CN103045573A - Cordyceps sinensis cytosine deaminase, coding gene and application thereof - Google Patents

Abstract

The invention relates to an enzyme for synthesizing uracil for cytosine or for synthesizing metabolic 5-methyl cytosine from thymidine, which is participated by hirsutella sinensis of Cordyceps sinensis which is Bailing producing strain, a gene for coding the enzyme and applications thereof, wherein the enzyme is a cytosine deaminase which has more than 90% of homology with an nucleotide sequence shown in SEQ ID No.1; and the coding gene has more than 90% of homology with a nucleotide sequence shown in SEQ ID No.2. The invention detailedly researches the metabolism approach of synthesis of uracil by cytosine and synthesis of 5-methyl cytosine by thymidine in principle. The clone DNA of the nucleotide sequence provided in the invention can be transferred into engineering bacteria by a transduction, conversion and conjugal transfer method. By regulating the expression of biosynthesis genes of uracil and 5-methyl cytosine, high expressivity of uracil and 5-methyl cytosine is endowed to a host. The invention provides an effective approach for raising the output of uracil and 5-methyl cytosine, which has a great application prospect.

Description

Cordyceps sinensis Isocytosine deaminase, encoding gene and application thereof

(1) technical field

The present invention relates to a kind of Isocytosine deaminase (cytosine deaminase) that sets out the anabolism uridylic and synthesize 5-methylcytosine from thymus pyrimidine from the participation cytosine(Cyt) of " hundred make " production bacterium Cordyceps sinensis China pilose spore, the gene of this enzyme of encoding and application thereof.

(2) background technology

Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is that Cordyceps fungus colonizes in stroma and the complex body on the larva corpse (comprising stroma and polypide) on lepidopteran (Lepidoptera) Hepialidae insect (the Hepialus armoricanus Oberthur) larva.Cordyceps sinensis is traditional fungi herb resource that a class is treasured, and has the various characteristics of meta-bolites and biological activity, shows huge application and development prospect at biomedicine field.Cordyceps sinensis with its multiple medicinal efficacy extensively, obviously receive much concern worldwide enjoys high praise.The traditional Chinese medical science thinks, Cordyceps sinensis enters lung kidney two warps, and is can tonifying lung cloudy, again can kidney-replenishing, and cure mainly and suffer from a deficiency of the kidney, impotence and seminal emission, soreness of waist and knee joint, weak after being ill, chronic cough is weak, phthisical cough phlegm blood, spontaneous sweatings etc. are unique a kind of simultaneously balances, regulate the Chinese medicine of negative and positive.Modern pharmacology confirms, and Cordyceps sinensis has the widely biological activity such as immunomodulatory, antibiotic, antitumor, anti-oxidant, anti-ageing, hypoglycemic blood fat, gonadotropic Effect.

Cordyceps fungus is a kind of ascomycetes, has Conidial Stage (anamorph) and thecaspore stage (teleomorph) in its life history.And what use in the actual productions such as artificial culture, liquid fermenting is the Cordyceps fungus in imperfect stage, thereby the evaluation of Anamorph of Cordyceps Sinensis is extremely important.Chinese scholars is being done a lot of work aspect Cordyceps Resources investigation, anamorph conclusive evidence, activeconstituents compartment analysis and the mechanism of action, the Application and Development.The Cordyceps sinensis China pilose spore has been proved to be the anamorph existence form of Cordyceps sinensis, has the activeconstituents identical with natural cordyceps and drug effect.

Natural cs has strict parasitics and special ecotope, therefore its output is very low, price is high.Wild cordyceps restricts scarcity of resources owing to factors such as being subjected to growing environment.Owing to making little progress at artificial culture in recent years, the research of wild cordyceps surrogate focuses mostly on liquid fermenting.Utilizing liquid submerged fermentation to cultivate Cordyceps mycelium, extract or fermented liquid, is a kind of effective way that solves Cordyceps sinensis medicine source.Chinese caterpillar fungus fermentation is produced the Chinese caterpillar fungus substitute, both can effectively protect these precious resources of Chinese caterpillar fungus, and not climate, geographical environment and the strict restriction of Chinese caterpillar fungus parasitic conditions is suitable for large-scale industrialization production again.Its composition of the substitute of producing such as mycelium is also similar to natural cs with drug effect, thereby is devoted to the fermentation culture of Cordyceps mycelium both at home and abroad always.The mycelia that the aweto cultured by artificial fermentation China pilose spore obtains, through toxicity, pharmacology, plant research, proof is basically identical with natural cs chemical constitution, pharmacological action, can replace natural cs to produce cordyceps product, to remedy the shortage of natural resources, by the optimization to fermentation condition, the amount of mycelial biomass and meta-bolites all is significantly improved.

