has anti-inflammatory and immunosuppressive activity, IL-2 is secreted by a Jurkat cell line induced by combination of phytohemagglutinin and phorbol 12-tetradecanoate-13-acetate, and the CD3 and CD28 monoclonal antibodies have obvious inhibitory action on stimulation of mouse T cells to generate cell factors IFN-gamma, but have no obvious cytotoxicity on Jurkat and splenocytes.

Description

Difunctional sesterterpene/diterpene synthase LcTPS2, coding gene and product and application thereof
Technical Field
The invention relates to the technical field of synthetic biology and natural medicinal chemistry, in particular to a bifunctional sesterterpene/diterpene synthase LcTPS2, a coding gene, a product thereof and application thereof.
Background
Terpenoids are natural products with the most varieties and the most abundant chemical structure changes, are widely present in higher plants and microorganisms, and have important economic and medicinal values. Macrocyclic terpenoids containing 12-membered rings and above are a class of terpenoid natural products with unique structures, and generally have significant defense functions as well as anticancer, antileukemic, anti-inflammatory and antibacterial activities.
In recent years, researchers have intensively studied the synthetic pathways of different terpenes by adopting functional genomics and metabonomics technologies, and a large amount of data support is provided for the synthetic biology research of terpenes. At present, a yeast engineering strain with high terpene yield is constructed by a synthetic biology method, so that the high-efficiency production of various target products is realized, and the overall production level of terpenes is effectively improved. Therefore, the method for synthesizing the terpenoid by adopting the synthetic biology strategy is expected to become an effective technical means for producing the plant source terpenoid. However, natural 14-/18-membered ring sesterterpenes have not been isolated from any organisms, terpene synthase encoding genes responsible for the cyclization of 14-/18-membered ring sesterterpenes and their uses have not been reported, and their anti-inflammatory and immunosuppressive activities have not been reported in the literature.
Disclosure of Invention
In view of the above, the invention aims to provide a bifunctional sesterterpene/diterpene synthase LcTPS2, a coding gene, a product thereof and application thereof, wherein a genetic engineering cell constructed by utilizing the bifunctional sesterterpene/diterpene synthase LcTPS2 coding gene is safe and stable, the production cycle is short, and the macrocyclic sesterterpene product has anti-inflammatory and immunosuppressive activities and can be applied to preparation of anti-inflammatory drugs and immunosuppressive drugs.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a difunctional sesterterpene/diterpene synthase LcTPS2, wherein the synthase LcTPS2 is as follows:
(1) protein formed by amino acid sequence shown in SEQ ID NO. 1;
(2) and (2) the derivative protein with the same function and with one or more amino acid residues through substitution and/or deletion and/or addition of the amino acid sequence shown in SEQ ID NO. 1.
Preferably, the coding gene of the bifunctional sesterterpene/diterpene synthase LcTPS2 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 obtained by substituting and/or deleting and/or adding one or more nucleotides and expresses the same functional protein.
The invention provides a recombinant vector, an expression cassette, a transgenic cell strain and a recombinant bacterium containing the difunctional sesterterpene/diterpene synthase LcTPS2 encoding gene.
The invention also provides a preparation method of the fermentation liquor containing the 14-/18-membered macrocyclic terpenoid, which comprises the following steps:
constructing a recombinant vector containing the coding gene, transforming the recombinant vector into escherichia coli or saccharomyces cerevisiae, and obtaining the fermentation broth after fermentation culture.
The invention also provides a preparation method of the transgenic plant, which comprises the following steps:
and constructing a recombinant vector of the coding gene, and transforming the recombinant vector into a plant to obtain a positive plant, namely the transgenic plant.
The invention also provides application of the difunctional sesterterpene/diterpene synthase LcTPS2 or the difunctional sesterterpene/diterpene synthase LcTPS2 coding gene in preparation of 14-/18-membered macrocyclic terpenoid with the following structural formula of 1-8
Figure BDA0003048434560000021
The invention also provides application of the 14-/18-membered macrocyclic terpenoid in preparing anti-inflammatory drugs or immunosuppressive drugs.
Preferably, the anti-inflammatory or immunosuppressive drug comprises a cytokine inhibitor.
The invention also provides a pharmaceutical composition which comprises pharmaceutically acceptable pharmaceutical excipients and one or more of the 14-/18-membered macrocyclic terpenoid.
Preferably, the pharmaceutical composition comprises a tablet, a capsule, a granule, an oral liquid preparation, an intravenous injection or an intramuscular injection.
