CN103031296B - Cordyceps sinensis dCMP (Deoxycytidine Monophosphate) deaminase, encoding gene and application of cordyceps sinensis dCMP deaminase - Google Patents

Abstract

The present invention relates to an enzyme derived from the "Bailing" production bacterium Cordyceps sinensis, which participates in the synthesis and metabolism of deoxyuridine acid from deoxycytidylic acid, the gene encoding the enzyme and its application. The enzyme is dCMP deaminase , having more than 90% homology of the amino acid sequence shown in SEQ ID No.1, and its coding gene has more than 90% homology of the nucleotide sequence shown in SEQ ID No.2; The metabolic pathway of nucleotide synthesis deoxyuridine has been studied in detail, and the cloned DNA comprising the nucleotide sequence provided by the present invention can be used to transfer in engineering bacteria by transduction, transformation, and transfer methods, and by regulating deoxyuridine The expression of the uridine biosynthesis gene endows the host with high expression of deoxyuridine, provides an effective way to expand the yield of deoxyuridine, and has great application prospects.

Description

Cordyceps sinensis dCMP deaminase, coding gene and application thereof

(1) Technical field

The present invention relates to a kind of dCMP deaminase (dCMP deaminase) from the "Bailing" production fungus Cordyceps sinensis which participates in the synthesis and metabolism of deoxyuridine from deoxycytidylic acid, the gene encoding the enzyme and its application.

(2) Background technology

Cordyceps sinensis (Berk.) Sacc. is a complex (including sub-seats and worm bodies) on the larvae of Cordyceps sinensis (Berk.) Sacc. . Cordyceps sinensis is a kind of precious traditional fungal medicinal resources, which has the characteristics of diverse metabolites and biological activities, and has shown great application and development prospects in the field of biomedicine. Cordyceps sinensis has attracted much attention for its extensive and obvious medicinal effects, and is highly respected all over the world. Chinese medicine believes that Cordyceps sinensis enters the two meridians of the lung and kidney, which can not only nourish the lung yin, but also nourish the kidney yang. The only traditional Chinese medicine that can balance and regulate Yin and Yang at the same time. Modern pharmacology has confirmed that Cordyceps sinensis has a wide range of biological activities such as immune regulation, antibacterial, anti-tumor, anti-oxidation, anti-aging, hypoglycemic and blood fat, and sex hormone-like effects.

Cordyceps sinensis is an ascomycete that has a conidium stage (anamorph) and an ascospore stage (sexual) in its life cycle. However, in actual production such as artificial cultivation and liquid fermentation, the asexual stage of Cordyceps sinensis is used, so the identification of the asexual form of Cordyceps sinensis is very important. Scholars at home and abroad have done a lot of work in the investigation of Cordyceps sinensis resources, confirmation of anamorphs, separation and analysis of active components, mechanism of action, development and application. Cordyceps sinensis has been proven to be the anamorphic form of Cordyceps sinensis, which has the same active ingredients and medicinal effects as natural Cordyceps sinensis.

Natural Cordyceps has strict parasitic properties and special ecological environment, so its output is very low and its price is high. Wild Cordyceps sinensis is limited by factors such as the growth environment, and resources are scarce. Due to the little progress in artificial cultivation in recent years, the research on wild Cordyceps substitutes has mostly focused on liquid fermentation. It is an effective way to solve the source of Cordyceps sinensis by using liquid submerged fermentation to cultivate the mycelia, extract or fermentation liquid of Cordyceps sinensis. Cordyceps fermented to produce substitutes of Cordyceps, which can effectively protect the precious resource of Cordyceps, and is not strictly restricted by climate, geographical environment and parasitic conditions of Cordyceps, and is suitable for industrialized large-scale production. The produced substitutes, such as mycelium, are also similar in composition and efficacy to natural Cordyceps sinensis, so domestic and foreign countries have been devoting themselves to the fermentation and cultivation of Cordyceps sinensis mycelium. The mycelium obtained by artificial fermentation of Cordyceps sinensis Trichosporum sinensis has been proved by toxicological, pharmacological and phytochemical studies to be basically consistent with the chemical composition and pharmacological effects of natural Cordyceps, and can replace natural Cordyceps to produce Cordyceps products to make up for the shortage of natural resources. By optimizing the fermentation conditions, the biomass of mycelium and the amount of metabolites were significantly increased.

In recent years, with the rapid development of natural product chemistry and modern chromatographic techniques, the research and development of Cordyceps products has gradually shifted from the direct use of Cordyceps raw materials or crude extracts to deeper research on functional metabolites. A lot of research has been done on the metabolites of Cordyceps sinensis at home and abroad. The metabolites mainly include nucleosides, polysaccharides, polypeptides, sterols, etc. The research on the biosynthesis and pharmacological effects of the product has achieved initial results.

