Disclosure of Invention
The invention provides four new-structure erythromycin analogues, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
the structural formula of the erythromycin analogue is shown as the formula (I):
the invention also provides a preparation method of the erythromycin analogue, which is prepared by fermenting Streptomyces sp.CB00271 strain, a genetically engineered strain Streptomyces sp.ZX02 or a genetically engineered strain Streptomyces sp.ZX06, wherein the genetically engineered strain Streptomyces sp.ZX02 is a genetically engineered strain obtained by knocking out an orf9 gene in a genome of Streptomyces sp.CB00271, and the genetically engineered strain Streptomyces sp.ZX06 is a genetically engineered strain obtained by knocking out an orf4 gene in a genome of Streptomyces sp.CB00271; the nucleotide sequence of the orf9 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the orf4 gene is shown as SEQ ID NO. 2.
Preferably, in the above-mentioned erythromycin analogue, the compound 3 is prepared by fermentation of Streptomyces sp.CB00271 strain, and the specific process is as follows:
s1, fermenting: inoculating Streptomyces sp.CB00271 spores to a culture medium containing 50mL of pancreatic protein soybean broth (TSB) for 1-2 days, inoculating the Streptomyces sp.CB00271 spores to an mR2A culture medium according to an inoculum size of 4% for continuous culture for 7 days, adjusting the pH of a fermentation broth to 10-11, continuing to react for 12 hours, adjusting the pH of the reacted fermentation broth to 3-4, separating out red precipitate, centrifuging to collect the precipitate and thalli, ultrasonically extracting the precipitate and thalli with a mixed solvent, and spin-drying the extract to obtain a crude extract;
S2, separating: dissolving the crude extract, passing through silica gel chromatographic column for the first time, eluting to obtain four components (Fr.1-Fr.4); subjecting Fr.3 to silica gel column for the second time to obtain 5 components (Fr.3.1-Fr.3.5); fr.3.2 was isolated using semi-preparative HPLC to give compound 3.
Preferably, the pH of the fermentation broth is adjusted to 10-11 with saturated NaOH solution.
Preferably, the pH of the fermentation broth is adjusted to 11 with saturated NaOH solution.
After experimental exploration, the supernatant product after 7 days of fermentation in the mR2A medium was found to be only compound 1 and a small amount of 3, and compound 3 was gradually increased with increasing fermentation days, because compound 1 was completely converted to compound 3 in 12 hours under alkaline conditions.
The structural formulas of all the compounds in the invention are shown as the formula (II):
preferably, the mixed solvent in S1 is a mixed solvent of dichloromethane and methanol, and the dichloromethane and methanol in the mixed solvent are mixed in a ratio of 1:1.
The solubility of the mixed solvent to the precipitate is obviously better than that of other pure solvents or mixed solvents with other proportions.
Preferably, the solvent in which the crude extract is dissolved is methylene chloride.
Preferably, the mesh number of the first silica gel chromatographic column in the step S2 is 300-400 mesh.
Preferably, the elution in S2 is gradient elution, and mixed solutions of dichloromethane and methanol in a mass ratio of 100:0, 99:1, 98:2, 97:3, 96:4, 95:5, 94:6, 93:7, 92:8, 91:9, and 90:10 are sequentially used for gradient elution.
According to different gradient elution components, sequentially eluting 10 mixed solvents in proportion, and maximally washing impurities and enriching target compounds.
Preferably, the mesh number of the second silica gel chromatographic column in the step S2 is 200 mesh.
The mesh numbers of the silica gel chromatographic columns are inconsistent in the two times, the first time is coarse separation, and the second time is fine separation.
Preferably, the mR2A medium consists of: 0.5g/L glucose, 0.5g/L peptone, 0.5g/L yeast extract, 0.5g/L acid hydrolyzed casein, 0.3g/L sodium pyruvate, 0.025g/L MgSO4 ·7H2 O、0.5g/L K2 HPO4 ，pH 7.0。
Preferably, in the above-mentioned erythromycin analogue, the compound 4 is prepared by fermenting a genetically engineered strain Streptomyces sp.zx02, and the specific process is as follows:
A. fermentation: inoculating a genetically engineered strain Streptomyces sp.ZX02 onto an R2A plate containing Streptomyces thiobacillus for 7-9 days, inoculating spores into a TSB culture medium for 1-2 days, inoculating into an mR2A culture medium according to an inoculum size of 4% for continuous culture for 7 days, regulating the pH of a fermentation liquor to 3-4, separating out a reddish brown precipitate, centrifugally collecting the precipitate and thalli, ultrasonically extracting by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;
B. Separating: dissolving the crude extract, passing through silica gel chromatographic column, eluting to obtain five components (Fr.1-Fr.5); fr.2 and Fr.3 were combined and passed through a silica gel column to give 5 fractions (Fr.2.1-Fr.2.5); fr.2.4 was further isolated, using semi-preparative HPLC, and compound 4 was finally isolated.
Preferably, fr.2.4 is further isolated using semi-preparative HPLC, while compound 5 is isolated.
The retention time of Compound 4 was 12.002min, its m/z 511.0875[ M+H ]]HRESIMS determined that its molecular formula is C25 H18 O12 The retention time of Compound 5 was 14.331min, which was (m/z 495.0923[ M+H)]In the (+), HRESIMS determines that the molecular formula is C25 H18 O11 . Based on the molecular weight and characteristic UV absorption, it was determined that compound 5 was 7,8-dideoxygriseorhodin C, and that compound 4 had one more hydroxyl group than compound 5, and that compound 4 was a novel erythromycin analogue by further analysis of C-spectrum and H-spectrum and comparison with the previous compounds, the structure of which was shown in FIG. 1.
Preferably, in the above-mentioned erythromycin analogue, the compounds 8 and 11 are prepared by fermenting a genetically engineered strain Streptomyces sp.ZX06, and the specific process is as follows:
C. fermentation: inoculating a genetically engineered strain Streptomyces sp.ZX06 onto an R2A plate containing Streptomyces thiobacillus for 7-9 days, inoculating spores into a TSB culture medium for 1-2 days, inoculating into an mR2A culture medium according to an inoculum size of 4% for continuous culture for 7 days, regulating the pH of a fermentation liquor to 3-4, separating out a reddish brown precipitate, centrifugally collecting the precipitate and thalli, ultrasonically extracting by using a mixed solvent, and spin-drying an extracting solution to obtain a crude extract;
D. Separating: dissolving the crude extract, subjecting to silica gel chromatographic column, eluting to obtain 8 components (Fr.1-Fr.8); fr.2 and Fr.3 were combined and passed through a silica gel column to give 5 fractions (Fr.2.1-Fr.2.5); combining fr.2.2 and fr.2.3 for further separation using semi-preparative HPLC, final isolation yields compound 10 and compound 11; the fr.4 and fr.5 were combined for further separation using semi-preparative HPLC, and finally isolated as compound 8 and compound 9.
