CN103045573B

CN103045573B - Cordyceps sinensis cytosine deaminase, coding gene and application thereof

Info

Publication number: CN103045573B
Application number: CN201210536936.7A
Authority: CN
Inventors: 郑裕国; 柳志强; 吴晖; 李邦良; 许静; 吴玲芳; 许峰; 薛亚平; 袁水金; 王鸿艳
Original assignee: Zhejiang University of Technology ZJUT; Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Current assignee: Zhejiang University of Technology ZJUT; Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Priority date: 2012-12-10
Filing date: 2012-12-10
Publication date: 2015-03-18
Anticipated expiration: 2032-12-10
Also published as: CN103045573A

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 participation cytosine(Cyt) producing bacterium Cordyceps sinensis China pilose spore from " hundred makes " to set out anabolism uridylic and synthesize the Isocytosine deaminase (cytosine deaminase) of 5-methylcytosine from thymus pyrimidine, 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 the stroma on lepidopteran (Lepidoptera) Hepialidae insect (Hepialus armoricanusOberthur) larva and the complex body (comprising stroma and polypide) on larva corpse.Cordyceps sinensis is traditional fungi herb resource that a class is treasured, and has the feature of meta-bolites and diverse biological activities, shows huge application and development prospect at biomedicine field.Cordyceps sinensis extensively, obviously receives much concern with its multiple medicinal efficacy, worldwide enjoys high praise.The traditional Chinese medical science is thought, Cordyceps sinensis enters lung kidney two warp, can tonifying lung cloudy, again can kidney-replenishing, 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, and phthisical cough phlegm blood, spontaneous sweating etc. are unique a kind of Chinese medicine that can balance simultaneously, regulate negative and positive.Modern pharmacology confirms, and Cordyceps sinensis has the biological activity widely such as immunomodulatory, antibacterial, 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 in the actual production such as artificial culture, liquid fermenting, use the Cordyceps fungus in imperfect stage, thus the qualification of Anamorph of Cordyceps Sinensis is extremely important.Chinese scholars is done a lot of work in Cordyceps Resources investigation, anamorph confirmation, activeconstituents compartment analysis and the mechanism of action, Application and Development.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, and price is high.Wild cordyceps owing to restricting by factors such as growing environments, scarcity of resources.Owing to making little progress on artificial culture in recent years, the research of wild cordyceps surrogate focused mostly on liquid fermenting.Utilizing liquid submerged fermentation to cultivate Cordyceps mycelium, extract or fermented liquid, is a kind of effective way solving Cordyceps sinensis medicine source.Chinese caterpillar fungus fermentation produces Chinese caterpillar fungus substitute, both can these precious resources of available protecting Chinese caterpillar fungus, and the restriction that climate, geographical environment and Chinese caterpillar fungus parasitic conditions are not strict again, is suitable for industrialization scale operation.The substitute produced is as also similar to natural cs with drug effect in its composition of mycelium, is thus devoted to the fermentation culture of Cordyceps mycelium both at home and abroad always.The mycelia that aweto cultured by artificial fermentation China pilose spore obtains, through toxicity, pharmacology, plant research, prove with natural cs chemical constitution, pharmacological action basically identical, natural cs can be replaced to produce cordyceps product, to make up the shortage of natural resources, by the optimization to fermentation condition, the amount of mycelial biomass and meta-bolites is all significantly improved.

In recent years, along with the develop rapidly of natural product chemistry and modern chromatographic techniques, in worm grass product research and development, progressively turn to deeper functional metabolic Study on product by the direct utilization of Chinese caterpillar fungus raw material or crude extract.Large quantifier elimination is done to Chinese caterpillar fungus meta-bolites both at home and abroad, meta-bolites mainly comprises several large compounds such as nucleosides, polysaccharide, polypeptide, sterol, and wherein the representative research of functional metabolic product in biosynthesizing, pharmacological action etc. such as miazines nucleosides, Cordyceps polysaccharide, N.F,USP MANNITOL wins initial success.

Ucleosides is one of topmost active substance of Chinese caterpillar fungus, and wherein miazines nucleosides comprises cytidine(C and uridine.Uridine (uracil riboside), also known as uridine (uIidine), has another name called and to be muttered ribosyl uridylic by snow riboside, dihydro-pyrimidin nucleosides, 1-β-D-.Cytidine(C (cytosine riboside) has another name called cytidine (cytidine), cytidine, 1-β-D-ribofuranosyl cytidine.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., nucleic acid medicine shows irreplaceable effect at antiviral, anti-tumor aspect in recent years.