Nucleosides are one of the most important active substances of Cordyceps, and pyrimidine nucleosides include cytidine and uridine. Uridine nucleoside (uracil riboside) is also called uridine (uIidine), also known as snow riboside, dihydropyrimidine nucleoside, 1-β-D-ribocopyranosyl uracil. Cytosine riboside (cytosine riboside) is also known as cytidine (cytidine), cytosine, 1-β-D-nucleofuranoside cytosine. Pyrimidine nucleosides are multipurpose nucleosides that can be used as additives in health products and food. As beauty and anti-ultraviolet radiation cosmetics, it has gradually become a necessity in people's life. As a drug, it has been clinically used in many diseases such as central nervous system, urinary system, metabolism and cardiovascular disease. In the United States, nucleic acid drugs have been used in antiviral, It has shown irreplaceable effect in anti-tumor.

Due to the important physiological functions of pyrimidine nucleosides and their analogs, there have been many studies on the production of pyrimidine nucleosides by microorganisms at home and abroad. However, Cordyceps sinensis, which is an important synthetic metabolite of pyrimidine nucleosides, still only stays in the analysis of metabolite components and efficacy research, and the research on related genes and proteins in the synthetic metabolic pathway of pyrimidine nucleosides in Cordyceps sinensis is still rare.

(3) Contents of the invention

The purpose of the present invention is to conduct in-depth research on the enzyme and its coding gene for synthesizing and metabolizing deoxyuridine acid of "Bai Ling" producing fungus Cordyceps sinensis Trichosporum sinensis, and to provide "Bailing" producing fungus Cordyceps sinensis participating in deoxycytidic acid Enzymes, coding genes and their applications for starting the synthesis of deoxyuridine.

The technical scheme adopted in the present invention is:

The present invention relates to a kind of dCMP deaminase derived from the "Bailing" production fungus Cordyceps sinensis, which participates in the synthesis and metabolism of deoxyuridine from deoxycytidylic acid. The enzyme has 90% of the amino acid sequence shown in SEQ ID No.1 For the above homology, preferably its amino acid sequence is shown in SEQ ID No.1 (denoted as pynP protein); this enzyme can catalyze deoxycytidine to prepare deoxyuridine. Due to the specificity of the amino acid sequence, any fragment or variant of the peptide protein containing the amino acid sequence shown in SEQ ID No.1, such as its conservative variant, biologically active fragment or derivative, as long as the fragment of the peptide protein or the peptide protein The variants have more than 90% homology with the aforementioned amino acid sequence, and all belong to the protection scope of the present invention. Specifically, the changes may include deletion, insertion or substitution of amino acids in the amino acid sequence; wherein, for conservative changes in variants, the replaced amino acids have similar structures or chemical properties to the original amino acids, such as replacing isoamino acids with leucine Leucine, variants may also have non-conservative changes, such as replacing glycine with tryptophan.

The pathway to obtain deoxyuridine from deoxycytidylate synthesis is as follows:

The invention also relates to the application of the enzyme in biocatalyzing the preparation of deoxyuridine from deoxycytidylate.

Further, the application is as follows: using the broken mixed solution of the wet bacteria obtained by fermentation and culture of the cells containing Cordyceps sinensis dCMP deaminase after the cells are broken, and using deoxycytidylic acid as the substrate, at a pH of 6.5 to 8.5 In the transformation system composed of the buffer solution, the transformation reaction was carried out at 30°C and 150rpm for 2~3h. After the reaction, the reaction solution was filtered, and the filtrate was the crude product containing deoxyuridine acid. The crude product was separated and purified to obtain deoxyuridine uridine acid.

The initial concentration of the substrate is 10g/L, the volumetric dosage of the catalyst is 100mL/g substrate, and the dry cell concentration of the catalyst is 6.7mg/mL.

The preparation method of the catalyst is as follows: the Cordyceps sinensis dCMP deaminase recombinant strain E. coliBL21/pET-28a/pynP is inoculated in 5 mL of LB liquid medium containing Kan resistance (50 mg/L), 37 ° C, 250 r/min Incubate overnight. Take 1mL of the culture, transfer it to 50mL of fresh LB liquid medium containing Kan resistance, and cultivate it at 37°C and 250r/min until the cell concentration OD600 is about 0.6-0.8, and add a certain concentration ( 240mg/ml) IPTG induction culture for 8h, take the induction culture medium and filter, collect the wet bacteria, weigh 0.5g of the collected wet bacteria, suspend in 15mL of phosphate buffer (50mM, pH8.0), ultrasonic break (power 350W , crushing for 2s, interval of 2s, total ultrasonic crushing for 5min), and the bacterial cell mixture obtained after ultrasonic crushing was used as the enzyme for catalysis.

The present invention also relates to the coding gene of the above-mentioned enzyme, i.e. dCMP deaminase gene, said gene has more than 90% homology of the nucleotide sequence shown in SEQ ID No.2, preferably its nucleotide sequence is as SEQ ID No. 2 (denoted as pynP gene). Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO: 2, as long as it has more than 90% homology with the polynucleotide, falls within the protection scope of the present invention. A polynucleotide variant refers to a polynucleotide sequence having one or more nucleotide changes. Variants of this polynucleotide may be biological variants or abiotic variants, including substitutional variants, deletion variants and insertional variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of a polynucleotide without substantially changing the function of the encoded peptide protein.