The invention starts from a Labiatae plant Leucosceptrum canum, clones and functionally identifies a terpene synthase LcTPS2 coding gene for synthesizing 14-/18-membered ring terpenoid, and the nucleotide sequence of the gene is shown in SEQ ID NO. 2. The nucleotide sequence is connected with different expression vectors to construct recombinant plasmids capable of being expressed in escherichia coli, saccharomyces cerevisiae and tobacco, and then the recombinant plasmids are converted into the escherichia coli, the saccharomyces cerevisiae or the ben-sienna to construct engineering cells, so that the 14-/18-membered ring terpenoid compound is heterogeneously synthesized by the escherichia coli, the saccharomyces cerevisiae and the ben-sienna. The gene engineering cell constructed by the invention is safe and stable, has short production period and shows important value in application and development. The macrocyclic sesterterpene product of the bifunctional sesterterpene/diterpene synthase provided by the invention has anti-inflammatory and immunosuppressive activities, and can be applied to preparation of anti-inflammatory drugs and immunosuppressive drugs. Experiments show that the sesterterpene compound provided by the invention has obvious inhibition effect on the combination of Phytohemagglutinin (PHA) and Phorbol 12-myristate 13-acetate (PMA) to induce the Jurkat cell line to secrete IL-2, and CD3 and CD28 monoclonal antibodies to stimulate mouse T cells to generate cytokine IFN-gamma, but has no obvious cytotoxicity on Jurkat and splenocytes.
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FIG. 1 is a PCR agarose gel electrophoresis of the gene encoding the bifunctional sesterterpene/diterpene synthase LcTPS2 in example 1, wherein lane M is DL2000DNAmarker and lane 1 is the target band;
FIG. 2 is a schematic structural diagram of an E.coli expression plasmid expressing a gene encoding a bifunctional sesterterpene/diterpene synthase LcTPS2 in example 2;
FIG. 3 is a total ion flow graph and mass spectrum of the enzymatic reaction product from gas chromatography-mass spectrometry (GC-MS) analysis of the enzymatic reaction product of example 4; wherein a is a total ion flow diagram of LcTPS2 with a diterpene precursor (geranylgeranyl pyrophosphate, GGPP) as a substrate; b is a total ion flow diagram of LcTPS2 with a sesquiterpene precursor (farnesyl diphosphate, FPP) as a substrate; c is the total ion flow diagram of LcTPS2 with monoterpene precursor (geranyl diphosphate, GPP) as substrate; d is the total ion flow graph of the control group; e is a mass spectrum of a specific compound synthesized by catalyzing a diterpene precursor by LcTPS 2;
FIG. 4 is a total ion flow diagram of GC-MS analysis of an engineered E.coli extract expressing a gene encoding a bifunctional sesterterpene/diterpene synthase LcTPS2 in example 5;
FIG. 5 is the structural diagram of the tobacco expression plasmid expressing the gene encoding the bifunctional sesterterpene/diterpene synthase LcTPS2 in example 6;
FIG. 6 is a total ion flow diagram of GC-MS analysis of a transgenic tobacco extract transiently expressing a gene encoding a bifunctional sesterterpene/diterpene synthase LcTPS2 in example 6.
Detailed Description
The invention provides a difunctional sesterterpene/diterpene synthase LcTPS2, wherein the synthase LcTPS2 is as follows: (1) protein formed by amino acid sequence shown in SEQ ID NO. 1; or (2) a derivative protein with the same function and with one or more amino acid residues substituted and/or deleted and/or added in the amino acid sequence shown in SEQ ID NO. 1. In the invention, the protein can catalyze geranylgeranyl pyrophosphate and geranylfarnesyl pyrophosphate to synthesize 14-/18-membered macrocyclic terpenes.
The invention provides a coding gene of a difunctional sesterterpene/diterpene synthase LcTPS2, which comprises the following components in part by weight: (a) a nucleotide sequence shown as SEQ ID NO. 2; or (b) a nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.2 and expresses the same functional protein. In the present invention, the open reading frame of the coding gene shown in SEQ ID NO.2 is 1659bp, coding 552 amino acids with a molecular weight of 64.2kD, and it was aligned by BLASTN analysis in NCBI, and the result showed 78.12% homology with germasene A synthsase (PatTpsCF2)/AY508728.1 of Pogostemon cablin (Pogostemon cablin) of Labiatae.
In the invention, the coding gene is a terpene synthase gene obtained by cloning from a Leucosceptrum micranthum, can specifically catalyze direct precursor GGDP of diterpene/sesterterpene or geranyl farnesyl pyrophosphate (GFPP) to synthesize 14-membered diterpene or 14-/18-membered sesterterpene, is obtained by cloning from plants for the first time, and enriches the diversity of terpene synthases by finding the enzyme. In the invention, the method for obtaining the cDNA sequence of the LcTPS2 encoding gene of the bifunctional sesterterpene/diterpene synthase is not particularly limited, and the conventional cDNA obtaining mode in the field can be adopted.