In recent years, along with the develop rapidly of natural product chemistry and modern chromatographic technique, to progressively turning to deeper functional meta-bolites research by the direct utilization of Chinese caterpillar fungus raw material or crude extract in the worm grass product research and development.The Chinese caterpillar fungus meta-bolites has been done a large amount of research both at home and abroad, meta-bolites mainly comprises several large compounds such as nucleosides, polysaccharide, polypeptide, sterol, and wherein the representative researchs of functional meta-bolites at aspects such as biosynthesizing, pharmacological actions such as miazines nucleosides, Cordyceps polysaccharide, N.F,USP MANNITOL win initial success.

Ucleosides is one of topmost active substance of Chinese caterpillar fungus, and wherein the miazines nucleosides comprises cytidine(C and uridine.Uridine (uracil riboside) claim again uridine (uIidine), and another name is by snow riboside, dihydro-pyrimidin nucleosides, the 1-β-D-ribosyl uridylic of muttering.Cytidine(C (cytosine riboside) has another name called cytidine (cytidine), cytidine, 1-β-D-furans nucleosides cytosine(Cyt).Pyrimidine nucleoside is multiduty nucleosides, can be used as the additive of healthcare products and food.It also becomes the requisite of people's life gradually as beauty treatment and anti-ultraviolet radiation makeup, as medicine, clinical application is in nervus centralis, uropoiesis, metabolism and many-sided disease such as cardiovascular, in the U.S., the nucleic acid medicine has shown irreplaceable effect at antiviral, anti-tumor aspect in recent years.

Because the important physiological action of pyrimidine nucleoside and analogue thereof, related microorganism is produced the existing a lot of researchs of miazines nucleosides both at home and abroad.Yet as the Cordyceps fungus of important anabolism miazines nucleosides, also only rest in the research of meta-bolites composition analysis and effect, also rarely found to the research of genes involved and albumen in the Cordyceps fungus miazines nucleosides metabolic pathway of synthesizing.

(3) summary of the invention

The object of the invention is that enzyme and the encoding gene thereof of " hundred make " being produced bacterium Cordyceps sinensis China pilose spore anabolism uridylic or 5-methylcytosine further investigate, and provides " hundred makes " to produce bacterium Cordyceps sinensis China pilose spore participation cytosine(Cyt) set out anabolism uridylic or thymus pyrimidine set out enzyme, encoding gene and the application thereof of anabolism 5-methylcytosine.

The technical solution used in the present invention is:

The present invention relates to a kind ofly produce bacterium Cordyceps sinensis China pilose spore from " hundred make " and participate in the set out Isocytosine deaminase of anabolism uridylic or 5-methylcytosine of cytosine(Cyt) or thymus pyrimidine, described enzyme has the 90% above homology of aminoacid sequence shown in the SEQ ID No.1, and preferably its aminoacid sequence (is designated as pynL albumen) shown in SEQ ID No.1; But this enzyme catalysis cytosine(Cyt) prepares uridylic, or the catalysis thymus pyrimidine prepares 5-methylcytosine.Because the singularity of aminoacid sequence; any fragment or its variant that contains the peptide protein of aminoacid sequence shown in the SEQ ID No.1; such as its examples of conservative variations, bioactive fragment or derivative; as long as the fragment of this peptide protein or peptide protein variant and aforementioned amino acid sequence homology all belong to the row of protection domain of the present invention more than 90%.Concrete described change can comprise amino acid whose disappearance, insertion or replacement in the aminoacid sequence; Wherein, for the conservative property change of variant, the amino acid of replacing has the structure similar to original acid or chemical property, and as replacing Isoleucine with leucine, variant also can have non-conservation and change, as replacing glycine with tryptophane.

The path that is obtained uridylic or 5-methylcytosine by cytosine(Cyt) or thymus pyrimidine anabolism is as follows:

The invention still further relates to described enzyme prepares uridylic or biocatalysis thymus pyrimidine at the biocatalysis cytosine(Cyt) and prepares application in the 5-methylcytosine.

Further, described being applied as: to contain the broken mixed solution of wet thallus after cytoclasis that Cordyceps sinensis Isocytosine deaminase thalline fermentation culture obtains as catalyzer, take cytosine(Cyt) or thymus pyrimidine as substrate, be in the transformation system that consists of of 6.5 ~ 8.5 buffered soln in pH, conversion reaction 2 ~ 3h under 30 ℃, 150rpm condition, after reaction finishes, with reacting liquid filtering, get the crude product that filtrate being contains uridylic or 5-methylcytosine, described crude product separation and purification obtains uridylic or 5-methylcytosine.

The starting point concentration of described substrate is 10g/L, and the volumetric usage of described catalyzer is the 100mL/g substrate, and the dry mycelium concentration of described catalyzer is 6.7mg/mL.