The gene can be used to construct a genetically engineered bacterium that can biocatalyze deoxycytidylic acid to prepare deoxyuridine acid, so as to expand the output of deoxyuridine acid or its derivatives. The recombinant carrier of the ammonia enzyme gene is transformed into Escherichia coli, and the obtained recombinant genetically engineered bacteria are induced and cultured, and the culture solution is separated and purified to obtain bacterial cells containing Cordyceps sinensis dCMP deaminase.

The gist of the present invention is to provide the amino acid sequence shown in SEQ ID NO: 1 and the nucleotide sequence shown in SEQ ID NO: 2. In the case of known amino acid sequence and nucleotide sequence, the amino acid sequence and The acquisition of nucleotide sequences, as well as related vectors and host cells, are obvious to those skilled in the art.

The bacterial strain capable of providing the Cordyceps sinensis dCMP deaminase and its coding gene of the present invention is Hirsutella Sinensis L0106, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC No: M 2011278, which has been It is disclosed in the previously applied patent CN102373190A.

The beneficial effects of the present invention are mainly reflected in: the present invention has studied in detail the metabolic pathway of deoxycytidylic acid synthesis deoxyuridine in principle, and the cloned DNA comprising the nucleotide sequence provided by the present invention can be used for transduction The method of transformation, transformation, and transfer is transferred into engineering bacteria, and by regulating the expression of deoxyuridine biosynthesis genes, the host is endowed with high expression of deoxyuridine, which provides a good way for expanding the output of deoxyuridine or its derivatives It is an effective way and has great application prospects.

(4) Description of drawings

Figure 1 is the formaldehyde denaturing gel electrophoresis pattern of the total RNA of the "Bailing" production fungus Cordyceps sinensis;

Figure 2 is an annotated diagram of pyrimidine metabolic pathway;

Fig. 3 is the gel electrophoresis figure of dCMP deaminase gene PCR amplification product;

Figure 4 is the physical map of the cloning vector pMD18-T Vector and the expression vector pET-28a;

Figure 5 is the physical map of the recombinant cloning plasmid pMD18-T/pynP;

Figure 6 is a schematic diagram of the construction process of the recombinant expression plasmid pET-28a/pynP;

Figure 7 is the physical map of the recombinant expression plasmid pET-28a/pynP;

Figure 8 is the SDS-PAGE diagram of dCMP deaminase protein.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Embodiment 1: The cultivation of "Bailing" production fungus Cordyceps sinensis

Source of the strain: Firstly, natural Cordyceps sinensis was collected from Qinghai, and brought back to Hangzhou for isolation and screening, and the L0106 strain was obtained, and the strain was identified as Hirsutella Sinensis, which was preserved in a typical culture in China Collection Center, the preservation number is CCTCC No: M 2011278, which has been disclosed in the previously applied patent CN102373190A.

Inoculate the strain on the slant, and the medium formula (this is the liquid formula before solidification, prepared according to the following proportions before making the slant) is glucose 2.0% (w/v, 1% means that 100mL medium contains 1g , the same below), corn flour 1.0%, potato juice 0.5%, dextrin 0.5%, yeast powder 0.5%, bran 1.0%, silkworm chrysalis powder 2.0%, peptone 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05% , agar powder 1.0%, and water as the balance, cultivated at 12-16°C for 25 days; then inoculated the strains in the fermentation medium, and the medium formula was glucose 1.0%, molasses 1.0%, silkworm chrysalis powder 0.5%, soybean cake powder 1.0%, yeast extract 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, the balance is water, placed on a shaker, and cultivated at a temperature of 12-16°C for 25 days. The liquid is separated, and the solid is placed in a sterile device for later use.

Example 2: Extraction of total RNA of "Bailing" production fungus Cordyceps sinensis

The total RNA was extracted with TRIzol reagent, and the steps were as follows: 1) Grinding with liquid nitrogen: Take 1 g of fresh bacteria and put it into a mortar, add liquid nitrogen repeatedly to grind until powdery, and divide into pre-cooled 1.5mL centrifuge tubes, Add 1mL TRIzol reagent, mix well, and let stand on ice for 5min to completely separate the nucleic acid-protein complex. 2) RNA isolation: add 0.2mL chloroform, shake vigorously for 15s, let stand on ice for 2~3min, centrifuge at 12000rpm at 4°C for 15min, separate the layers, and take the upper aqueous phase, about 600μL. 3) RNA precipitation: add 500 μL of isopropanol, let stand on ice for 10 minutes, centrifuge at 12000 rpm at 4°C for 10 minutes, and discard the supernatant. 4) RNA washing: add 1 mL of 75% (v/v) ethanol, suspend the pellet, let stand on ice for 10 min, centrifuge at 4°C, 7500 rpm for 15 min; repeat the above washing steps, and wash again. 5) Dissolving RNA: Place the centrifuge tube on ice, open and dry for 5-10 minutes, add appropriate amount of DEPC water to dissolve.