The invention also provides application of the compounds 3,4,8 and 11 in preparing antitumor drugs.
The invention uses mR2A culture medium to ferment the original strain Streptomyces sp.CB00271, and the secondary metabolite is analyzed by UPLC-MS. Analysis was performed by the UPLC program and the analysis showed that multiple erythromycin analogs could be produced in the original strain. Peak 3, which has a retention time at 13.342min and a characteristic UV absorbance (λmax 238nm,309nm and 508 nm), was analyzed by HRLCMS and its m/z was 523.0876[ M+H ]]Peak 6 at retention time 14.051min and with characteristic UV absorbance values (λmax 236nm,316nm and 496 nm), which is (m/z 555.31[ M+H)]In the presence of a peak 9 with characteristic UV absorption values (lamda max 231nm,312nm and 512 nm) at retention time 14.431min (m/z 553.0982[ M+H) ]+ ) Peak 1 at retention time 15.590min and with characteristic UV absorbance values (λmax 239nm,313nm and 513 nm), which is (m/z 537.1035[ M+H)]In the presence of a peak 7 with characteristic UV absorption values (λmax239nm, 313nm and 513 nm) at a retention time of 15.98min (m/z 509.0714[ M+H)]In the presence of a peak 2 with characteristic UV absorption values (λmax 230nm,312nm and 504 nm) at retention time of 16.976min (m/z 523.0792[ M+H)]+ ). According to the molecular weight and the characteristic ultraviolet absorption, the compound 1 can be determined to be beta-Yuxithromycin, the compound 2 is gamma Yuxithromycin, the compound 6 is heliquimycin, the compound 7 is Yuxithromycin CA1, and the compound 10 is 3' -hydroxy-beta-Yuxithromycin. Compound 3, like compound 2, has a molecular weight that differs from compound 1 by 1 methylene group, and may be a novel erythromycin derivative. By taking samples of different fermentation days and performing HPLC analysis, as shown in FIG. 4, it can be seen that the supernatant product was only compound 1 and a small amount of compound 2 on fermentation day 2, and that as the fermentation day increases, compound 3 gradually increased, and that the pH of the fermentation broth also gradually increased. After 7 days of fermentation broth collection, the fermentation broth is centrifuged at high speed and then taken upThe supernatant was adjusted to pH 11 with 1M NaOH, left at room temperature for 12 hours, sampled at various time points and subjected to HPLC detection, and the result was shown in FIG. 5, in which compound 1 was completely converted into compound 3 in 12 hours under alkaline conditions, and in combination with molecular weight analysis therebetween, it was estimated that compound 1 could be stripped off one methylene group to form compound 3 under alkaline conditions. In order to determine the structural formula of the other compound 3, 8L scale fermentation and subsequent in-vitro conversion are adopted to enrich the compound 3, and a chloroform-methanol system is utilized to carry out silica gel column separation and HPLC-UV preparation to obtain a pure product of the compound 3; through nuclear magnetic resonance analysis and in vitro transformation analysis, the structure of the compound 3 is shown in figure 1, and the compound 3 is beta-erythromycin acid which is a novel erythromycin analogue.
The Streptomyces sp.CB00271 of the original strain of the invention was added 20mM Mg2+ Can grow well and produce more spores on MS plates. When the strain was tested for sensitivity to different antibiotics, it was found that the strain was resistant to the addition of 20mM Mg2+ And MS containing 50. Mu.g/mL of aplastic or 20. Mu.g/mL of thiostrepton did not grow, the strain grew well on medium containing 40. Mu.g/mL of nalidixic acid; and E.coli S17-1 naphthyridoneacid is extremely sensitive, so that the method can use the apramycin and the thiostrepton as screening marks for screening mutant strains. The shuttle plasmid pSET152 of the escherichia coli and the Streptomyces is utilized to carry out indirect transfer between the species of the escherichia coli and the Streptomyces, the combined Streptomyces and the escherichia coli are covered by the apramycin and the nalidixic acid, and more zygotes grow on a flat plate after about 4 days, which indicates that the Streptomyces sp.CB00271 has better joint transfer property. The establishment of the genetic operation system lays a solid foundation for the genetic engineering transformation of the strain.
The invention determines the biosynthesis gene cluster of the erythromycin in CB00271 by carrying out total gene sequencing and secondary metabolite biosynthesis gene cluster analysis on the Streptomyces sp.CB00271 of the original strain and subsequent gene knockout experiments. By comparing and analyzing the reported erythromycin gene cluster, we find the jade in CB00271 The erythromycin gene cluster is different from the known gene cluster, compared with the reported erythromycin gene cluster rubDSM2012 (GenBank accession no.AF293355),rubCB00271 The gene cluster is provided with a plurality of genes, wherein the gene cluster comprises an anthraquinone oxidoreductase gene orf9; compared to the reported griseoviridin gene cluster grh (GenBank accession No. AF509565), rubCB00271 Several unique modified enzyme genes are included in the gene cluster, including one ester cyclase gene orf4, as detailed in Table 1. Later, 2 engineering strains Streptomyces sp.ZX02 and Streptomyces sp.ZX06 which can directionally produce the erythromycin analogues are obtained by knocking out the two special genes orf9 and orf 4. The strain was fermented with mR2A medium and analyzed for secondary metabolites by UPLC-MS. Analysis by the UPLC program showed that two Yuxithromycin analogues 4 and 5 could be produced in the Streptomyces sp.ZX02 mutant in a targeted manner. Peak 4 with retention time at 12.002min and characteristic UV absorbance (λmax 239nm,313nm and 510 nm) analyzed by HRLCMS, m/z 511.0875[ M+H ]]+ Peak 5 at retention time 14.331min and with characteristic UV absorbance values (λmax 232nm,315nm and 502 nm), which is (m/z 495.0923[ M+H)]+ ). Based on molecular weight and characteristic uv absorption, compound 5 can be determined to be 7,8-dideoxygriseorhodin C. In order to determine the structural formula of the other compound, 6L scale fermentation is adopted, and a chloroform methanol system is utilized to carry out silica gel column separation and HPLC-UV preparation to obtain a pure product of the compound 4; by nuclear magnetic resonance analysis, we determined that compound 4 was a novel erythromycin analog (fig. 1).