The preparation method of described catalyzer is: Cordyceps sinensis Isocytosine deaminase recombinant bacterium E. coli BL21/pET-28a/pynL is inoculated in the LB liquid nutrient medium that 5mL contains Kan resistance (50mg/L) 37 ℃, 250r/min overnight incubation.Get the 1mL culture, its transfer in the fresh LB liquid nutrient medium that contains the Kan resistance of 50mL 37 ℃, 250r/min are cultured to cell concentration OD ₆₀₀Be about about 0.6～0.8, the IPTG inducing culture 8h that adds finite concentration (240mg/ml) in the culture, getting inducing culture liquid filters, collect wet thallus, the wet thallus 0.5g that takes by weighing collection suspends with phosphate buffered saline buffer (50mM, pH8.0) 15mL, and the thalline mixed solution that obtains after the ultrasonication (power 350W, broken 2s, interval 2s, altogether ultrasonication 5min) is as the catalysis enzyme.

The invention still further relates to the encoding gene of above-mentioned enzyme, i.e. cytosine deaminase gene, described gene has the 90% above homology of nucleotide sequence shown in the SEQ ID No.2, and preferably its nucleotide sequence (is designated as the pynL gene) shown in SEQ ID No.2.Because the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQ ID NO:2 as long as itself and this polynucleotide have 90% above homology, all belongs to the row of protection domain of the present invention.The variant of described polynucleotide refers to a kind of polynucleotide sequence that one or more Nucleotide change that has.The variant of these polynucleotide can make living displacement varient or the varient of non-life, comprises replacing varient, deletion mutation body and inserting varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of polynucleotide, but can be from the function of the peptide protein that changes in fact its coding.

Described gene can be used for making up can the biocatalysis cytosine(Cyt) or thymus pyrimidine prepare the genetic engineering bacterium of uridylic or 5-methylcytosine, to enlarge the output of uridylic and 5-methylcytosine or derivatives thereof.Described being applied as: make up the recombinant vectors that contains described cytosine deaminase gene, described recombinant vectors is converted in the intestinal bacteria, the recombination engineering bacteria that obtains carries out inducing culture, and the nutrient solution separation and purification obtains to contain the somatic cells of Isocytosine deaminase.

Main points of the present invention have been to provide the nucleotide sequence shown in the aminoacid sequence shown in the SEQ ID NO:1 and the SEQ ID NO:2, in the situation that known this aminoacid sequence and nucleotide sequence, the acquisition of this aminoacid sequence and nucleotide sequence, and the acquisition of related vector, host cell, all be apparent to those skilled in the art.

The bacterial strain that Cordyceps sinensis Isocytosine deaminase of the present invention and encoding gene thereof can be provided is China pilose spore (Hirsutella Sinensis) L0106, this culture presevation is at Chinese Typical Representative culture collection center, deposit number is CCTCC No:M 2011278, formerly discloses among the patent CN102373190A of application.

Beneficial effect of the present invention is mainly reflected in: the present invention studies in great detail the synthetic uridylic of cytosine(Cyt) or the synthetic 5-methylcytosine pathways metabolism of thymus pyrimidine on principle, the cloned DNA that comprises nucleotide sequence provided by the present invention can be used for by transduction, transform, change in the engineering bacteria in conjunction with the method that shifts, by regulating the expression of uridylic or 5-methylcytosine biosynthesis gene, give the high expression level of host's uridylic or 5-methylcytosine, for the output that enlarges uridylic and 5-methylcytosine or derivatives thereof provides effective way, has the major application prospect.

(4) description of drawings

Fig. 1 is the denaturing formaldehyde gel electrophoresis figure that " hundred make " produces the total RNA of bacterium Cordyceps sinensis China pilose spore;

Fig. 2 is pyrimidine metabolic approach annotated map;

Fig. 3 is cytosine deaminase gene pcr amplification product gel electrophoresis figure;

Fig. 4 is cloning vector pMD18-T Vector and expression vector pET-28a physical map;

Fig. 5 is restructuring cloned plasmids pMD18-T/pynL physical map;

Fig. 6 is recombinant expression plasmid pET-28a/pynL building process schematic diagram;

Fig. 7 is recombinant expression plasmid pET-28a/pynL physical map;

Fig. 8 is Isocytosine deaminase protein SDS-PAGE figure.

(5) embodiment

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

Embodiment 1: " hundred make " produces the cultivation of bacterium Cordyceps sinensis China pilose spore

Bacterium source: at first gather natural cordyceps from Qinghai, and it is taken back Hangzhou carry out separation screening, obtained the L0106 bacterial strain, and be China pilose spore (Hirsutella Sinensis) through this bacterial strain of strain identification, this culture presevation is at Chinese Typical Representative culture collection center, deposit number is CCTCC No:M 2011278, formerly discloses among the patent CN102373190A of application.