Example 3: Sequencing of the RNA sample of "Bailing" production fungus Cordyceps sinensis

After extracting the total RNA from the sample, the mRNA was enriched with Oligo(dT) magnetic beads. Add fragmentation buffer to break mRNA into short fragments (200~700bp), use mRNA as a template, use six base random primers (random hexamers) to synthesize the first cDNA strand, then synthesize the second cDNA strand, and then pass QiaQuick PCR After the kit is purified and eluted with EB buffer, end repair is performed, polyA is added and sequencing adapters are connected, and then agarose gel electrophoresis is used for fragment size selection, and finally PCR amplification is performed, and the built sequencing library is sequenced with IlluminaGA IIx . The original image data obtained by sequencing is converted into sequence data by base calling, that is, raw data or raw reads. The reads containing only the adapter sequence in the original sequencing reads were removed for subsequent analysis.

Example 4: Short-read RNA sequence assembly of the "Bailing" production fungus Cordyceps sinensis

Use the short reads assembly software SOAPdenovo (Li, Zhu et al. De novoassembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010, 20: 265-272.) to do de novo transcriptome assembly. SOAPdenovo first connects reads with a certain length of overlap into longer N-free Contig fragments. Then compare the reads back to Contig, determine the different Contigs from the same transcript and the distance between these Contigs through paired-end reads, SOAPdenovo connects these Contigs together, and the unknown sequence in the middle is represented by N, thus obtaining Scaffold. Further use paired-endreads to fill holes in the Scaffold, and finally obtain a Unigene sequence with the least N and no longer extension at both ends. Finally, the Unigene sequence was compared with the protein database nr, Swiss-Prot, KEGG, and COG by blastx (evalue<0.00001), and the protein with the best alignment result was used to determine the sequence direction of the Unigene. If there are conflicts in the comparison results between different libraries, the sequence direction of Unigene is determined according to the priority of nr, Swiss-Prot, KEGG and COG, and the Unigene software ESTScan (Iseli, Jongeneel) that cannot be compared with the above four libraries et al. ESTScan: a program for detecting, evaluating, and reconstructing potential coding regions in EST sequences[J]. In Proceedings of 9th InternationalConference on Intelligent Systems for Molecular Biology. AAAIPress, Menlo Park, 1-CA, 9pp. 19 .) predict its coding region and determine the orientation of the sequence. For the Unigene whose sequence direction can be determined, the sequence from 5' to 3' direction is given, and for the Unigene whose sequence direction cannot be determined, the sequence obtained by the assembly software is given.

Example 5: Functional annotation of "Bailing" production fungus Cordyceps sinensis Unigene

The functional annotation information gives Unigene protein functional annotation, Pathway annotation, COG functional annotation and Gene Ontology (GO) functional annotation. First, the Unigene sequence is compared to the protein database nr, Swiss-Prot, KEGG and COG (evalue<0.00001) by blastx, and the protein with the highest sequence similarity to the given Unigene is obtained, thereby obtaining the protein function annotation information of the Unigene. According to the KEGG annotation information, the Pathway annotation of Unigene can be further obtained. Comparing Unigene and COG databases, predicting the possible functions of Unigene and performing functional classification statistics. According to nr annotation information, use Blast2GO software (Conesa, Gotz et al. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research[J]. Bioinformatics, 2005, 21(18): 3674-3676.) to get Unigene GO annotation information. After getting the GO annotations of each Unigene, use WEGO software (Ye, Fang et al. WEGO: a web tool for plotting GO annotations[J]. Nucleic Acids Research, 2006,34: 293-297.) to do GO for all Unigenes Functional classification statistics, to understand the gene function distribution characteristics of the species from a macro perspective.

Example 6: Analysis of Metabolic Pathway of Deoxyuridine Acid of "Bailing" Production Fungus Cordyceps sinensis

Figure 2 shows the pyrimidine metabolism (map00240) in the annotation of KEGG metabolic pathways. The annotated enzymes are the enzymes related to the metabolic pathway of Cordyceps sinensis, which is produced by "Bailing", which has been detected. It can be seen from the figure that the detection Arrived a Unigene of dCMP deaminase that synthesizes deoxyuridine from deoxycytidine. Through the online detection of the ORF Finder software in NCBI, the open reading frame (SEQ ID No.2) of this gene was found and the corresponding protein sequence (SEQ ID No.1) was obtained.

Example 7: Design of primers for the "Bailing" production fungus Cordyceps sinensis dCMP deaminase gene

Use the GENE RUNNER primer design software to design primers based on the predicted DNA sequence of the open reading frame of the gene, and use it to clone the dCMP deaminase gene of the "Bailing" production bacteria Chaemoria chinensis to synthesize and metabolize deoxyuridine acid. The primers were provided by Shanghai Sangong Synthesized by Bioengineering Co., Ltd., the primer sequences are listed below:

pynP gene: forward primer 5' AGCGAATTCATGCTCATTGGAATTTGCGG 3'

Reverse primer 5' AGGAAGCTTTCAAAAGAGGTCCATCTTTTC 3'

The length of pynP gene is 1143bp

Example 8: Preparation of first-strand cDNA of "Bailing" production fungus Cordyceps sinensis

First according to the method provided in embodiment 1 after cultivating Mortierella sinensis fermentation mycelium, then according to the method provided in Example 2, the total RNA of Mortierella sinensis is extracted, after obtaining the total RNA, carry out "100" as follows "Ling" production fungus Cordyceps sinensis cDNA first-strand synthesis, used for subsequent gene cloning experiments.