Analysis of the Streptomyces sp.ZX06 mutant strain using the same UPLC procedure as described above showed that the mutant strain produced 4 Yuxithromycin analogues 8/9/10/11, which were further HRLCMS analysis and had peak 8 at 12.869min retention time and characteristic UV absorbance values (λmax 231nm,308nm and 516 nm) with m/z 539.0822[ M+H ]]+ Peak 9 at retention time 13.979min and with characteristic UV absorbance values (λmax 231nm,312nm and 495 nm), which is (m/z 525.0663[ M+H)]+ ) At a retention time of 14.431min and with characteristic UV absorption values (lamda max 231nm,312nm and 512 nm)Peak 10 (m/z 553.0982[ M+H)]+ ) Peak 11 at retention time 15.421min and with characteristic UV absorbance values (λmax 230nm,312nm and 494 nm), which is (m/z 539.0822[ M+H)]+ ). Compounds 8 and 10 differ by one methylene group and compounds 9 and 11 differ by one methylene group. During the fermentation, four analogues were found to have a certain relationship, and when ZX06 was fermented for 2 days, the products were only compounds 10 and 11, and as the number of days of fermentation increased, and the pH of the broth became greater, compounds 8 and 9 increased gradually, as shown in fig. 2. When we collected 2 days of fermentation broth and centrifuged at high speed, the supernatant was taken, pH of the supernatant was adjusted to 11 with 1M NaOH, and left at room temperature for 24 hours, it was observed that compounds 10 and 11 could be completely converted into compounds 8 and 9 in 24 hours under alkaline conditions as shown in FIG. 3, and in combination with molecular weight analysis between 4, it was estimated that compounds 10 and 11 were separated from one methylene group under alkaline conditions to form compounds 8 and 9, respectively. Based on molecular weight and characteristic uv absorption, it was determined that compound 9 was erythromycin CA2 and compound 10 was 3' -hydroxy- β -erythromycin, of which compounds 8 and 11 should be new compounds. In order to determine the structures of the compounds corresponding to the two peaks, 8L of scale fermentation treatment is adopted, and the pure products of the compounds 8 and 11 are respectively obtained by silica gel column separation and HPLC-UV preparation through a chloroform methanol system; the structures of the compounds 8 and 11 are shown in figure 1 through nuclear magnetic resonance analysis and in-vitro transformation analysis, the compound 8 is 3 '-hydroxy-beta-erythromycin, the compound 11 is 3' -hydroxy-gamma-erythromycin, and both are novel erythromycin analogues.
The invention relates to a gene orf9 (the nucleotide sequence of which is shown as SEQ ID NO. 1) and an ester cyclase gene orf4 (the nucleotide sequence of which is shown as SEQ ID NO. 2) of which anthraquinone oxidoreductase in a Yuxithromycin biosynthesis gene cluster is responsible for post-oxidation modification, which are subjected to substitution and single mutation respectively, so that engineering strains for directionally producing Yuxithromycin analogs can be obtained. In the biosynthetic gene cluster of Yuxithromycin orf9 encodes a polypeptide of 325 amino acids, which is defined as anthraquinone oxidoreductase. orf4 encodes a 288 amino acid polypeptide, defined as an ester cyclase. To determine the specific role of Orf9 and Orf4 genes encoding proteins Orf9 and Orf4 in the biosynthesis of erythromycin, in-frame substitution was performed with a DNA fragment resistant to thiostrepton, respectively, so that Orf9 (fig. 6) and Orf4 (fig. 7) were inactivated. Transferring the knocked-out vector into E.coli S17-1 strain, performing indirect transfer with wild type CB00271 strain to obtain some zygotes, selecting ZX02 with correct resistance phenotype (thiostrepton tolerance, aplamycin sensitivity) and genotype, and fermenting ZX06 and wild type CB00271 strain simultaneously under the same condition, wherein the wild type strain is used as a positive control. Analysis of the broth extract of mutant strain ZX02 by HPLC revealed that the mutant strain produced 2 analogs of erythromycin (FIG. 8), and that the ZX06 mutant strain produced 4 analogs of erythromycin (FIG. 8). These data demonstrate that Orf9 and Orf4 proteins have important roles in the biosynthesis of erythromycin.
The nucleotide sequence or a part of the nucleotide sequence provided by the invention can be used for obtaining genes similar to orf9 and orf4 from other organisms by using a Polymerase Chain Reaction (PCR) method or a DNA containing 1 st to 978 th positions of the sequence shown by SEQ ID NO.1 or a DNA containing 1 st to 867 th positions of the sequence shown by SEQ ID NO.2 as probes by a Southern hybridization method and the like.
The nucleotide sequence provided by the invention or at least part of the nucleotide sequence can be modified or mutated in vitro and in vivo, including insertion, substitution or deletion, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutation, reconnection of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical agents, and the like.
Cloned genes comprising the nucleotide sequences provided herein, or at least a portion thereof, may be expressed in a foreign host by a suitable expression system to yield the corresponding enzyme or other higher biologically active substance or yield. Such exogenous hosts include E.coli, streptomyces, micromonospora, pseudomonas, bacillus, yeast, plants and animals, and the like.
Polypeptides comprising the amino acid sequences or at least parts of the sequences provided herein may still be biologically active or even have new biological activities after removal or substitution of certain amino acids, or may have improved yields or optimized protein dynamics or other properties aimed at.
The invention provides a way for directional use in genetically engineered microorganisms, which comprises DNA fragments or genes that can be used to construct mutants that produce daunorubicin or a derivative thereof in a targeted manner.
The invention also provides application of anthraquinone oxidoreductase Orf9 in the erythromycin biosynthesis gene cluster in directionally producing erythromycin analogues.
The discovery of the biosynthetic gene cluster of natural products and the development of gene knockout technology provide possibility and means for the genetic engineering of strains. Many researches have been successfully carried out on structural modification and transformation of natural products with more complicated structures or directional accumulation of certain target components through biosynthesis means, a series of derivatives or high-yield mutant strains with equivalent or stronger activity are constructed, good cases are provided for genetic engineering transformation of streptomycete, and new hopes are brought for solving the treatment of increasingly serious tumor diseases.
The new erythromycin analogue is discovered through fermentation of the original strain or the erythromycin is structurally modified or reformed through gene knockout to obtain more erythromycin structural analogues, so that the preclinical researches such as anticancer activity analysis and action mechanism of the natural products are accelerated, and the application and development of new antitumor drugs are accelerated.