With this bacterial classification inoculation in the inclined-plane, (this is the liquid formulations before solidifying to culture medium prescription, prepare afterwards again bevel in following ratio) be glucose 2.0%(w/v, contain 1g in the 1% expression 100mL substratum, down together), Semen Maydis powder 1.0%, murphy juice 0.5%, dextrin 0.5%, yeast powder 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, peptone 1.0%, sal epsom 0.05%, potassium primary phosphate 0.05%, agar powder 1.0%, surplus is water, cultivates 25 days at 12 ~ 16 ℃; Then with bacterial classification inoculation in fermention medium, culture medium prescription is glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal 0.5%, soybean cake powder 1.0%, yeast extract paste 0.5%, sal epsom 0.01%, potassium primary phosphate 0.02%, surplus is water, place on the shaking table, 12 ~ 16 ℃ of cultivations of temperature 25 days under aseptic condition, are carried out solid-liquid separation after cultivation finishes, and solid placed aseptic utensil, for subsequent use.

Embodiment 2: " hundred make " produces the extraction of the total RNA of bacterium Cordyceps sinensis China pilose spore

Extract total RNA with TRIzol reagent, step is specially: 1) liquid nitrogen grinding: get the new fresh thalli of 1g and put into mortar, repeatedly adding liquid nitrogen fully is ground to Powdered, divide and install in the 1.5mL centrifuge tube of precooling, add 1mL TRIzol reagent, mixing leaves standstill 5min on ice, and the nucleic acid-protein mixture is separated fully.2) RNA separates: add the 0.2mL chloroform, firmly shake mixing 15s, leave standstill 2 ~ 3min on ice, and 4 ℃, the centrifugal 15min of 12000rpm, the upper strata water is got in layering, approximately 600 μ L.3) RNA precipitation: add 500 μ L Virahols, leave standstill 10min on ice, 4 ℃, the centrifugal 10min of 12000rpm abandon supernatant.4) RNA washing: add 1mL 75%(v/v) ethanol will precipitate and hang, and leave standstill 10min on ice, 4 ℃, the centrifugal 15min of 7500rpm; Washing step above repeating is washed one time again.5) dissolving RNA: centrifuge tube is placed unlimited dry 5 ~ 10min on ice, add an amount of DEPC water dissolution.

Embodiment 3: " hundred make " produces the order-checking of bacterium Cordyceps sinensis China pilose spore RNA sample

After extracting the total RNA of sample, use with Oligo(dT) enrichment with magnetic bead mRNA.Add fragmentation buffer mRNA is broken into short-movie section (200 ~ 700bp), take mRNA as template, with the synthetic article one cDNA chain of hexabasic basic random primer (random hexamers), then synthesize second cDNA chain, pass through again QiaQuick PCR test kit purifying and add the EB buffer solution elution and do terminal reparation afterwards, add polyA and connect sequence measuring joints, then carrying out clip size with agarose gel electrophoresis selects, carry out at last pcr amplification, the sequencing library of building up checks order with Illumina GA IIx.The raw image data that order-checking obtains is converted into sequence data through base calling, i.e. raw data or raw reads.Remove the reads that only contains the adaptor sequence among the primitive sequencer reads, standby with subsequent analysis.

Embodiment 4: " hundred make " produces the short sequence assembling of reading of bacterium Cordyceps sinensis China pilose spore RNA

Use short reads composite software SOAPdenovo(Li, Zhu et al. De novo assembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010,20:265-272.) do and transcribe group and from the beginning assemble.The reads that SOAPdenovo at first will have certain-length overlap is linked to be the longer Contig fragment that does not contain N.Then reads is compared back Contig, determine from the different Contig of same transcript and the distance between these Contig by paired-end reads, SOAPdenovo connects together these Contig, and middle unknown nucleotide sequence represents with N, so just obtains Scaffold.Further utilize paired-end reads that Scaffold is done filling-up hole and process, obtain at last containing N minimum, the Unigene sequence that two ends can not prolong again.At last, Unigene sequence and albumen database nr, Swiss-Prot, KEGG and COG are blastx compare (evalue＜0.00001), get the sequence direction that the best albumen of comparison result is determined Unigene.If the comparison result between the different sink is contradictory, then press nr, Swiss-Prot, the priority of KEGG and COG is determined the sequence direction of Unigene, with above four storehouses all to less than Unigene software ESTScan(Iseli, Jongeneel 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, CA, pp. 1999,138-148.) predict its coding region and determine the direction of sequence.Provide its sequence from 5' to the 3' direction for the Unigene that can determine the sequence direction, provide the sequence that composite software obtains for the Unigene that can't determine the sequence direction.