Using PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa) to reverse transcribe the first strand of cDNA from Total RNA, the experimental steps are as follows:

1) Prepare the following mixtures in Microtube tubes.

2) Denaturation and annealing operations are conducive to the denaturation of template RNA and the specific annealing of reverse transcription primers and templates, which can improve the efficiency of reverse transcription reactions. Therefore, denaturation and annealing reactions are performed on a PCR instrument, and the conditions are set as follows:

65°C, 5 minutes

3) After annealing, centrifuge for a few seconds to collect the mixture of template RNA/primers and so on at the bottom of the Microtube tube.

4) Prepare the following reverse transcription reaction solution in the above Microtube tube.

5) Carry out the reverse transcription reaction on the PCR instrument according to the following conditions.

42℃ 15～30min

70℃ for 15 minutes

In general, there is a PolyA structure at the 3' end of eukaryotic mRNA, and the number of A bases varies from ten to several hundred. Using this structure, Oligo (dT) primers can be used to reverse transcriptase. Next, use mRNA as a template to synthesize the first strand of cDNA. This invention uses the sequence of the dT region (provided in PrimeScript 1st Strand cDNA Synthesis Kit) independently developed by TaKaRa as a primer. If the integrity of the obtained mRNA is good, then through reverse transcription The process can obtain the first-strand cDNA of all enzyme protein coding genes in the species.

Example 9: Cloning, expression and detection of protein activity of the functional gene dCMP deaminase pynP of the synthetic and metabolic deoxyuridine acid gene dCMP deaminase pynP of "Bailing" production fungus Cordyceps sinensis

1. PCR amplification of dCMP deaminase pynP gene

Using the first strand of cDNA obtained in Example 8 as a template, use the pynP gene primer synthesized in Example 7 5' AGC GAA TTC ATG CTC ATT GGA ATT TGC GG 3', 5' AGG AAG CTT TCA AAA GAG GTC CAT CTT TTC3' performs Pfu DNA polymerase PCR amplification reaction, and the reaction conditions are set as follows:

Pfu PCR amplification reaction system:

Pfu DNA Ploymerase PCR amplification conditions:

2. Gel electrophoresis detection of dCMP deaminase pynP gene PCR product

The specific detection method is as follows: 1) Heat the prepared 0.9% agarose gel in a microwave oven to dissolve it evenly; 2) Take 15mL of the gel, and when the gel is cooled to about 50°C, add 1μL of the staining solution Gold view, After mixing evenly, pour it into the electrophoresis gel plate, remove the air bubbles and insert the spotting comb; 3) After the gel solidifies, carefully take out the spotting comb, and put the gel plate into the electrophoresis tank (the end of the spotting hole is close to the negative pole of the electrophoresis tank ), add TAE electrophoresis buffer to the electrophoresis tank; 4) Take 5 μL of the sample and add 1.5 μL of 6×Loading Buffer and 4 μL of ddH ₂ O to mix, then load the sample with a pipette gun, the loading volume is 10 μL; 5) Connect the electrophoresis The power line between the tank and the electrophoresis instrument, the positive pole is red, and the negative pole is black; 6) Turn on the power and start electrophoresis, the highest voltage does not exceed 5 V/cm; 7) It can be terminated when the sample runs through 2/3 of the rubber plate Electrophoresis; 8) After cutting off the power, take out the gel and put it into a gel imager for observation and taking pictures.

Transcriptome sequencing predicted the size of the dCMP deaminase pynP gene to be 1143bp, and the results of agarose gel electrophoresis showed that the dCMP deaminase pynP gene had been successfully amplified, and the actual size was consistent with the predicted size, as shown in Figure 3.

3. Base A treatment and purification of dCMP deaminase pynP gene PCR product

Since the end of the Pfu DNA polymerase PCR product is blunt, it needs to be treated with base A after gel recovery and purified before it can be used for T-vector ligation. The gel recovery product plus base A system is as follows:

A base was added to the PCR machine at 72°C for 20 min, and finally purified with the AxyPrep PCR cleaning kit.

4. Connection of dCMP deaminase pynP gene and cloning vector

The cloning vector pMD18-T Vector was purchased from TaKaRa Company (TaKaRa code D101A). Its physical map is shown in Figure 4. The dCMP deaminase pynP gene was connected with the cloning vector to construct the recombinant plasmid pMD18-T/pynP. The physical map is shown in Figure 5. The system and connection conditions are as follows.

Connection system:

Connection conditions: 16°C, 16h; inactivation: 65°C, 15min.

5. Transformation of dCMP deaminase recombinant plasmid pMD18-T/pynP

The recombinant plasmid pMD18-T/pynP was transformed into Escherichia coli E. coli JM109 to construct recombinant bacteria E. coli JM109/pMD18-T/pynP carrying dCMP deaminase pynP gene. The specific steps are: 1) Transfer 10 μL of the reaction system to the competent cell E. coli JM109, and ice bath for 30 minutes; 2) Heat shock: 42°C, 90s; 3) Ice bath: 2-3min; 4) Add 800 μL of liquid LB , 37°C, 250rpm, 1h; 5) Coating plate (with Amp resistance); 6) Cultivate overnight in a 37°C incubator.