Due to the important physiological action of pyrimidine nucleoside and analogue thereof, domestic and international related microorganism produces the existing a lot of research of miazines nucleosides.But 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 Cordyceps fungus miazines nucleosides metabolic pathway of synthesizing.

(3) summary of the invention

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

The technical solution used in the present invention is:

The present invention relates to a kind of Isocytosine deaminase of set out from " hundred make " production bacterium Cordyceps sinensis China pilose spore participation cytosine(Cyt) or thymus pyrimidine anabolism uridylic or 5-methylcytosine, described enzyme has aminoacid sequence more than 90% homology shown in SEQ ID No.1, and preferably its aminoacid sequence (is designated as pynL albumen) as shown in SEQ ID No.1; This enzyme can prepare uridylic by catalysis cytosine(Cyt), or 5-methylcytosine prepared by catalysis thymus pyrimidine.Due to the singularity of aminoacid sequence; the fragment of any peptide protein containing aminoacid sequence shown in SEQ ID No.1 or its variant; 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 sequences homology, more than 90%, all belong to the row of scope.Concrete described change can comprise amino acid whose disappearance, insertion or replacement in aminoacid sequence; Wherein, the conservative property for variant changes, and the amino acid replaced has the structure similar to original acid or chemical property, and as replaced Isoleucine with leucine, variant also can have non-conservation and change, as replaced glycine with tryptophane.

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

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

Further, described is applied as: with the broken mixed solution of wet thallus after cytoclasis obtained containing Cordyceps sinensis Isocytosine deaminase thalline fermentation culture for catalyzer, with cytosine(Cyt) or thymus pyrimidine for 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 uridylic or 5-methylcytosine, described crude isolate purified acquisition uridylic or 5-methylcytosine.

The starting point concentration of described substrate is 10g/L, and the volumetric usage of described catalyzer is 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. coliBL21/pET-28a/pynL is inoculated in 5mL and contains in the LB liquid nutrient medium of Kan resistance (50mg/L), 37 DEG C, 250r/min overnight incubation.Get 1mL culture, transferred in 50mL fresh containing the LB liquid nutrient medium of Kan resistance in 37 DEG C, 250r/min is cultured to cell concentration OD ₆₀₀be about about 0.6 ~ 0.8, the IPTG inducing culture 8h of finite concentration (240mg/ml) is added in culture, get induction broth to filter, collect wet thallus, wet thallus 0.5g phosphate buffered saline buffer (50mM, pH8.0) 15mL taking collection suspends, and the thalline mixed solution that ultrasonication (power 350W, broken 2s, interval 2s, altogether ultrasonication 5min) obtains afterwards is as catalysis enzyme.

The invention still further relates to the encoding gene of above-mentioned enzyme, i.e. cytosine deaminase gene, described gene has nucleotide sequence more than 90% homology shown in SEQ ID No.2, and preferably its nucleotide sequence (is designated as pynL gene) as shown in SEQ ID No.2.Due to the singularity of nucleotide sequence, shown in any SEQ ID NO:2, the variant of polynucleotide, as long as itself and this polynucleotide have more than 90% homology, all belongs to the row of scope.The variant of described polynucleotide refers to a kind of polynucleotide sequence having one or more Nucleotide and change.The variant of these polynucleotide can make raw displacement varient or the varient of non-life, comprises and replaces varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of polynucleotide, disappearance or insertion, but can not from the function of peptide protein changing in fact its coding.

Described gene can be used for building biocatalysis cytosine(Cyt) or thymus pyrimidine can prepare the genetic engineering bacterium of uridylic or 5-methylcytosine, to expand the output of uridylic and 5-methylcytosine or derivatives thereof.Described is applied as: build the recombinant vectors containing described cytosine 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 Isocytosine deaminase.

Main points of the present invention there are provided the nucleotide sequence shown in the aminoacid sequence shown in SEQ ID NO:1 and SEQ ID NO:2, when this aminoacid sequence known and nucleotide sequence, the acquisition of this aminoacid sequence and nucleotide sequence, and related vector, host cell acquisition, be all apparent to those skilled in the art.