Embodiment 5: " hundred make " produces bacterium Cordyceps sinensis China pilose spore Unigene functional annotation

Functional annotation information provides protein function note, Pathway note, COG functional annotation and the Gene Ontology(GO of Unigene) functional annotation.At first, by blastx with the Unigene sequence alignment to albumen database nr, Swiss-Prot, KEGG and COG(evalue＜0.00001), obtain having the albumen of highest serial similarity with given Unigene, thereby obtain the protein function annotation information of this Unigene.Can further obtain the Pathway note of Unigene according to the KEGG annotation information.Unigene and COG database are compared, and the possible function of prediction Unigene is also done the function statistic of classification to it.According to the 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.) obtain the GO annotation information of Unigene.After obtaining the GO note of each Unigene, with WEGO software (Ye, Fang et al. WEGO:a web tool for plotting GO annotations[J]. Nucleic Acids Research, 2006,34:293-297.) all Unigene are done GO functional classification statistics, from the gene function distribution characteristics of these species of macroscopic view understanding.

Embodiment 6: " hundred make " produces bacterium Cordyceps sinensis China pilose spore uridylic and the 5-methylcytosine pathways metabolism is analyzed

Fig. 2 is the pyrimidine metabolic (map00240) in the KEGG pathways metabolism note, the enzyme of note is that " hundred make " that has detected produced bacterium Cordyceps sinensis China pilose spore pyrimidine metabolic approach relevant enzymes, as can be seen from the figure, detected from the synthetic uridylic of cytosine(Cyt) or from 1 Unigene of Isocytosine deaminase of the synthetic 5-methylcytosine of thymus pyrimidine.Detect online by the ORF Finder software among the NCBI, found out the open reading frame (SEQ ID No.2) of this gene and obtained corresponding protein sequence (SEQ ID No.1).

Embodiment 7: " hundred make " produces bacterium Cordyceps sinensis China pilose spore cytosine deaminase gene design of primers

The gene open reading frame dna sequence dna design primer that uses GENE RUNNER primer-design software to obtain according to prediction, be used for the cytosine deaminase gene that clone's " hundred make " produces bacterium China pilose spore anabolism uridylic or 5-methylcytosine, primer is given birth to worker's biotechnology company limited by Shanghai and is synthesized, and primer sequence is following listed:

PynL gene: forward primer 5 ' ATAGAATTCATGGGCAACGACACGACGGCG 3 '

Reverse primer 5 ' AGGAAGCTTTCACGTTGCCCGCATCTTGATTC 3 '

The pynL mrna length is 1971bp

Embodiment 8: " hundred make " produces the preparation of bacterium Cordyceps sinensis China pilose spore cDNA the first chain

After the method that provides according to embodiment 1 is first turned out sutella sinensis fermented mycelium, the method that provides according to embodiment 2 is again carried out the extraction of total RNA to China pilose spore, obtain being undertaken synthesizing of " hundred make " production bacterium Cordyceps sinensis China pilose spore cDNA the first chain by following behind total RNA, be used for follow-up each gene clone experiment.

Adopt synthetic cDNA the first chain of PrimeScript 1st Strand cDNA Synthesis Kit test kit (TaKaRa) reverse transcription from Total RNA, experimental procedure is as follows:

1) the following mixed solution of preparation in the Microtube pipe.

2) sex change, annealing operation are conducive to the sex change of template ribonucleic acid and the specificity annealing of reverse transcription primer and template, can improve reverse transcription reaction efficient, so carry out sex change, annealing reaction at the PCR instrument, condition setting is as follows:

65℃，5 min

3) the centrifugal several seconds made the mixed solution of template ribonucleic acid/primer etc. be gathered in Microtube pipe bottom after annealing finished.

4) the following inverse transcription reaction liquid of preparation in above-mentioned Microtube pipe.

5) on the PCR instrument, carry out reverse transcription reaction by following condition.

42℃ 15～30 min

70℃ 15 min

Generalized case, at eukaryote mRNA 3 ' end a PolyA structure is arranged, the quantity of A base does not wait to hundreds of is individual ten, utilize this structure can utilize Oligo (dT) primer, under the effect of ThermoScript II, synthetic cDNA the first chain take mRNA as template, the present invention adopts the sequence (providing among the PrimeScript 1st Strand cDNA Synthesis Kit) in the dT zone of being developed alone by TaKaRa to be primer, if the mRNA integrity that obtains is better, can obtain so cDNA first chain of all zymoprotein encoding genes in the species by the reverse transcription process.