The invention also provides application of the nucleotide sequence shown as SEQ ID NO.2 in preparing a compound of the erythromycin analogue.
The application is that four compounds in the erythromycin analogue can be directionally produced after the nucleotide sequence shown as SEQ ID NO.2 is deleted.
In a word, the information containing the post-modification gene and the protein related to the biosynthesis of the Yuxithromycin provided by the invention can help people understand the biosynthesis mechanism of anthraquinone natural products, and provides possibility for obtaining engineering strains by further utilizing a genetic modification mode. The gene and the protein thereof provided by the invention can also be used for searching and finding compounds or genes and proteins which can be used in medicine, industry or agriculture.
The streptomycete is Streptomyces sp.CB00271 and is preserved in China center for type culture collection (China center for type culture Collection) on 6-month 2 days, wherein the address is eight-path 299 of Wuhan university in Wuchang district of Wuhan, hubei province, and the preservation number is CCTCC M2020176.
The beneficial effects are that:
the compounds 3 and 8 prepared by the invention have better tumor inhibition activity, and can be used for preparing antitumor drugs. Especially, the activity of the compound 3 is far better than that of a similar compound 1, and is also far better than that of a positive anti-tumor western medicine control group, so that the application of the compound 3 in the future anti-tumor medicine is proved to have very good prospect. Provides greater possibility for accelerating the preclinical researches such as anticancer activity analysis, action mechanism and the like of the natural products and the application and development of new antitumor drugs.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. All percentages below refer to mass percentages. The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1Streptomyces sp.CB00271 cultivation and establishment of genetic manipulation System and Gene Cluster analysis
Solid culture of 1.1CB00271:
streptomyces sp.CB00271 is isolated from forest soil of Yunnan, and the strain is stored on an R2A culture medium inclined plane, wherein the R2A culture medium comprises the following components: 0.5g/L glucose, 0.5g/L peptone, 0.5g/L yeast extract, 0.5g/L acid hydrolyzed casein, 0.3g/L sodium pyruvate, 0.025g/L MgSO4 ·7H2 O、0.5g/L K2 HPO4 And 20g/L agar, pH7.0, and sterilized for use.
Establishment of 1.2Streptomyces sp.CB00271 genetic manipulation System
We studied the growth and sporulation of Streptomyces sp.CB00271 on different media and found it to contain 20mM Mg2+ The yield of spores on MS culture medium is good. Meanwhile, the sensitivity of Streptomyces sp.CB00271 to various antibacterial drugs was studied, and the antibacterial drugs tested include: sensitivity to Apramycin (Apramycin, am), nalidixic acid, thiostrepton (Tsr), etc., streptomyces sp.CB00271 was found to be sensitive to Apramycin (50 μg/mL) and Thiostrepton (20 μg/mL). Nalidixic acid (40 μg/mL) is non-toxic to Streptomyces sp.CB00271. The strain E.coli S17-1 is very sensitive to nalidixic acid (40. Mu.g/mL). This work demonstrates that we can introduce a thiostrepton resistance gene at the time of gene replacement mutation and that it can be screened for zygotes with thiostrepton and nalidixic acid at the time of conjugal transfer. The feasibility of this genetic manipulation was confirmed by performing conjugation transfer with E.coli containing S17-1 of the integrative plasmid pSET152 and Streptomyces sp.CB00271, and covering the conjugation plate with apramycin and nalidixic acid, and more spliceosomes were found to occur about 4 days. Laying a good foundation for the development of subsequent experiments.
Gene cluster analysis of 1.3CB00271
The biosynthesis gene cluster of erythromycin in CB00271 was determined by whole gene sequencing and secondary metabolite biosynthesis gene cluster analysis of the original strain Streptomyces sp.CB00271, followed by gene knockout experiments. By comparative analysis with the reported Yuxithromycin gene cluster, we found that the Yuxithromycin gene cluster in CB00271 is different from the known gene cluster, compared with the reported Yuxithromycin gene cluster rubDSM2012 (GenBank accession no.AF293355),rubCB00271 The gene cluster is provided with a plurality of genes, wherein the gene cluster comprises an anthraquinone oxidoreductase gene orf9; compared to the reported griseoviridin gene cluster grh (GenBank accession No. AF509565), rubCB00271 Several unique modified enzyme genes are included in the gene cluster, including one ester cyclase gene orf4, as detailed in Table 1.
Table 1 comparison of CB00271 Yuxithromycin biosynthesis Gene Cluster analysis
Example 2Streptomyces sp.CB00271 fermentation analysis and subsequent Scale fermentation, separation purification and structural identification of Yuxithromycin analog
CB00271 produces a plurality of erythromycin analogs having a retention time at 13.342min and a peak 3 with characteristic UV absorbance values (λmax238 nm,309nm and 508 nm) of m/z 523.0876[ M+H ]++, molecular formula is C26 H18 O12 The method comprises the steps of carrying out a first treatment on the surface of the Peak 6 at retention time 14.051min and with characteristic UV absorbance values (λmax 236nm,316nm and 496 nm), m/z 555.31[ M+H ]]++, molecular formula is C26 H18 O14 The method comprises the steps of carrying out a first treatment on the surface of the Peak 10 at retention time 14.431min and with characteristic UV absorbance values (lamda max 231nm,312nm and 512 nm), which is (m/z 553.0982[ M+H)]+ ) Molecular formula C27 H20 O13 The method comprises the steps of carrying out a first treatment on the surface of the Peak 1 at retention time 15.590min and with characteristic UV absorbance values (λmax 239nm,313nm and 513 nm), which is (m/z 537.1035[ M+H)](+) molecular formula C27 H20 O12 The method comprises the steps of carrying out a first treatment on the surface of the At the retention timePeak 2 at 16.076min and having characteristic UV absorbance values (λmax 230nm,312nm and 504 nm), which is (m/z 523.0792[ M+H)](+) molecular formula C26 H18 O12 . Based on the molecular weight and characteristic uv absorption and retention time, it was determined that compound 1 was β -erythromycin, 2 was γerythromycin, compound 6 was heliomycin, compound 10 was 3' -hydroxy- β -erythromycin, and compound 3, like 2, was 1 methylene different from compound 1, possibly a new derivative of erythromycin. By taking samples of different fermentation days and performing HPLC analysis, as shown in FIG. 6, it can be seen that the supernatant product was only compound 1 and a small amount of compound 2 on fermentation day 2, and that as the fermentation day increases, compound 3 gradually increased, and that the pH of the fermentation broth also gradually increased. When we collected 7 days of fermentation broth and centrifuged at high speed, we took the supernatant, adjusted the pH of the supernatant to 11 with 1M NaOH, left at room temperature for 12 hours, sampled at different time points and tested by HPLC, and as shown in FIG. 7, compound 1 was completely converted into compound 3 in 12 hours under alkaline conditions, and then combined with molecular weight analysis between the two, it was estimated that compound 1 could be stripped off one methylene group to form compound 3 under alkaline conditions.