The invention provides a recombinant vector, an expression cassette, a transgenic cell strain and a recombinant bacterium containing the difunctional sesterterpene/diterpene synthase LcTPS2 encoding gene.
The invention also provides a preparation method of the fermentation liquor containing the 14-/18-membered macrocyclic terpenoid, which comprises the following steps:
constructing a recombinant vector containing the coding gene, transforming the recombinant vector into escherichia coli or saccharomyces cerevisiae, and obtaining the fermentation broth after fermentation culture. In the present invention, the basic vector of the recombinant vector is not particularly limited, and in the present embodiment, the basic vector is preferably pCold TF. In the present invention, the type of Escherichia coli or Saccharomyces cerevisiae is not particularly limited, and in the examples of the present invention, Escherichia coli is preferably a strain Rosetta (DE3) or Escherichia coli BL21(DE 3). The method for constructing the recombinant vector and the method for transforming the recombinant vector are not particularly limited in the present invention, and a method which is conventional in the art may be used.
The invention also provides a preparation method of the transgenic plant, which comprises the following steps:
constructing a recombinant vector of the coding gene, and transforming the recombinant vector into a plant to obtain a positive plant, namely the transgenic plant. In the present invention, the basic vector of the recombinant vector is not particularly limited, and in the examples of the present invention, the basic vector is preferably a binary vector pEAQ-HT. The method for constructing the recombinant vector of the present invention is not particularly limited, and a method which is conventional in the art may be used. In the present invention, the method of transformation is not particularly limited, and the transformation in the examples of the present invention is preferably Agrobacterium transfection.
The invention also provides application of the difunctional sesterterpene/diterpene synthase LcTPS2 or the difunctional sesterterpene/diterpene synthase LcTPS2 coding gene in preparation of 14-/18-membered macrocyclic terpenoid with the following structural formula of 1-8.
Figure BDA0003048434560000061
In the present invention, the 14-/18-membered macrocyclic terpenoid is preferably a 14-membered diterpene or a 14-/18-membered sesterterpene. In the invention, the difunctional sesterterpene/diterpene synthase LcTPS2 and the coding gene thereof synthesize 14-membered diterpene or 14-/18-membered sesterterpene by specifically catalyzing direct precursor GGDP or GFPP of diterpene/sesterterpene.
The invention also provides application of the 14-/18-membered macrocyclic terpenoid in preparing anti-inflammatory drugs or immunosuppressive drugs. In the present invention, the 14-/18-membered macrocyclic terpenoid is preferably a compound having the structural formula 1, the structural formula 3, the structural formula 5, and the structural formula 6 described above. In the present invention, the anti-inflammatory agent or immunosuppressive agent preferably includes a cytokine inhibitor.
The invention also provides a pharmaceutical composition which comprises pharmaceutically acceptable pharmaceutical excipients and one or more of the 14-/18-membered macrocyclic terpenoid. In the present invention, the dosage form of the pharmaceutical composition preferably includes tablets, capsules, granules, oral liquid preparations, intravenous injections or intramuscular injections. The pharmaceutical excipients are not particularly limited, and conventional pharmaceutical excipients in the field can be adopted according to the dosage form.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
In the following examples, unless otherwise specified, all methods are conventional.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Obtaining a cDNA sequence of a difunctional sesquiterpene/diterpene synthase LcTPS2 encoding gene and analyzing bioinformatics:
obtaining RNA from flos Agkistrodon according to molecular cloning manual, and using SMARTTMRespectively carrying out reverse transcription on primers 5 '-CDS primer, SMART IITM A oligonucleotide and 3' -CDS primer in an RACE cDNA Amplification Kit to synthesize cDNA, and carrying out PCR Amplification by taking specific primer pairs LcTPS2-3RACE-out, LcTPS2-3RACE-in, LcTPS2-5RACE-out and LcTPS2-5RACE-in as primersAnd carrying out agarose gel electrophoresis on the amplification product to obtain the attached figure 1, and recovering and purifying the amplification product to obtain the full-length cDNA sequence of the LcTPS2 gene.