Embodiment 9: " hundred make " produces the detection of bacterium Cordyceps sinensis China pilose spore anabolism uridylic or 5-methylcytosine functional gene Isocytosine deaminase pynL gene cloning, expression and protein vigor

1, the pcr amplification of Isocytosine deaminase pynL gene

CDNA the first chain that obtains in the embodiment 8 is as template, carry out Pfu archaeal dna polymerase pcr amplification reaction with pynL gene primer 5 ' ATA GAA TTC ATG GGC AAC GAC ACG ACG GCG 3 ', 5 ' AGG AAG CTT TCA CGT TGC CCG CAT CTT GAT TC 3 ' synthetic among the embodiment 7, condition setting is as follows:

Pfu pcr amplification reaction system:

Pfu DNA Ploymerase pcr amplification condition:

2, Isocytosine deaminase pynL gene PCR product gel electrophoresis detection

Concrete detection method is: 0.9% the sepharose that 1) will prepare makes its dissolving evenly with microwave-oven-heating; 2) get the 15mL gel, when treating that gel is cooled to 50 ℃ of left and right sides, add 1 μ L staining fluid Gold view, pour on the treatments of Electrophoretic Slab Gels after mixing, remove and insert the point sample comb behind the bubble; 3) after gel solidifies, carefully take out the point sample comb, offset plate is put into electrophoresis chamber (point sample hole one end is near the negative pole of electrophoresis chamber), in electrophoresis chamber, add the TAE electrophoretic buffer; 4) get 5 μ L samples and then add 6 * Loading Buffer, 1.5 μ L and ddH ₂Using liquid-transfering gun loading, applied sample amount after O 4 μ L mix is 10 μ L; 5) supply lead between connection electrophoresis chamber and the electrophoresis apparatus, just very red, negative pole is black; 6) power-on, the beginning electrophoresis, maximum voltage is no more than 5 V/cm; 7) when sample ran offset plate 2/3 the time can stop electrophoresis; 8) cut off the electricity supply after, gel taken out puts into the gel imaging instrument and observe, take pictures.

The size of transcribing group order-checking prediction Isocytosine deaminase pynL gene is 1971bp, and agarose gel electrophoresis result (Fig. 3) shows and successfully amplified Isocytosine deaminase pynL gene, and actual size and prediction in the same size are seen Fig. 3.

3, the base A that adds of Isocytosine deaminase pynL gene PCR product processes and purifying

Because Pfu archaeal dna polymerase PCR product end is flush end, so just can be used for the connection of T carrier after after glue reclaims, also need adding base A processing, purifying.It is as follows that glue recovery product adds base A system:

72 ℃ add A base 20 min in the PCR instrument, use at last AxyPrep PCR cleaning agents box purifying.

4, Isocytosine deaminase pynL gene and cloning vector is connected

Cloning vector pMD18-T Vector is available from TaKaRa company (TaKaRa code D101A), its physical map is seen Fig. 4, Isocytosine deaminase pynL gene is connected construction recombination plasmid pMD18-T/pynL with cloning vector, physical map is seen Fig. 5, and linked system and condition of contact are as follows.

Linked system:

Condition of contact: 16 ℃, 16h; Deactivation: 65 ℃, 15min.

5, the conversion of Isocytosine deaminase recombinant plasmid pMD18-T/pynL

Recombinant plasmid pMD18-T/pynL changed among the intestinal bacteria E. coli JM109 make up the recombinant bacterium E. coli JM109/pMD18-T/pynL that carries respectively Isocytosine deaminase pynL gene.Concrete steps are: 1) 10 μ L reaction systems are gone among the competent cell E. coli JM109 ice bath 30min; 2) thermal shock: 42 ℃, 90s; 3) ice bath: 2-3min; 4) add 800 μ L liquid LB, 37 ℃, 250rpm, 1h; 5) spread plate (containing the Amp resistance); 6) 37 ℃ of incubator overnight incubation.

6, the positive restructuring of Isocytosine deaminase E. coli JM109/pMD18-T/pynL screening

Single bacterium colony is carried out bacterium colony PCR, whether change goal gene over to checking.Bacterium colony PCR experiment flow: add with toothpick picking list bacterium colony and to contain in the 1.5mL centrifuge tube of 50 μ L sterilized waters, boiling water bath 30min, then centrifugal with supernatant as template, carry out pcr amplification, the PCR program setting is Taq enzymatic amplification general procedure.Adopt at last 0.9% agarose gel electrophoresis detection bacterium colony PCR product.

7, the order-checking of Isocytosine deaminase recombinant plasmid pMD18-T/pynL

After the detected positive recombinant bacterium liquid LB culture medium culturing of bacterium colony PCR spent the night, get 4mL bacterium liquid and extract plasmid, the operation instructions that method provides by AxyPrep plasmid DNA small volume of reagent box.Order-checking is finished by Shanghai Sani's bio tech ltd.Through sequence verification, sequence SEQ ID No.2 recombinates to pMD18-T/pynL.