To identify the structure of compound 3, we performed a large scale fermentation of 8L and subsequent in vitro transformations to enrich compound 3, as follows: a portion of CB00271 spores was inoculated into a medium containing 50mL of TSB at 30℃for 1-2 days, inoculated into mR2A medium at 4% of the inoculum size, and placed on a 30℃shaker at 220rpm for further 7 days. After fermentation of CB00271 for 7 days, the pH of the broth was adjusted to 11 with saturated NaOH solution and kept on shaker overnight. Then, the pH of the fermentation broth after the reaction is adjusted to 3-4 by 1M hydrochloric acid, and then red precipitate is separated out, the high-speed centrifugation is carried out, and the supernatant is discarded to collect the precipitate and thalli. Dissolving and extracting the precipitate and thallus collected by centrifugation with mixed solvent of dichloromethane and methanol for several times until the extract is colorless, centrifuging, mixing the soluble parts, and concentrating under vacuum to obtain crude extract. The crude extract was dissolved in a small amount of dichloromethane and subjected to 300-400 mesh silica gel column chromatography, eluting with a gradient of dichloromethane and methanol, to give four components (fr.1-fr.4). Fr.3 was further purified by 200 mesh silica gel column chromatography in this order to obtain 5 fractions (Fr.3.1-Fr.3.3). Fr.3.2 was further isolated using semi-preparative HPLC to give compound 3. Further through analysis of C spectrum and H spectrum and comparison with the previous compounds, the structure of the compound 3 is shown in figure 1, and the compound 3 is a novel erythromycin analogue which can be generated by hydrolyzing beta-erythromycin under alkaline conditions, so that the compound 3 is named as beta-erythromycin acid.
Construction of orf9 and orf4 Gene knockout plasmids in example 3Streptomyces sp.CB00271
Construction of orf9 knockout plasmid in 3.1Streptomyces sp.CB00271
2 pairs of primers were designed using S sp.CB00271 total DNA as template
pD9F：5′-AATGTGAACACGCCTCCTCGACCCGCAG-3′(SEQ ID NO.3)；
pD9R:5'-GCGGCCGCGGATCCTCTAGAGGCAAGACCACCTTGTCGCG-3' (SEQ ID NO. 4); and
pD10F：5′-ACGACGGCCAGTGCCAAGCTTGGCGAAGACCGAGATGC-3′(SEQ ID NO.5)；
pD10R:5'-CAACCGATAAAAGGCGCACGAGCTGATG-3' (SEQ ID NO. 6); the method is used for PCR amplification of the homologous arms, purifying and recovering the specific products with the length of 1.5kb, and designing a pair of primers:
p9TF：5′-CGTGCGCCTTTTATCGGTTGGCCGCGAG-3′(SEQ ID NO.7)；
p9TR:5'-CGAGGAGGCGTGTTCACATTCGAACGGTCTCTG-3' (SEQ ID NO. 8); the kit is used for amplifying the thiostrepton resistance gene box, and the specific products with the length of 0.9kb are purified and recovered. The construction method was performed by using a strategy of seamless cloning, referring to (TSV-S1) Trelief SoSoo Cloning Kit instruction manual. The left and right homologous arms, the resistance genes and the pOJ260 vector tangential by HindIII and XbaI enzymes are subjected to seamless connection, the linked products are transformed into competent E.coli DH5 alpha according to the concentration of 3:3:3:1, monoclonal is selected for colony verification, positive clone extraction plasmids are subjected to enzyme digestion verification and sequencing verification, and plasmids with correct sequencing are orf9/10 gene knockout plasmids pYLW003 for subsequent gene knockout.
Construction of orf4 knockout plasmid in 3.2Streptomyces sp.CB00271
With S sp.cb00271 total DNA as template, 2 pairs of primers were designed separately:
pD4UPF：5′-AATGTGAACAGGCGTCGTGGAACAGC-3′(SEQ ID NO.9)；
pD4UPR:5'-GCGGCCGCGGATCCTCTAGAGGATGTCGCGGCCCGA-3' (SEQ ID NO. 10); and
pD4DNF：5′-ACGACGGCCAGTGCCAAGCTTGGACTGGCCCATGGTGAC-3′(SEQ ID NO.11)；
pD4DNR (5'-GCCAACCGATAAAAGGTCTCCGAGCACTGG-3' (SEQ ID NO. 12); for the PCR amplification of the left and right homology arms, the recovery of specific products of 1.3kb in length was purified, while designing a pair of primers:
p4TF：5′-CTCGGAGACCTTTTATCGGTTGGCCGCGAG-3′(SEQ ID NO.13)；
p4 TR: 5'-CACGACGCCTGTTCACATTCGAACGGTCTCTG-3' (SEQ ID NO. 14); the kit is used for amplifying the thiostrepton resistance gene box, and the specific products with the length of 0.9kb are purified and recovered. The construction method was performed by using a strategy of seamless cloning, referring to (TSV-S1) Trelief SoSoo Cloning Kit instruction manual. The left and right homologous arms, the resistance genes and the pOJ260 vector tangential by HindIII and XbaI enzymes are subjected to seamless connection, the linked products are converted into competent E.coli DH5 alpha according to the concentration of 3:3:3:1, monoclonal is selected for colony verification, positive clone extraction plasmid is subjected to enzyme digestion verification and sequencing verification, and the plasmid with correct sequencing is orf9 gene knockout plasmid pYLW010 and is used for subsequent gene knockout.