6. Screening of dCMP deaminase E. coli JM109/pMD18-T/pynP positive recombinant bacteria

Colony PCR does not need to extract genomic DNA, but directly uses the DNA exposed after bacterial pyrolysis as a template for PCR amplification. This method is simple and fast, and can quickly identify whether the colony is a positive colony containing the target plasmid. more common. In the experiment, the corresponding single colony inoculated into the liquid medium was subjected to colony PCR to verify whether the target gene was transferred. First, pick a single colony with a toothpick and add it to a 1.5 mL centrifuge tube containing 50 μL of sterile water, put it in a boiling water bath for 30 min, then centrifuge the supernatant as a template, and perform PCR amplification. The PCR program is set to the general program of Taq enzyme amplification. Finally, 0.9% agarose gel electrophoresis was used to detect colony PCR products.

7. Sequencing of dCMP deaminase recombinant plasmid pMD18-T/pynP

After culturing the positive recombinant bacteria detected by colony PCR in liquid LB medium overnight, take 4 mL of the bacterial liquid to extract the plasmid, according to the operating instructions provided by the AxyPrep Plasmid DNA Mini Kit. Sequencing was performed by Shanghai Sunny Biotechnology Co., Ltd. After sequencing verification, the sequence SEQ ID No.2 has been recombined into pMD18-T/pynP.

8. Construction of dCMP deaminase recombinant expression plasmid pET-28a/pynP

According to the principle of exogenous gene expression in Escherichia coli and the comparison of the pET-28a expression vector and dCMP deaminase pynP gene restriction site, the EcoRI and Hind III double restriction sites were determined, and the recombinant large intestine The bacillus E. coli JM109/pMD18-T/pynP was cultured on a liquid LB test tube shaker and the recombinant plasmid was extracted.

The recombinant plasmid pMD18-T/pynP of the dCMP deaminase pynP gene and the expression vector pET-28a were digested with EcoRI/Hind III restriction endonucleases at 37°C for 6 hours respectively. The digestion system was as follows:

EcoRI/HindⅢ double enzyme digestion system:

After enzyme digestion, inactivate at 65°C for 15 minutes, and then recover and purify with Axygen DNA Gel Extraction Kit.

The dCMP deaminase pynP gene and the expression vector pET-28a were digested and purified, and then ligated with T4 ligase overnight at 16°C to construct the recombinant expression plasmid pET-28a/pynP. The construction process is shown in Figure 6, and the dCMP obtained The map of the deaminase recombinant expression plasmid pET-28a/pynP is shown in Figure 7. The connection system consists of the following:

Connection system:

9. Transformation of dCMP deaminase recombinant expression plasmid pET-28a/pynP and screening of positive single clones

The two dCMP deaminase recombinant expression plasmids pET-28a/pynP constructed were heat-shock transformed into E. coli BL21 host bacteria, and then coated with kanamycin (Kan) resistance (50mg/L) Incubate overnight at 37°C on LB agar plates. Randomly select a single colony from the plate, perform PCR amplification with primers for each functional gene, and select positive clones.

10. Induced expression of dCMP deaminase recombinant bacteria E. coli BL21/pET-28a/pynP

The identified positive monoclonals were inoculated in 5 mL of LB liquid medium containing Kan resistance (50 mg/L), and cultured overnight at 37 °C and 250 r/min. Take 1 mL of the culture and transfer it to 50 mL of LB liquid medium containing Kan resistance (50 mg/L) at 37°C and 250 r/min to cultivate until the cell concentration OD600 is about 0.6-0.8. A certain concentration (240mg/L) of IPTG was added to the culture to induce culture for 8h. The bacteria were collected for electrophoresis analysis and enzyme activity detection.

11. SDS-PAGE analysis of expression products of dCMP deaminase recombinant bacteria E. coli BL21/pET-28a/pynP

E. coli BL21 bacteria transformed with empty vector and recombinant bacteria without induction agent IPTG were used as controls. After the recombinant bacteria identified as positive were induced and cultivated by IPTG for a certain period of time (8h), 0.5mL of the induced culture was taken, collected by centrifugation, resuspended in 50μL of distilled water, added 50μL of loading buffer, mixed evenly, and boiled for 10min. SDS-PAGE electrophoresis analysis, Figure 8 is the SDS-PAGE figure of the dCMP deaminase protein pynP expressed by recombinant bacteria E. coli BL21/pET-28a/pynP (the amino acid sequence is shown in SEQ ID No.1 after sequencing verification) .