8, the structure of Isocytosine deaminase recombinant expression plasmid pET-28a/pynL

The experimental basis foreign gene is in the principle of expression in escherichia coli, and expression vector pET-28a and Isocytosine deaminase pynL gene restriction enzyme site comparison situation, determined EcoR I and Hind III double enzyme site, and recombination bacillus coli E. coli JM109/pMD18-T/pynL has been carried out the cultivation of liquid LB test tube shaker, recombinant plasmid extraction.

The recombinant plasmid pMD18-T/pynL of Isocytosine deaminase pynL gene and expression vector pET-28a with EcoR I/Hind III restriction enzyme 37 ℃ respectively enzyme cut and process 6h, it is as follows that enzyme is cut system:

EcoR I/Hind III double digestion system:

Enzyme is cut and is finished rear 65 ℃ of deactivation 15min, then respectively with Axygen dna gel recovery test kit reclaim, purifying.

Isocytosine deaminase pynL gene and expression vector pET-28a spend the night with 16 ℃ of connections of T4 ligase enzyme behind double digestion, purifying again, make up recombinant expression plasmid pET-28a/pynL, its building process is seen Fig. 6, makes up the Isocytosine deaminase recombinant expression plasmid pET-28a/pynL collection of illustrative plates that obtains and sees Fig. 7.Linked system is composed as follows:

Linked system:

9, the screening of the conversion of Isocytosine deaminase recombinant expression plasmid pET-28a/pynL and positive monoclonal

Two Isocytosine deaminase recombinant expression plasmid pET-28a/pynL heat shock that will build respectively is converted in the E. coli BL21 Host Strains, then be applied on the LB agar plate that contains kantlex (Kan) resistance (50mg/L) 37 ℃ of overnight incubation.Random choose list bacterium colony from the flat board carries out pcr amplification with the primer of each functional gene, selects positive colony.

10, the abduction delivering of Isocytosine deaminase recombinant bacterium E. coli BL21/pET-28a/pynL

Be inoculated in the LB liquid nutrient medium that 5mL contains Kan resistance (50mg/L) 37 ℃, 250r/min overnight incubation with being accredited as positive mono-clonal.Get the 1mL culture, its transfer in the fresh LB liquid nutrient medium that contains Kan resistance (50mg/L) of 50mL 37 ℃, 250r/min are cultured to cell concentration OD600 and are about about 0.6～0.8.The IPTG inducing culture 8h that adds respectively finite concentration (240mg/L) in the culture.Collecting thalline surveys for electrophoresis analysis and enzyme biopsy.

11, Isocytosine deaminase recombinant bacterium E. coli BL21/pET-28a/pynL expression product SDS-PAGE analyzes

With the E. coli BL21 bacterium that changes empty carrier over to and the recombinant bacterium that do not add inductor IPTG in contrast.Be accredited as positive recombinant bacterium behind IPTG inducing culture certain hour (8h), get 0.5mL inducing culture thing, centrifugal collection thalline, be resuspended in the 50 μ L distilled water, add 50 μ L sample-loading buffers, boil 10min behind the mixing, carry out the SDS-PAGE electrophoretic analysis, Fig. 8 is that the Isocytosine deaminase albumen pynL(that recombinant bacterium E. coli BL21/pET-28a/pynL expresses is shown in the SEQ ID No.1 through its aminoacid sequence of sequence verification) SDS-PAGE figure.

12, the protein-active of Isocytosine deaminase recombinant bacterium E. coli BL21/pET-28a/pynL detects

The preparation of enzyme liquid: the recombinant bacterium E. coli BL21/pET-28a/pynL wet thallus 0.5g(dry weight 0.1g that takes by weighing collection) use respectively phosphate buffered saline buffer (50mM, pH8.0) 15mL to suspend, ultrasonication (power 350W, broken 2s, interval 2s, common ultrasonication 5min) is as the catalysis enzyme.

Isocytosine deaminase pynL transformation system: transform adding E. coli BL21/pET-28a/pynL ultrasonication thalline 10mL, 0.1g cytosine(Cyt) or 0.1g thymus pyrimidine in the bottle at 50mL, 30 ℃, 150r/min conversion reaction 2 ~ 3h, after transforming end, the centrifuging and taking supernatant is standby with subsequent detection.

Detection method: high performance liquid chromatography detection by quantitative uridylic and cytosine(Cyt).Pre-treatment: after conversion fluid was centrifugal, supernatant liquor was crossed 0.22 μ m microporous membrane and is used for the high performance liquid chromatography detection.Condition: chromatographic column: Agilent C18 post (4.6mm * 250mm i.d.5 μ m); Column temperature: 35 ℃; Sampling volume: 20 μ L; Flow velocity 1mL/min; Detect wavelength 260nm; Moving phase: A: ultrapure water; B: methyl alcohol.Gradient elution: 0min, 15%B keeps 3min; 3.0-3.5min, 15-24%B; 3.5min 24% keeps 5min; 8.5-9.0min, 24-35%B; 35%B keeps 6min; 15.0-16.0min, 35-85%B; 85%B keeps 6min; 22.0-22.5min, 85%-15%B; 15%B keeps 5min.