EXAMPLE 4 construction of orf9 Gene-deletion mutant Streptomyces sp.ZX02
Transferring the constructed knockdown plasmid pYLW003 into E.colli S17-1 to obtain E.colli S17-1 transferred with the knockdown plasmid pYLW003, which is named as E.colli S17-1/pJu2014/pYLW003, and performing conjugation transfer with a wild strain. The bonding transfer process is as follows: s sp.CB00271 spore 2 tube (about 10) frozen in-80℃refrigerator9 Spores), washed 2 times with LB, resuspended in 1mL LB medium, heat-shocked in a water bath at 50℃for 10min, and then allowed to stand to room temperature to serve as a conjunctive transfer recipient. Transforming the constructed recombinant plasmid into E.coli S17-1 from the strain containing two antibioticsAm, km) were inoculated on 3mL of LB medium (containing the corresponding antibiotics) by picking up the monoclonal, shaking culture at 37℃at 230rpm for 12-15 hours, inoculating 500. Mu.l of the bacterial liquid on 50mL of LB medium (containing the corresponding antibiotics), shaking culture at 37℃at 230rpm for 4-8 hours to OD600 Reaching 0.3-0.4. The bacterial liquid was poured into a 50mL sterilizing centrifuge tube, and centrifuged at 4000rpm for 10min to collect bacterial cells. The cells were washed twice with 25mL of LB medium, and resuspended in 2mL of LB medium to be used as a conjugal transfer donor. Heat-shocked S sp.CB00271 spores and E.coli S17-1 (containing recombinant plasmid) bacterial liquid are mixed according to the following ratio of 1:1 volume, after centrifugation to remove a portion of the supernatant, the mixture was applied to M S medium (containing 20mM MgCl)2 ) Negative and positive controls were made simultaneously. Only 20 μ l S sp.cb00271 spores were plated on M S medium used as positive and negative controls. After the M S culture medium coated with the bacterial liquid is placed in a constant temperature incubator at 30 ℃ for culturing for 18-20 hours, the surface escherichia coli is removed by sterile water, 50mg/L of Am (or 20mg/L of Tsr) and 40mg/L of nalidixic acid are covered on the joint transfer culture medium and the negative control, 40 mug/mL of nalidixic acid is covered on the positive control, and the culture medium and the negative control are placed on an ultra clean bench for purging for 2-3 hours until the surface is dried. The culture medium is placed in a 30 ℃ incubator for inversion culture, and white zygote single colonies grow after 4-7 days. Double crossover mutant strain ZX02 was identified by its resistance phenotype to antibodies (AmS &TsrR) And genotypes, wherein PCR verification of orf9 gene deletion was performed using primers:
pYZ9F：5′-CCCGCGCTTCCATCAGC-3′(SEQ ID NO.15)；
pYZ9R:5'-ACGTCCTGCGGGTCGAG-3' (SEQ ID NO. 16); the PCR amplification reaction was performed to confirm that the orf9 gene deletion mutant S sp.CB00271 was designated as the double crossover knockout strain Streptomyces sp.ZX02.
The construction process of orf4 gene deletion mutant strain is the same as that of Streptomyces sp.ZX02, and the primers are verified:
pYZ4F：5′-TCATGGCGTGGGCAGTCC-3′(SEQ ID NO.17)；
pYZ4R:5'-CCGGAACGGAGGGACTC-3' (SEQ ID NO. 18), from which an orf4 gene deletion mutant S sp.CB00271 was obtained, designated as double crossover knockout strain Streptomyces sp.ZX06.
EXAMPLE 5 fermentation of wild-type Strain with double-crossover mutant strains Streptomyces sp.ZX02 and Streptomyces sp.ZX06 and HPLC analysis thereof
Both the wild strain and the double-exchanged mutant strain are cultured on a R2A solid culture medium plate at 30 ℃ for 7-9 days, wherein the plate of the double-exchanged mutant strain simultaneously contains the S.thiostrepton with the final concentration of 20 mug/mL; selecting appropriate amount of spores, inoculating into a triangular flask containing 50mL TSB culture medium, and culturing on a shaking table at 30deg.C and 220rpm for 1-2 days; inoculating the strain into a fermentation medium mR2A in an inoculum size of 4%, wherein the composition of the fermentation medium mR2A comprises: 5 parts of soluble starch, 5 parts of glucose, 2.5 parts of peptone, 2.5 parts of yeast powder, 0.5 part of acid hydrolyzed casein, 0.3 part of sodium pyruvate and 0.25 part of MgSO4 ·7H2 O,0.5 part K2 HPO4 pH 7.0. The cells were incubated at 30℃and 220rpm on a shaker for 7 days. 50mL of fermentation liquor is subjected to pH adjustment to 3-4 by using 1M HCl, after uniform mixing, reddish brown precipitate is separated out, the fermentation liquor is subjected to centrifugal collection of precipitate and thalli, the centrifugal precipitate and thalli are subjected to ultrasonic extraction for a plurality of times by using a mixed solvent of dichloromethane and methanol until the extract is colorless, the extract is combined to obtain a mixed liquor, the mixed liquor is concentrated and dried, the volume is re-fixed to 50mL, and after centrifugation at 12000rpm for 5min, the supernatant is taken for HPLC analysis. HPLC analysis conditions and procedures were as follows: mobile phase a was 99.9% deionized water and 0.1% formic acid; mobile phase B was 100% acetonitrile at a flow rate of 1.0mL/min, uv detector wavelength of 310nm, linear gradient analysis procedure: 0-1 min, 95% A;1-18 minutes, 95% a to 5%A;18-20 minutes, 5%A;20-20.5 minutes, 5%A to 95% a;20.5-25 minutes, 95% A. The HPLC analysis results are shown in FIGS. 4 and 5. From the figure, it can be seen that the ZX02 mutant produced only 2 erythromycin analogues (fig. 4); ZX06 produced 4 Yuxithromycin analogues (FIG. 5).
Example 6Streptomyces sp.ZX02 mutant Large Scale fermentation, separation purification and Structure identification of Yuxithromycin analog
Firstly, the mutant strain ZX02 is inoculated on an R2A plate containing 20 mug/mL of S.thiostrepton, after 7-9 days of spore growth is good, part of spores are inoculated on a culture medium containing 50mL of TSBShaking culture at 30deg.C for 1-2 days, inoculating into mR2A culture medium according to 4% inoculum size, shaking culturing at 30deg.C with 220rpm for 7 days, and fermenting for 6L. And then, regulating the pH of the fermentation liquor to 3-4 by using 1M HCl, uniformly mixing, separating out reddish brown precipitate, centrifugally collecting the precipitate and thalli from the fermentation liquor, ultrasonically extracting the centrifugally precipitated and thalli for a plurality of times by using a mixed solvent of dichloromethane and methanol until the extracting solution is colorless, combining the extracting solutions, mixing the obtained mixed solutions, and enriching and separating target compounds. The crude extract was dissolved in a small amount of dichloromethane and subjected to 300-400 mesh silica gel column chromatography, eluting with a gradient of dichloromethane and methanol, to give five components (fr.1-fr.5). Fr.2 and Fr.3 were combined and further purified sequentially by 200 mesh silica gel column chromatography to give 5 fractions (Fr.2.1-Fr.2.5). Fr.2.4 was further isolated using semi-preparative HPLC, resulting in isolation of 4 and 5. The retention time of Compound 4 was 12.002min, its m/z 511.0875[ M+H ] ]+ HRESIMS determined that its molecular formula is C25 H18 O12 The retention time of Compound 5 was 14.331min, which was (m/z 495.0923[ M+H)]+ ) HRESIMS determined that its molecular formula is C25 H18 O11 . Based on the molecular weight and characteristic UV absorption, it was determined that compound 5 was 7,8-dideoxygriseorhodin C, and that compound 4 had one more hydroxyl group than compound 5, and that compound 4 was a novel erythromycin analogue by further analysis of C-spectrum and H-spectrum and comparison with the previous compounds, the structure of which was shown in FIG. 1.