Primer and method for producing the same Sequence of Numbering
LcTPS2-3race-out TCCGCAATGAAGGAAGACTTTGAATGGCTAA SEQ ID NO.3
LcTPS2-3race-in GTTATGAGGTTGAGAAGGAGAGGG SEQ ID NO.4
LcTPS2-5race-out CCCCTCTCCTTCTCAACCTCATAACTACC SEQ ID NO.5
LcTPS2-5race-in CCTTGCCAATTCCTTAAGTGCTTT SEQ ID NO.6
Through analysis, the open reading frame of the LcTPS2 coding gene of the bifunctional sesterterpene/diterpene synthase is 1659bp (SEQ ID NO.2), 552 amino acids (SEQ ID NO.1) are coded, the molecular weight is 64.2kDa, and the amino acid sequence coded by the LcTPS2 gene of the bifunctional sesterterpene/diterpene synthase comprises typical DDXXD and NSE/DTE conserved motifs. The homology search using BLASTN was conducted by placing the gene encoding bifunctional sesterterpene/diterpene synthase LcTPS2 in NCBI and comparing the gene at nucleotide level, and the result showed 78.12% homology with germasene A synthsase (PatTpsCF2)/AY508728.1 of Pogostemon cablin (Pogostemon cablin) belonging to the family Labiatae. In addition, the Target P software predicts that the candidate bifunctional sesterterpene/diterpene synthase is a chloroplast targeting protein containing 6 amino acid transit peptide.
Example 2
Constructing an expression vector of the coding gene LcTPS2 of the bifunctional sesterterpene/diterpene synthase:
PCR amplification was carried out using LcTPS2 gene cDNA synthesized in example 1 as a template, LcTPS2F: 5'-GGTACCATGGCTGCTCCAATCTCTGCAAACC-3' (SEQ ID NO.7) and LcTPS2R: 5'-CTGCAGCTAAATCTTAATTTGATCGATGAAC-3' (SEQ ID NO.8) as forward and reverse primers, using high fidelity enzyme PrimeSTAR HS DNA Polymerase, and the PCR system was 50. mu.L.
The PCR amplification reaction system is as follows:
5×PrimeSTAR HS Buffer 10μL
dNTP Mixture(2.5mM each) 4μL
Primer F 1μL
Primer R 1μL
Template cDNA 0.5μL
PrimeSTAR HS DNA Polymerase 0.5μL
deionized water 33μL
The reaction procedure for PCR amplification was: 10sec at 98 ℃, 15sec at 60 ℃, 2min at 72 ℃ and 35 cycles. And (5) recovering and purifying the product after the procedure is finished.
The purified product and the pCold TF vector are subjected to double digestion by restriction endonucleases KpnI and PstI, reacted at 37 ℃ for 3 hours, subjected to 1% agarose gel electrophoresis to detect the size of a band, and subjected to product recovery and purification. The PCR product after enzyme digestion is connected with a vector pCold TF, namely the cDNA of the coding gene of the difunctional sesquiterpene/diterpene synthase LcTPS2 is cloned to a recombinant vector pCold TF/LcTPS2 on a pCold TF expression vector containing an HIS label at the N terminal, and the structural formula is shown in figure 2. The recombinant vector pCold TF/LcTPS2 is transformed into Escherichia coli DH5 alpha, the Escherichia coli DH5 alpha is coated on an LB solid plate added with ampicillin (100 mu g/mL) for screening, the LB solid plate is cultured in a constant temperature incubator at 37 ℃ overnight until a single colony grows out, the single colony is picked up for PCR and enzyme digestion verification, and a positive clone is selected for DNA sequencing verification.
Example 3
Protein-induced expression and solubility analysis of bifunctional sesterterpene/diterpene synthase LcTPS 2:
the recombinant pCold TF/LcTPS2 strain with correct sequencing is picked to extract plasmids, the plasmids are transformed into an escherichia coli expression strain Rosetta (DE3), LB solid plates added with ampicillin (100 mu g/mL) and chloramphenicol (34 mu g/mL) are used for screening, single clones are randomly picked for colony PCR verification, the single clones which are verified to be correct are inoculated into 6mL LB liquid culture medium containing ampicillin and chloramphenicol resistance, and shake culture is carried out at 37 ℃ for overnight. Inoculating the activated bacteria into 100mL LB liquid culture medium according to the proportion of 1:100, and performing shake culture at 37 ℃ until OD600The value is about 0.5. 5mL of the bacterial liquid was taken asControl, 95. mu.L of 0.3mM IPTG was added and induction was carried out overnight at 16 ℃. Preparing a related protein purification buffer, centrifuging the low-temperature induced bacterial liquid at 4 ℃ and 12000rpm for 10min, discarding the supernatant, adding 7mL of buffer1(20mM Tris-HCl pH8.0, 500mM NaCl, 10mM imidazole) into the precipitate, and re-suspending the thalli; ultrasonically crushing thallus on ice for 10 min; 100 μ L of the sample was retained as a control, and the remaining samples were centrifuged at 12000rpm for 10min at 4 ℃ to transfer the supernatant, and 500 μ L of Ni-NTA Agarose (purchased from Qiagen) was added and incubated at 4 ℃ for 1 h; adding the protein solution containing Ni-NTA Agarose into a Polypropylene column (purchased from Qiagen company), sequentially eluting by 6mL of buffer1, 6mL of buffer 2(20mM Tris-HCl pH8.0, 500mM NaCl, 20mM imidazole) and 3mL of buffer 3(320mM Tris-HCl pH8.0, 500mM NaCl, 250mM imidazole), simultaneously collecting eluates, wherein the eluent of the buffer3 is purified protein, adding isovolumetric glycerol, uniformly mixing, storing at-20 ℃, and performing protein solubility analysis by 10% SDS-PAGE according to related operation steps, wherein the result shows that the LcTPS2 is soluble protein and the size of the protein is between 95KDa and 140 KDa.