12. Detection of protein activity of dCMP deaminase recombinant bacteria E. coli BL21/pET-28a/pynP

Preparation of enzyme solution: Weigh 0.5 g (dry weight 0.1 g) of the collected recombinant bacteria E. coli BL21/pET-28a/pynP wet cells, suspend them in 15 mL of phosphate buffer (50 mM, pH 8.0), and ultrasonically break ( Power 350W, crushing 2s, interval 2s, total ultrasonic crushing 5min) used as catalytic enzyme.

dCMP deaminase pynP transformation system: Add 10mL of E. coli BL21/pET-28a/pynP ultrasonically disrupted bacteria, 0.1g of deoxycytidylic acid in a 50mL transformation bottle, 30℃, 150r/min transformation reaction for 2~3h, transformation After the end, centrifuge to take the supernatant for subsequent detection.

Detection method: quantitative detection of deoxyuridine acid by high performance liquid chromatography. Pre-treatment: After the transformation solution is centrifuged, the supernatant is passed through a 0.22 μm microporous membrane for detection by high performance liquid chromatography. Conditions: Chromatographic column: Agilent C18 column (4.6mm×250mm i.d.5μm); column temperature: 35°C; injection volume: 20μL; flow rate 1mL/min; detection wavelength 260nm; mobile phase: A: ultrapure water; B: methanol . Gradient elution: 0min, 15%B, hold for 3min; 3.0-3.5min, 15-24%B; 3.5min, 24% for 5min; 8.5-9.0min, 24-35%B; 35%B for 6min; 15.0 -16.0min, 35-85%B; 85%B for 6min; 22.0-22.5min, 85%-15%B; 15%B for 5min.

Preparation of the standard curve: Accurately weigh the standard deoxyuridine acid, about 1.00mg-2.00mg, and dilute to volume with ultrapure water. Prepare 6 gradients (1μg/mL, 30μg/mL, 60μg/mL, 90μg/mL, 120μg/mL, 150μg/mL) for each standard. Then the samples were loaded sequentially, and each concentration was repeated 5 times. Take a certain volume of the standard solution with the highest concentration and mix them together, then make up the volume, and then analyze the optimal conditions for the detection of deoxyuridine acid by HPLC.

After the detection and calculation of the above chromatographic conditions, we draw the following conclusions: the maximum specific activity of dCMP deaminase expressed by dCMP deaminase recombinant bacteria E. coli BL21/pET-28a/pynP is 820mol/ min/mg, the substrate conversion rate was 98%.

EQUENCE LISTING

the

<110> Zhejiang University of Technology, Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd.

the

<120> Cordyceps sinensis dCMP deaminase, coding gene and application thereof

the

<130>

the

<160> 2

the

<170> PatentIn version 3.5

the

<210> 1

<211> 380

<212> PRT

<213> Hirsutella Sinensis

the

<400> 1

the

Met Leu Ile Gly Ile Cys Gly Gly Ile Cys Ser Gly Lys Lys Thr Val

1 5 10 15

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the

Ala Lys Tyr Leu Val Glu His His Gly Phe Lys Leu Leu His Leu Thr

20 25 30

the

the

Gly His Arg Gln Ala Ser Phe Ser Ser Ala Ser Glu Leu Gly Ser Pro

35 40 45 45

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the

Asp Pro Leu Ser Pro Arg Pro Arg Ser Pro Pro Pro Ala Gly Ala Leu

50 55 60 60

the

the

Gly Ser Ser Ala Leu Thr Thr Gly Asn Asp Asp Gly Trp Gln Ser Ser

65 70 75 80

the

the

Ser Asp Ser Ser Ser Leu Ala Leu Arg Pro Ser Pro Gly Gly Ser Ser Leu

85 90 95

the

the

Cys Phe Ala Thr Ala Glu Ala Leu Leu Glu Phe Val Thr Thr Arg Trp

100 105 110

the

the

Gly Gly Arg Phe Val Thr Thr Asn Ile Pro Thr Glu Ala Ala Leu Asp

115 120 125

the

the

Val Leu Leu Arg Arg Pro Phe Phe Leu Leu Leu Ser Val Asp Ala Pro

130 135 140

the

the

Leu Thr Val Arg Trp Arg Arg Phe Gln Gln Arg Gly Gly Asp Ser Ala

145 150 155 160

the

the

Ala Gly Val Ser Leu Asp Asp Phe Val Ala Arg Ser Asp Ala His Leu

165 170 175

the

the

Tyr Asp Ala Asp Arg Gly Leu Gln Pro Leu Ile Ser Arg Ala Ser Val

180 185 190

the

the

Arg Leu Leu Asn Thr Ser Ser Ser Ser Leu Ala His Leu Tyr Ala Thr Leu

195 200 205

the

the

Gly Lys Leu Asp Ile Pro Asn Pro Asp Arg Leu Arg Pro Gly Trp Asp

210 215 220

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Thr Tyr Phe Met Ala Leu Ala Ser Leu Ala Ala Gln Arg Ser Asn Cys

225 230 235 240

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Met Lys Arg Arg Val Gly Cys Val Leu Val Gly Arg Glu Arg Arg Val

245 250 255

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Ile Ser Thr Gly Tyr Asn Gly Thr Pro Arg Gly Leu Arg Asn Cys Ala

260 265 270

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Asp Gly Gly Cys Pro Arg Cys Asn Ala Gly His Gly Ser Gly Val Gly

275 280 285

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Leu Ala Thr Cys Leu Cys Ile His Ala Glu Glu Asn Ala Leu Leu Glu