Example 7Streptomyces sp.ZX06 mutant fermentation analysis and subsequent Large Scale fermentation, isolation and purification and Structure identification of Yuxithromycin analog
ZX06 produced 4 Yuxithromycin analogues, which were further analysed by HRLCMS and had peak 8 at retention time 12.869min with characteristic UV absorbance values (lamda max 231nm,308nm and 516 nm), m/z 539.0822[ M+H ]]+ Molecular formula C26 H18 O13 The method comprises the steps of carrying out a first treatment on the surface of the Peak 9 at retention time 13.979min and with characteristic UV absorbance values (lamda max 231nm,312nm and 495 nm), m/z 525.0663[ M+H ]]+ Molecular formula C25 H16 O13 The method comprises the steps of carrying out a first treatment on the surface of the Peak 10 at retention time 14.431min and with characteristic UV absorbance values (lamda max 231nm,312nm and 512 nm), M/z553.0982[ M+H ]]+ Molecular formula C27 H20 O13 The method comprises the steps of carrying out a first treatment on the surface of the Peak 11 at retention time 15.421min and with characteristic UV absorbance values (λmax 230nm,312nm and 494 nm), m/z 539.0822[ M+H ] ]+ Molecular formula C26 H18 O13 . Based on molecular weight and characteristic ultraviolet absorption, it was determined that compound 9 was rubrovycin CA2 and compound 10 was 3' -hydroxy- β -erythromycin; compound 8 and compound 10 differ by one methylene group, and compound 9 and compound 11 also differ by one methylene group. During the fermentation, four analogues were found to have a certain relationship, as in fig. 2, when zx06 was fermented for 2 days, the products were only compound 10 and compound 11, and as the number of days of fermentation increased, and the pH of the fermentation broth became greater, compound 8 and compound 9 gradually increased. When we collected 2 days of fermentation broth and centrifuged at high speed, the supernatant was taken, pH of the supernatant was adjusted to 11 with 1M NaOH, and left at room temperature for 24 hours, samples were taken at different time points for HPLC detection, and as a result, as shown in FIG. 3, under alkaline conditions, compound 10 and compound 11 could be completely converted into compound 8 and compound 9 within 24 hours, and then, in combination with molecular weight analysis between 4, it was estimated that compound 10 and compound 11 were separated from each other under alkaline conditions to form a methylene group to form compound 8 and compound 9.
The fermentation method and the extraction method for the ZX06 mutant strain are the same as the treatment method for the ZX04 mutant strain. The isolation procedure for compounds in ZX06 mutants was as follows: the crude extract was dissolved in a small amount of dichloromethane and subjected to 300-400 mesh silica gel column chromatography, eluting with a gradient of dichloromethane and methanol, to give 8 fractions (fr.1-fr.8). Fr.2 and Fr.3 were combined and further purified sequentially by 200 mesh silica gel column chromatography to give 5 fractions (Fr.2.1-Fr.2.5). Combining fr.2.2 and fr.2.3 for further separation using semi-preparative HPLC, final isolation yields compound 11 and compound 10; the fr.4 and fr.5 were combined for further separation using semi-preparative HPLC, and finally isolated compound 9 and compound 8. Through analysis of C spectrum and H spectrum and the interconversion relation between the C spectrum and the H spectrum, the compound 9 can be determined to be the erythromycin CA2, and the compound 10 is 3' -hydroxy-beta-erythromycin; wherein the structures of the compound 8 and the compound 11 are shown in figure 2, the compound 8 is 3 '-hydroxy-beta-erythromycin acid, and the compound 11 is 3' -hydroxy-gamma-erythromycin, which are novel erythromycin analogues.
Table 2 Nuclear magnetic data for Compounds 3/4/11
The structural formulas of compounds 1-11 are shown in FIG. 1, respectively.
Example 8 anti-tumor analysis of Compound 1/2/3/4/8/9/10/11
Tumor cell lines were used for the experiments, namely HeLa (human cervical cancer cells), A549 (human small cell lung cancer cells) and Caco-2 (human colon adenocarcinoma cells).
The experimental method adopts a conventional CCK-8 method. Collecting cells in logarithmic phase, adjusting cell suspension concentration to make the density of the cells to be tested 2×103 ～5×103 Each well (cell density depends on the cell type), 100. Mu.L of each well was cultured in a 96-well plate, and placed at 37℃and 5% CO2 After 24h incubation, the medium was aspirated using a row gun, and then drugs of different concentration gradients (100. Mu.M, 50. Mu.M, 25. Mu.M, 10. Mu.M, 1. Mu.M, 0.1. Mu.M, 0.01. Mu.M) formulated with medium were added, each concentration being parallel to 3 duplicate wells and incubated for 72h. The medium was aspirated off, 100. Mu.L of medium solution containing 10% CCK-8 was added to each well and incubated for 1-2h until the medium turned orange in color, the plates were removed and the absorbance OD of each well was measured at 450nm on an ELISA. The experiment required the setting of zeroing wells (blank medium) and control wells (containing the same Cells, medium without drug) and positive control wells (bleomycin,) with PBS added to the border wells. Cell viability% = (dosing OD value-blank OD value)/(control OD value-blank OD value) ×100%. Taking the average value of 3 compound holes in each group, drawing a cell growth inhibition curve, and calculating IC50 Values. The results are shown in Table 3.