Example 4
In vitro enzyme activity test and product analysis of the bifunctional sesterterpene/diterpene synthase LcTPS 2:
preparing a Reaction solution 10 × Reaction buffer: 1mM Tris-HCl pH 7.4, 50mM MgCl21M KCl, 20mM DTT, stored at-20 ℃. Taking 40 mu L of purified protein, 20 mu L of 10 × Reaction buffer, 10 mu L of GPP or FPP or GGPP, supplementing deionized water to 200 mu L, mixing the components uniformly, standing at 30 ℃ for Reaction for 3h, adding equal volume of n-hexane for extraction for 3 times, centrifuging at 12000rpm for 10min, taking organic phase nitrogen and concentrating to 50 mu L, and detecting by GC-MS to obtain the attached figure 3.
GC-MS chromatographic conditions: HP-5MS quartz capillary (30 m.times.250 μm.times.0.25 μm); setting a column temperature program: the initial temperature is 80 ℃, and the temperature is kept for 0 min; heating to 220 deg.C at 15 deg.C/min, and maintaining for 0 min; heating to 270 deg.C at 4 deg.C/min, and maintaining for 2 min; the sample is injected in a non-split mode, the sample injection amount is 6 mu L, the carrier gas is helium, the helium flow is 3mL/min, and the column front pressure is 40 KPa.
GC-MS mass spectrum conditions: the ion source EI has the temperature of 250 ℃, the electron energy of 70ev and the mass range of 35-550 amu.
As can be seen from the attached FIG. 3, compared with the control group (d), when the LcTPS2 uses a diterpene precursor (geranylgeranyl pyrophosphate) as a substrate, 1 specific component (shown as a) is synthesized, mass spectrometry is carried out on the specific component to obtain a mass spectrum shown as e, and the ion peak m/z 272 shows that the product is a diterpene compound, further proving that the LcTPS2 is a diterpene synthase. Whereas when LcTPS2 uses GPP and sesquiterpene precursor FPP as precursors, no specific product is synthesized (shown as c and b, respectively), indicating that the LcTPS2 has no function of synthesizing monoterpene or sesquiterpene.
Example 5
Heterologous expression of bifunctional sesterterpene/diterpene synthase LcTPS2 in escherichia coli:
the recombinant pCold TF/LcTPS2 plasmid was co-transformed with pET-MmGFDPS (methanogenic archaea GFPP synthase), pBbA5c-MevT-MBIS plasmid (which integrated the 7 genes responsible for the stepwise synthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate from acetyl-CoA and FPP synthase gene in the mevalonate pathway) into E.coli BL21(DE3) strain, pColdTF plasmid was co-transformed with pET-MmGFDPS, pBbA5c-MevT-MBIS plasmid into E.coli BL21(DE3) strain as a control strain, transformed colonies were screened using LB solid plates containing ampicillin (100. mu.g/mL), kanamycin (50. mu.g/mL) and chloramphenicol (34. mu.g/mL), single clones were selected for validation, the validated single clones were inoculated into 6mL LB medium containing the same antibiotics, cultured at 37 ℃ at 200rpm, and again into overnight culture medium containing the same antibiotics according to a ratio of 1:100, shake culture at 37 ℃ to OD600The value is about 0.5, 0.3mM Isopropyl-beta-D-Thiogalactoside (IPTG) is added, induction is carried out for 24 hours at 18 ℃, the temperature is adjusted to 25 ℃, culture is continued for 3 days, petroleum ether is extracted, an organic layer is taken, reduced pressure evaporation and concentration are carried out to obtain a reaction sample with the enzyme activity in the escherichia coli, the sample is analyzed and detected by GC-MS to obtain a figure 4, and the detection conditions refer to example 4.
Wherein the petroleum ether extract is subjected to silica gel column chromatography, and gradient elution with petroleum ether ethyl acetate (100: 0, 80: 1, 60: 1, 0: 1) is carried out from 60: part 1 gives compound 8; 100: performing silica gel column chromatography on part 0, and sequentially obtaining compounds 7, 3, 2, 4 and 5 by using petroleum ether as a mobile phase; 60: part 1 is further subjected to silica gel column chromatography (petroleum ether-ethyl acetate 60: 1, v/v), RP18 column chromatography (methanol-water 75%, v/v) and thin layer chromatography (petroleum ether-ethyl acetate 5: 1, v/v) to give compound 6; 60: part 1 was subjected to silica gel column chromatography (petroleum ether-ethyl acetate 30: 1, v/v), RP18 column chromatography (methanol-water 80%, v/v) and thin layer chromatography (petroleum ether-ethyl acetate 5: 1, v/v) to give compound 1.