290 295 300

the

the

Ala Gly Arg Glu Arg Ile Arg Asp Gly Ala Val Leu Tyr Cys Asp Thr

305 310 315 320

the

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Cys Pro Cys Leu Thr Cys Ser Ile Lys Ile Cys Gln Val Gly Ile Ser

325 330 335

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Glu Val Val Tyr Ala His Gly Tyr Ser Met Asp Lys Glu Thr Ala Ala

340 345 350

the

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Val Phe Ser Gln Ala Gly Val Lys Leu Arg Gln Phe Val Pro Pro Pro

355 360 365

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Asn Gly Leu Ile His Leu Glu Lys Met Asp Leu Phe

370 375 380

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the

<210> 2

<211> 1143

<212> DNA

<213> Hirsutella Sinensis

the

<400> 2

atgctcattg gaatttgcgg aggcatctgc tccggcaaga agacggtggc caagtacctc 60

the

gtcgagcatc acggtttcaa gctgctccat ctcacggggc accgccaggc gtcgttcagc 120

the

tccgcctcgg agctcggggag cccggaccct ctgtctcctc gtcctcgctc ccctccaccg 180

the

gccggcgcct tgggctcgag cgccctgact accggcaacg acgatggctg gcagtcgtcg 240

the

tcggactcgt cgctggccct gcgcccatcg cctggcggca gcagtctttg cttcgccacg 300

the

gccgaggcgc tgctcgagtt tgtcacgacg cgctggggcg gccgctttgt gacgaccaac 360

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atccccaccg aggcggccct cgacgtgctg ctgcgccgtc cctttttcct gctgctatcc 420

the

gtcgacgcgc cgctgacggt caggtggcgg cgcttccagc agcgcggcgg cgactcggcg 480

the

gcaggcgtca gcctcgacga ctttgtcgcg cgcagcgacg cgcacctgta cgacgcggac 540

the

cggggcctgc agccgctcat ctcgcgcgcg tccgtcaggc tgctcaacac gtcgtcgtcg 600

the

ctggcccacc tctacgccac gctcggcaag ctcgacatcc ccaacccgga ccgcctgcgc 660

the

ccaggctggg acacgtactt tatggcactc gcgtcgctgg ccgcccagcg ctccaactgc 720

the

atgaagcgac gggtcgggtg cgtcctcgtc ggccgcgagc ggcgcgtcat cagcacgggc 780

the

tacaacggca cgccgcgggg cctccgcaac tgcgccgacg gcggctgtcc gcgctgcaac 840

the

gccggccacg gctccggcgt cgggctggcc acgtgcctct gcatccacgc cgaggagaac 900

the

gccctgctcg aggccggccg cgagcgcatc agagacgggg ccgtcctcta ctgcgacacg 960

the

tgcccgtgcc tgacgtgcag catcaagatt tgccaggtgg gcatcagcga ggtcgtctac 1020

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gcccacggct acagcatgga caaggagacg gccgccgtct ttagccaggc gggcgtcaag 1080

the

ctgcggcagt ttgtgccgcc gcccaatggt ctgattcacc tggaaaagat ggacctcttt 1140

the

tga 1143

the

the

Claims (8)

1. a Cordyceps sinensis dCMP desaminase, is characterized in that the aminoacid sequence of described enzyme is for shown in SEQ ID No.1.

2. the application in deoxyuridylic acid prepared by Cordyceps sinensis dCMP desaminase at biocatalysis deoxycytidylic acid(dCMP) as claimed in claim 1.

3. apply as claimed in claim 2, it is characterized in that described being applied as: with the broken mixed solution of wet thallus after cytoclasis obtained containing Cordyceps sinensis dCMP desaminase thalline fermentation culture for catalyzer, take deoxycytidylic acid(dCMP) as substrate, be in the transformation system of buffered soln formation of 6.5 ~ 8.5 in pH, at 30 DEG C, conversion reaction 2 ~ 3h under 150rpm condition, after reaction terminates, by reacting liquid filtering, get filtrate and be crude product containing deoxyuridylic acid, described crude isolate purified acquisition deoxyuridylic acid.

4. apply as claimed in claim 3, it is characterized in that the starting point concentration of described substrate is 10g/L, the volumetric usage of described catalyzer is 100mL/g substrate, and the dry mycelium concentration of described catalyzer is 6.7mg/mL.

5. the gene of enzyme described in claim 1 of encoding.

6. gene as claimed in claim 5, is characterized in that the nucleotides sequence of described gene is classified as shown in SEQ ID No.2.

7. as described in one of claim 5 ~ 6, gene is building and can prepare application in the genetic engineering bacterium of deoxyuridylic acid by biocatalysis deoxycytidylic acid(dCMP).

8. apply as claimed in claim 7, it is characterized in that described being applied as: build the recombinant vectors containing described Cordyceps sinensis dCMP deaminase gene, described recombinant vectors is converted in intestinal bacteria, the recombination engineering bacteria obtained carries out inducing culture, and nutrient solution separation and purification obtains the somatic cells containing Cordyceps sinensis dCMP desaminase.