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

Beneficial effect of the present invention is mainly reflected in: the present invention studies in detail cytosine(Cyt) synthesis uridylic or thymus pyrimidine synthesis 5-methylcytosine pathways metabolism principle, the cloned DNA comprising nucleotide sequence provided by the present invention can be used for by transduction, transform, the method of Conjugative tiansfer proceeds in engineering bacteria, 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 expanding uridylic and 5-methylcytosine or derivatives thereof provides effective way, there is major application prospect.

(4) accompanying drawing explanation

Fig. 1 is the denaturing formaldehyde gel electrophorogram that " hundred make " produces bacterium Cordyceps sinensis China pilose spore total serum IgE;

Fig. 2 is pyrimidine metabolic pathway 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

Below in conjunction with specific embodiment, the present invention is described further, 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: first gather natural cordyceps from Qinghai, and taken back Hangzhou and carry out separation screening, obtain L0106 bacterial strain, and be China pilose spore (Hirsutella Sinensis) through this bacterial strain of strain identification, this culture presevation is in China typical culture collection center, deposit number is CCTCC No:M 2011278, discloses in the patent CN102373190A of previously application.

By this strain inoculation in inclined-plane, (this is the liquid formulations before solidification to culture medium prescription, by following proportions well after bevel again) be glucose 2.0%(w/v, 1% represents in 100mL substratum containing 1g, 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%, magnesium sulfate 0.05%, potassium primary phosphate 0.05%, agar powder 1.0%, surplus is water, cultivates 25 days at 12 ~ 16 DEG C; Then by strain 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%, magnesium sulfate 0.01%, potassium primary phosphate 0.02%, surplus is water, be placed on shaking table, temperature 12 ~ 16 DEG C is cultivated 25 days, after cultivation terminates aseptically, carries out solid-liquid separation, and solid is placed in sterilized equipment, for subsequent use.

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

Total serum IgE is extracted 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 is fully ground to Powdered, be dispensed in the 1.5mL centrifuge tube of precooling, add 1mL TRIzol reagent, mixing, leaves standstill 5min on ice, nucleic acid-protein mixture is separated completely.2) RNA is separated: add 0.2mL chloroform, firmly concussion mixing 15s, and leave standstill 2 ~ 3min on ice, 4 DEG C, the centrifugal 15min of 12000rpm, layering, gets upper strata aqueous phase, about 600 μ L.3) RNA precipitation: add 500 μ L Virahols, leaving standstill 10min on ice, 4 DEG C, the centrifugal 10min of 12000rpm, abandon supernatant.4) RNA washing: add 1mL 75%(v/v) ethanol, hangs precipitation, leaves standstill 10min on ice, 4 DEG C, the centrifugal 15min of 7500rpm; Repeat washing step above, then wash one time.5) RNA is dissolved: be placed in by centrifuge tube and open wide dry 5 ~ 10min on ice, add appropriate DEPC water dissolution.

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

After extracting sample total serum IgE, with Oligo(dT) enrichment with magnetic bead mRNA.Add fragmentation buffer and mRNA is broken into short-movie section (200 ~ 700bp), take mRNA as template, Article 1 cDNA chain is synthesized with hexabasic base random primer (random hexamers), then Article 2 cDNA chain is synthesized, do end reparation after adding EB buffer solution elution through QiaQuick PCR kit purifying, add polyA and connect sequence measuring joints again, then clip size selection is carried out with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library Illumina GA IIx built up checks order.The raw image data obtained that checks order is converted into sequence data through base calling, i.e. raw data or raw reads.Only containing the reads of adaptor sequence in removing primitive sequencer reads, standby with subsequent analysis.

Embodiment 4: " hundred make " production bacterium Cordyceps sinensis China pilose spore RNA is short reads sequence assembling

Use short reads composite software SOAPdenovo(Li, Zhu et al. De novoassembly of human genomes with massively parallel short read sequencing [J]. Genome Res, 2010,20:265-272.) do transcript profile and from the beginning assemble.First the reads with certain length overlap is linked to be the longer Contig fragment not containing N by SOAPdenovo.Then Contig is returned in reads comparison, determine from the distance between the different Contig of same transcript and these Contig by paired-end reads, these Contig connect together by SOAPdenovo, and middle unknown nucleotide sequence N represents, so just obtains Scaffold.Utilize paired-end reads to do filling-up hole process to Scaffold further, finally obtain containing N minimum, the Unigene sequence that two ends can not extend again.Finally, Unigene sequence and albumen database nr, Swiss-Prot, KEGG and COG are done blastx comparison (evalue<0.00001), get the sequence direction that the best albumen of comparison result determines Unigene.If the comparison result between different sink is contradictory, then press nr, Swiss-Prot, the priority of KEGG and COG determines 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.For determining that the Unigene in sequence direction provides the sequence in its direction from 5' to 3', for determining the sequence that the Unigene in sequence direction provides composite software and obtains.