As can be seen from FIG. 4, compared with the control group, 8 specific components were present in the in vivo enzymatic activity reaction sample of E.coli BL21(DE3) strain co-transformed with the recombinant pCold TF/LcTPS2 plasmid and pET-MmGFDPS, pBbA5c-MevT-MBIS plasmid.
Example 6
Heterologous expression of bifunctional sesterterpene/diterpene synthase LcTPS2 in nicotiana benthamiana:
the bifunctional sesterterpene/diterpene synthase LcTPS2 encoding gene is cloned into a binary vector pEAQ-HT by utilizing a homologous recombination technology to form an expression vector pEAQ-HT/LcTPS2, and the structural schematic diagram is shown in figure 5. Designing a proper Primer by using Primer5, and designing a Primer pair LcTPS2-Agel-F for constructing a pEAQ-HT/LcTPS2 expression vector: 5'-TTCTGCCCAAATTCGCGACCGGTATGGCTGCTCCAATCTCTGCAAACC-3' (SEQ ID NO.9) and LcTPS 2-Xhol-R: 5'-TGAAACCAGAGTTAAAGGCCTCGAGCTAAATCTTAATTTGATCGATGAAC-3' (SEQ ID NO. 10). Amplification was performed using the high fidelity enzyme PrimeSTAR HS DNA Polymerase, 50. mu.L PCR system.
The PCR amplification system is as follows:
5×PrimeSTAR HS Buffer 10μL
dNTP Mixture(2.5mM each) 4μL
Primer F 1μL
Primer R 1μL
Template cDNA 0.5μL
PrimeSTAR HS DNA Polymerase 0.5μL
deionized water 33μL
The reaction procedure for PCR amplification was: 10sec at 98 ℃, 15sec at 64 ℃, 2min at 72 ℃ and 35 cycles. And (5) recovering and purifying the product after the procedure is finished.
The arabidopsis geranyl farnesyl pyrophosphate synthase At-GFDPS2 is constructed on a pEAQ-HT carrier by using the same method to obtain pEAQ-HT/At-GFDPS 2. Respectively transforming agrobacterium LBA 4404 with vectors pEAQ-HT/LcTPS2 and pEAQ-HT/At-GFDPS2 with successful DNA sequencing verification, picking out a single clone for PCR verification, adding the single clone into LB liquid culture medium with proper volume (rifampicin: 25 mu g/mL, streptomycin: 50 mu g/mL, kanamycin: 50 mu g/mL) for culture until OD is achieved600The cells were centrifuged at 4000rpm for 10min at a value of 2, the supernatant was discarded, and the cells were collected and washed with MMA buffer (10mM MES pH5.6, 10mM MgCl)2100. mu.M acetosyringone) to OD600The value is about 0.8, the two bacteria are mixed uniformly in equal volume and are kept stand for 1-3h at room temperature. Sucking bacteria liquid by a sterile injector, injecting the young leaves of the Nicotiana benthamiana, pouring enough water on the Nicotiana benthamiana one day before injection, culturing the injected Nicotiana benthamiana in a greenhouse (16 h/light; 8h/dark,22 ℃), collecting the leaves when culturing for the sixth day, extracting with acetone, extracting with petroleum ether, concentrating by reduced pressure evaporation, and analyzing and detecting the sample by GC-MS to obtain the extractFIG. 6, see example 4 for assay conditions.
As can be seen from FIG. 6, the tobacco (i) injected with Agrobacterium containing pEAQ-HT/LcTPS2 synthesized 7 specific components with retention times consistent with the compound of example 5, compared to the tobacco (ii) injected with Agrobacterium containing pEAQ-HT/At-GFDPS2 only.
Example 7
The compounds 1 to 8 of example 5 were tested for their immunosuppressive activity in vitro:
the C57BL/6 mice were purchased from Beijing Wittingle laboratory animal technology, Jurkat cells (human T lymphocyte leukemia cells) were purchased from the cell bank of the China academy of sciences' typical culture Collection, PHA, PMA, Lipopolysaccharide (LPS), cyclosporin (Cyclosporine A, CsA) were purchased from sigma, MTS was purchased from promega, CD3/CD28 monoclonal antibody, murine IFN-MI and human IL-2 detection kits were purchased from BD Bioscience, RPMI-1640 medium, fetal bovine serum was purchased from Biological Industries.