Table 3 test of antitumor Activity of Compound 1/2/3/4/8/9/10/11 analysis results
As can be seen from table 2, the compounds 3 and 8 have better tumor inhibition activity and can be used for preparing antitumor drugs. Especially, the activity of the compound 3 is far better than that of a similar compound 1, and is also far better than that of a positive anti-tumor western medicine control group, so that the application of the compound 3 in the future anti-tumor medicine is proved to have very good prospect.
SEQUENCE LISTING
<110> Changsha Tianci biological medicine technology Co., ltd
<120> Yuxithromycin analogue, preparation method and application thereof
<130> 1
<160> 18
<170> PatentIn version 3.5
<210> 1
<211> 978
<212> DNA
<213> Synthesis
<400> 1
atgcgtatcg tccgccacca cgcgttcggg gcccccgacg tcctgcgggt cgaggaggcg 60
ccccgccccg aacccggtgc gggccaggtc ctgatccgga ccgaggccgt cggtgtgaac 120
ttcgccgaat gccagcgccg tcagggcatc ccggtcggcg gccccgccac cctgccgggc 180
tcgccgggcg gcgatgtcgc cggagtcgtc gaggcgctcg gcgagggggt gagcgaggtg 240
agcgtcggcg accgcgtcgt caccggcgca cccttcgacg cctacgccga atacgtcgtc 300
gcgcgggccg actggctctt tccggtcccg gagtccatcg acgcggccca ggccacgtcg 360
atgccgatcc ccgcgcagac ggcgtaccac gtcatcgcga cggcggcccg gctcgccagg 420
ggtgaatcga tgctgatcac ggcagcggcc ggtggtatcg ggcacctgct cgtccaactg 480
ggcaaggcgc tcggcgccgg ccgggtgatc gctgccgcgg gcagcgcggc caagctggac 540
ttcgtccgcg ggctgggcgc cgacgtcgcg atcaactaca ccgacgagga ctgggacgag 600
caggtcaggg cggccaccga cggccgtggc gccgatgtgg tgctggagac cgtgggcggc 660
gacatcctgc ggcggtcggt cggtctgatc gccccgttcg ggcggatggt ggtctacggc 720
acggccggag gggaactgcc tgccgtggag atcgcggaca tcttcgacaa ccggacggtg 780
atgggcttca gcatgtgggg cgtgatggcc catcgcccgc aggagatggc ggacggcgcc 840
aaagccctgc tcgacctggt gggatccggc cgggtgagcc cggtgatcca cgcccggatc 900
ccgctcgacc gggccggtgc ggcgcacgcc ctgatggagg cccggtccca gctcggccgg 960
gtggtgctgg tgccctga 978
<210> 2
<211> 867
<212> DNA
<213> Synthesis
<400> 2
atgacgatcc cgatccccgc ggagagcagg gcgcgcaacg ccgggctggt ccgcggtctg 60
tacgacgtgc tttaccggcg gcaccggccg gacgaggccg ggcggctgtt ccacgacgcc 120
catgtcgacc acgacccgcg ggtggggccc ggcccctccg ggccgttgcg gcactgggag 180
tcgatgcgcg cggcctttcc cgggctgcgc gcccgtatcg gccgtctggt cgcggacgag 240
gaccgggtca tgctgttcgc ggcctggacc ggacggactc cggccggtga cccgttcgag 300
ctgcacacgg ccgagttgta ccggatcgcg gagggccgga tcgccgagca ctggaatgtc 360
accgaccggg acgtgctcgc cgggcatgtc ccggacgtgc cggacgcgct cgtggggccg 420
gagccggccg tgctgcacgg tctccacagc gaggacgagc gggccaacgc ggcggtggtg 480
ctcgacgcgt accgggtggt gttcagcgag caccggctgg atctggccga ccgctactac 540
caccgggact accggcacca caacgcgcgt acggaccggg tgcccgacgg cctggaggcg 600
ttcaccgcgt tcttcgccga caacctggcc gccttccccg atctggccgt caccgtcgag 660
cacgtggtcg ccgccgggga ccgggtgatg gtcttcgtca cctggaccgg aacgctcacc 720
ggcgcctgga gcggtggtgg cccgtccggg ctgccgttgg tgatgcgcac ctgcgaccac 780
ttccggctgg aggacgggaa ggtctccgag cactgggagg tcgtcgacta tctgccggtc 840
cggcgtgccg gactgcccac gccatga 867
<210> 3
<211> 28
<212> DNA
<213> Synthesis
<400> 3
aatgtgaaca cgcctcctcg acccgcag 28
<210> 4
<211> 40
<212> DNA
<213> Synthesis
<400> 4
gcggccgcgg atcctctaga ggcaagacca ccttgtcgcg 40
<210> 5
<211> 38
<212> DNA
<213> Synthesis
<400> 5
acgacggcca gtgccaagct tggcgaagac cgagatgc 38
<210> 6
<211> 28
<212> DNA
<213> Synthesis
<400> 6
caaccgataa aaggcgcacg agctgatg 28
<210> 7
<211> 28
<212> DNA
<213> Synthesis
<400> 7
cgtgcgcctt ttatcggttg gccgcgag 28
<210> 8
<211> 33
<212> DNA
<213> Synthesis
<400> 8
cgaggaggcg tgttcacatt cgaacggtct ctg 33
<210> 9
<211> 26
<212> DNA
<213> Synthesis
<400> 9
aatgtgaaca ggcgtcgtgg aacagc 26
<210> 10
<211> 36
<212> DNA
<213> Synthesis
<400> 10
gcggccgcgg atcctctaga ggatgtcgcg gcccga 36
<210> 11
<211> 39
<212> DNA
<213> Synthesis
<400> 11
acgacggcca gtgccaagct tggactggcc catggtgac 39
<210> 12
<211> 30
<212> DNA
<213> Synthesis
<400> 12
gccaaccgat aaaaggtctc cgagcactgg 30
<210> 13
<211> 30
<212> DNA
<213> Synthesis
<400> 13
ctcggagacc ttttatcggt tggccgcgag 30
<210> 14
<211> 32
<212> DNA
<213> Synthesis
<400> 14
cacgacgcct gttcacattc gaacggtctc tg 32
<210> 15
<211> 17
<212> DNA
<213> Synthesis
<400> 15
cccgcgcttc catcagc 17
<210> 16
<211> 17
<212> DNA
<213> Synthesis
<400> 16
acgtcctgcg ggtcgag 17
<210> 17
<211> 18
<212> DNA
<213> Synthesis
<400> 17
tcatggcgtg ggcagtcc 18
<210> 18
<211> 17
<212> DNA
<213> Synthesis
<400> 18
ccggaacgga gggactc 17