The making of typical curve: accurately take by weighing uridylic (or cytosine(Cyt)) standard substance, about 1.00mg-2.00mg, ultrapure water constant volume.Each standard substance preparation 6 gradient (1 μ g/mL, 30 μ g/mL, 60 μ g/mL, 90 μ g/mL, 120 μ g/mL, 150 μ g/mL).Then loading successively, each concentration repeats for 5 times.After the reference liquid of getting the certain volume maximum concentration mixes, constant volume, then HPLC analyzes the optimal conditions that uridylic (cytosine(Cyt)) detects.

Detect and calculate through above-mentioned chromatographic condition, we draw to draw a conclusion: the high specific enzyme of the Isocytosine deaminase that Isocytosine deaminase recombinant bacterium E. coli BL21/pET-28a/pynL is expressed (Specific Activity) alive is lived for ratio enzyme maximum in 144mol/min/mg(cytosine(Cyt) and the thymus pyrimidine), substrate conversion efficiency is respectively 89%, 91%.

SEQUENCE LISTING

＜110〉Zhejiang Polytechnical University, Zhongmei Huadong Pharmaceutical Co., Ltd. Hangzhou

＜120〉Cordyceps sinensis Isocytosine deaminase, encoding gene and application thereof

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 105

<212> PRT

<213> Hirsutella Sinensis

<400> 1

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

20 25 30

Gly Ser Glu Phe Glu Leu Gly Thr Arg Gly Ser Ser Arg Asp Tyr Arg

35 40 45

Ile His Val Leu Leu Gly Gly Val Asp Gln Leu His Gly Asp Glu Leu

50 55 60

Glu Ala Ala Val Leu Glu Ala Arg Asp Asp Val Thr Asp Glu Ala Thr

65 70 75 80

Ser Asp Ala Ile Gly Leu Asp Gly Asp Glu Gly Pro Leu Val Asp Val

85 90 95

Thr Arg His Asp Glu Arg Phe Asn Asn

100 105

<210> 2

<211> 318

<212> DNA

<213> Hirsutella Sinensis

<400> 2

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctcggtacc 120

cggggatcct ctagagatta tagaattcat gtgctccttg gaggggtgga tcagcttcat 180

ggcgacgagc ttgaagccgc ggttctcgaa gcgagagatg atgttaccga cgaggccacg 240

tcggacgcca tcgggcttga cggcgatgaa ggtccgctcg ttgatgttac cagacatgat 300

gaaagattta acaattga 318

Claims (10)

1. a Cordyceps sinensis Isocytosine deaminase is characterized in that described enzyme has the 90% above homology of aminoacid sequence shown in the SEQ ID No.1.

2. Cordyceps sinensis Isocytosine deaminase as claimed in claim 1, the aminoacid sequence that it is characterized in that described enzyme is shown in the SEQ ID No.1.

3. the Cordyceps sinensis Isocytosine deaminase prepares application in uridylic or the 5-methylcytosine at biocatalysis cytosine(Cyt) or thymus pyrimidine as claimed in claim 1.

4. application as claimed in claim 3, it is characterized in that described being applied as: to contain the broken mixed solution of wet thallus after cytoclasis that Cordyceps sinensis Isocytosine deaminase thalline fermentation culture obtains as catalyzer, take cytosine(Cyt) or thymus pyrimidine as substrate, be in the transformation system that consists of of 6.5 ~ 8.5 buffered soln in pH, conversion reaction 2 ~ 3h under 30 ℃, 150rpm condition, after reaction finishes, with reacting liquid filtering, get the crude product that filtrate being contains uridylic or 5-methylcytosine, described crude product separation and purification obtains uridylic or 5-methylcytosine.

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

6. gene of the described enzyme of claim 1 of encoding.

7. gene as claimed in claim 6 is characterized in that described gene has the 90% above homology of nucleotide sequence shown in the SEQ ID No.2.

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

As gene as described in one of claim 6 ~ 8 make up can the biocatalysis cytosine(Cyt) or thymus pyrimidine prepare application in the genetic engineering bacterium of uridylic or 5-methylcytosine.

10. application as claimed in claim 9, it is characterized in that described being applied as: make up the recombinant vectors that contains described Cordyceps sinensis cytosine deaminase gene, described recombinant vectors is converted in the intestinal bacteria, the recombination engineering bacteria that obtains carries out inducing culture, and the nutrient solution separation and purification obtains to contain the somatic cells of Cordyceps sinensis Isocytosine deaminase.

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