Preparing a sample to be tested: 8 compounds of example 5 were dissolved in DMSO to prepare 20mM stock solutions.
The specific method comprises the following steps:
inoculation of Jurkat cells (2X 10)5And/well) adding 1-8 compounds into a 96-well plate, setting the concentrations of the compounds 1-8 to be five concentrations of 40 mu M, 20 mu M, 10 mu M, 5 mu M and 2.5 mu M respectively, adding 1 mu g/mL PHA and 10ng/mL PMA as stimulation groups at the same time, grouping the stimulation groups into non-stimulation groups, incubating the stimulation groups in an incubator for 24 hours, collecting supernatant, and detecting the content of IL-2 in the supernatant by Enzyme-Linked immunosorbent Assay (ELISA). In addition, 20. mu.L of MTS reagent was added to each well of unstimulated cells, and OD was measured after incubation at 37 ℃ for 1 hour490To evaluate the toxicity of the compounds on Jurkat cells. And simultaneously setting a CsA positive control group, a DMSO negative control group and a blank control group. Calculating the inhibitory activity of the compound on the PHA and PMA to induce the Jurkat cells to generate IL-2 and the toxicity of the compound on the Jurkat cells, and analyzing the half inhibitory concentration IC by using GraphPad Prism software50The results are shown in Table 1.
Inhibition (%) - (a450 (negative control) -a450 (experimental group))/(a 450 (negative control) -a450 (blank)) × 100%;
cytotoxicity (%) ═ a490 (negative control) -a490 (experimental group))/(a 490 (negative control) -a490 (blank)) × 100%.
TABLE 1 Compounds 1-8 for inhibiting the cytokine IL-2 production by Jurkat cells induced by the combination of PHA and PMA and toxicity to Jurkat cells
Figure BDA0003048434560000131
aA positive control;
bthe concentration of the compound for measuring the inhibition rate is 40 mu M;
cthe concentration of the compound for measuring the inhibition rate is 1 mu M;
dthe concentration of the compound for detecting cytotoxicity is 40 mu M;
ethe concentration of the cytotoxic compound was 1. mu.M.
As can be seen from Table 1, the compounds 1, 3, 5 and 6 of example 5 have significant inhibitory effects on the cytokine IL-2 production induced by Jurkat cells by the combination of PHA and PMA, but have no significant cytotoxic activity on Jurkat cells, indicating that the four compounds have anti-inflammatory and immunosuppressive effects.
Splenocytes were isolated aseptically from 6-8 week female C57BL/6 mice, T cells were isolated by nylon pilar method, and then inoculated with T cells (4X 10)5/well) in a 96-well plate (coated with 5 mug/mL CD3 monoclonal antibody), adding 1-8 compounds and 2 mug/mL CD28 monoclonal antibody respectively, placing at 37 ℃/5% CO2Culturing in an incubator, wherein the concentrations of the compounds 1-8 are respectively set to five concentrations of 20 MuM, 10 MuM, 5 MuM, 2.5 MuM and 1.25 MuM. And collecting cell supernatant after 48 hours, and detecting the secretion condition of IFN-gamma in the supernatant by an ELISA method. The CsA positive control group, the DMSO negative control group, the unstimulated group and the blank control group are simultaneously arranged in the experiment. The inhibitory activity of the compounds on the secretion of IFN-. gamma.by T cells was calculated and the median inhibitory concentration IC was analyzed using GraphPadprism software50The results are shown in Table 2.
The inhibition rate (%) - (a450 (negative control) -a450 (experimental group))/(a 450 (negative control) -a450 (blank)) × 100%.
Splenocytes were aseptically isolated from 6-8 week female C57BL/6 mice and inoculated with splenocytes (5X 10)5/well) in 96-well plates, then 20. mu.M compound was added, placed at 37 ℃/5% CO2Culturing in an incubator. After 48h, 10. mu.L of CCK-8 reagent was added to each well, and OD was determined after incubation at 37 ℃ for 4h450To evaluate the toxicity of the compounds on splenocytes. And simultaneously setting a CsA positive control group, a DMSO negative control group and a blank control group. Spleen cytotoxicity of the compounds was calculated and the results are shown in table 2.
Cytotoxicity (%) - (a450 (negative control) -a450 (experimental group))/(a 450 (negative control) -a450 (blank)) × 100%.
TABLE 2 inhibitory Activity of compounds 1-8 against CD3/CD28 mAbs for stimulating production of cytokine IFN-. gamma.by mouse T cells and toxicity to splenocytes
Figure BDA0003048434560000141
Figure BDA0003048434560000151
aThe concentration of the compound for measuring the inhibition rate is 20 mu M;
bthe concentration of the compound for measuring the inhibition rate is 1 mu M;
cthe concentration of the compound for detecting cytotoxicity is 20 mu M;
dthe concentration of the compound for determining cytotoxicity is 10. mu.M
As can be seen from Table 2, compounds 1 and 3 of example 5 have significant inhibitory effects on the production of the cytokine IFN-. gamma.by mouse T cells stimulated by the monoclonal antibody CD3/CD28, but have no significant cytotoxic activity against splenocytes, indicating that the two compounds have anti-inflammatory and immunosuppressive effects.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Kunming plant institute of Chinese academy of sciences
<120> bifunctional sesterterpene/diterpene synthase LcTPS2, coding gene and product and application thereof
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225 230 235 240
Gln Met Leu His Lys Glu Glu Leu His Glu Val Ser Arg Trp Trp Lys
245 250 255
Glu Leu Asp Leu Ile Ser Glu Leu Pro Tyr Ala Arg Asp Arg Val Val
260 265 270
Glu Cys Phe Phe Tyr Ala Val Gly Val Tyr His Glu Pro Gln Tyr Ser
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Thr Glu Ala Ile Glu Arg Trp Gly Ile Glu Glu Ile Asp Thr Leu Pro
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530 535 540
Leu Phe Ile Asp Gln Ile Lys Ile
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atggctgctc caatctctgc aaaccaacaa acgtctcatc gccctctcgc caatttctca 60
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gattatgaag atgaagccta tgatttgtac acggttgctc ttcattttcg actgttcagg 360
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cagactgcga ctggtattga gtgttatatg aaggaaaatg gagtgacaaa agaagaggca 1440
atgagcaagt tctctgaaat ggctacaaat gcatggaagg atattacaga agagtgttta 1500
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Claims (10)

1. Bifunctional sesterterpene/diterpene synthase LcTPS2, characterized in that the synthase LcTPS2 is:
(1) protein formed by amino acid sequence shown in SEQ ID NO. 1;
(2) and (2) the derivative protein with the same function and with one or more amino acid residues through substitution and/or deletion and/or addition of the amino acid sequence shown in SEQ ID NO. 1.
2. The gene encoding the bifunctional sesterterpene/diterpene synthase LcTPS2 of claim 1, characterized in that the coding gene 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 obtained by substituting and/or deleting and/or adding one or more nucleotides and expresses the same functional protein.
3. A recombinant vector, an expression cassette, a transgenic cell strain and a recombinant bacterium containing the coding gene LcTPS2 of the bifunctional sesterterpene/diterpene synthase of claim 2.
4. A method for preparing fermentation liquor containing 14-/18-membered macrocyclic terpenoid is characterized by comprising the following steps:
constructing a recombinant vector containing the coding gene of claim 2, transforming the recombinant vector into escherichia coli or saccharomyces cerevisiae, and obtaining the fermentation broth after fermentation culture.
5. A method for preparing a transgenic plant is characterized by comprising the following steps:
constructing a recombinant vector containing the coding gene of claim 2, and transforming the recombinant vector into a plant to obtain a positive plant, namely the transgenic plant.
6. Use of the bifunctional sesterterpene/diterpene synthase LcTPS2 of claim 1 or the bifunctional sesterterpene/diterpene synthase LcTPS2 encoding gene of claim 2 for the preparation of 14-/18-membered macrocyclic terpenoids having the following structural formulae 1-8.
Figure FDA0003048434550000011
7. Use of a 14-/18-membered macrocyclic terpenoid according to claim 6 for the preparation of an anti-inflammatory or immunosuppressive drug.
8. The use of claim 7, wherein the anti-inflammatory or immunosuppressive drug comprises a cytokine inhibitor.
9. A pharmaceutical composition comprising pharmaceutically acceptable pharmaceutical excipients and one or more of the 14-/18-membered macrocyclic terpenoid of claim 6.
10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition comprises a tablet, a capsule, a granule, an oral liquid, an intravenous injection, or an intramuscular injection.
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CN111621508A (en) * 2020-06-11 2020-09-04 云南中烟工业有限责任公司 Tobacco terpene synthase NtTPS7 gene and vector and application thereof
CN113462707A (en) * 2021-08-18 2021-10-01 中国热带农业科学院热带作物品种资源研究所 Mangifera indica terpene synthase gene TPS2 and application thereof
CN114107233A (en) * 2021-10-27 2022-03-01 武汉臻智生物科技有限公司 Phosetrene synthetase gene, high-yield strain and application
CN116555238A (en) * 2023-06-09 2023-08-08 中国中医科学院中药研究所 Use of SoSTPS1 as a sesquiterpene synthase
CN116574718A (en) * 2023-06-09 2023-08-11 中国中医科学院中药研究所 Use of SoSTPS2 as a sesquiterpene synthase

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