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

Functional annotation information provides the protein function annotation of Unigene, Pathway annotation, COG functional annotation and Gene Ontology(GO) functional annotation.First, by blastx by Unigene sequence alignment to albumen database nr, Swiss-Prot, KEGG and COG(evalue<0.00001), obtain the albumen with given Unigene with highest serial similarity, thus obtain the protein function annotation information of this Unigene.The Pathway annotation of Unigene can be obtained further according to KEGG annotation information.Compared by Unigene and COG database, the function that prediction Unigene is possible also does function statistic of classification to it.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.) the GO annotation information of Unigene is obtained.After obtaining the GO annotation 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.) GO functional classification statistics is done, from the gene function distribution characteristics being macroscopically familiar with these species to all Unigene.

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

Fig. 2 is the pyrimidine metabolic (map00240) in KEGG pathways metabolism annotation, the enzyme annotated is that " hundred make " detected produces bacterium Cordyceps sinensis China pilose spore pyrimidine metabolic pathway relevant enzymes, as can be seen from the figure, detect from cytosine(Cyt) synthesis uridylic or Isocytosine deaminase 1 Unigene from thymus pyrimidine synthesis 5-methylcytosine.By the ORF Finder software on-line checkingi in NCBI, have found the open reading frame (SEQ IDNo.2) of this gene and obtain corresponding protein sequence (SEQ ID No.1).

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

Use GENE RUNNER primer-design software according to predicting the gene open proofreading dna primers obtained, the cytosine deaminase gene of bacterium China pilose spore anabolism uridylic or 5-methylcytosine is produced for clone's " hundred make ", primer by Shanghai Sheng Gong biotechnology company limited synthesize, primer sequence as follows listed by:

PynL gene: forward primer 5 ' ATAGAATTCATGGGCAACGACACGACGGCG 3 '

Reverse primer 5 ' AGGAAGCTTTCACGTTGCCCGCATCTTGATTC 3 '

PynL mrna length is 1971bp

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

After the method first provided according to embodiment 1 turns out sutella sinensis fermented mycelium, the method provided according to embodiment 2 again carries out the extraction of total serum IgE to China pilose spore, carry out by following the synthesis that " hundred make " produces bacterium Cordyceps sinensis China pilose spore cDNA first chain, for follow-up each gene clone experiment after obtaining total serum IgE.

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

1) in Microtube pipe, following mixed solution is prepared.

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, and can improve reverse transcription reaction efficiency, so carry out sex change, annealing reaction in PCR instrument, condition setting is as follows:

65℃，5 min

3) annealing terminates the rear centrifugal several seconds mixed solution of template ribonucleic acid/primer etc. is gathered in bottom Microtube pipe.

4) in above-mentioned Microtube pipe, following inverse transcription reaction liquid is prepared.

5) in PCR instrument, reverse transcription reaction is carried out by following condition.

42℃ 15～30 min

70℃ 15 min

Generalized case, a PolyA structure is had at eukaryote mRNA 3 ' end, the quantity of A base is not at ten to hundreds of etc., utilize this structure can utilize Oligo (dT) primer, under the effect of ThermoScript II, take mRNA as templated synthesis cDNA first chain, the present invention adopts the sequence in the dT region developed alone by TaKaRa (providing in PrimeScript 1st Strand cDNA Synthesis Kit) to be primer, if the mRNA integrity obtained is better, cDNA first chain of all zymoprotein encoding genes in species so can be obtained by process of reverse-transcription.

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

1, the pcr amplification of Isocytosine deaminase pynL gene

With cDNA first chain obtained in embodiment 8 for 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 ' the AGG AAG CTT TCA CGT TGC CCG CAT CTT GAT TC 3 ' of synthesis in 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 detected through gel electrophoresis

Concrete detection method is: 1) make it be uniformly dissolved the sepharose microwave-oven-heating of prepare 0.9%; 2) get 15mL gel, when gel is cooled to about 50 DEG C, add 1 μ L staining fluid Gold view, pour into after mixing on treatments of Electrophoretic Slab Gels, after removing bubble, insert point sample comb; 3), after gel sets, the careful point sample that takes out is combed, and offset plate is put into electrophoresis chamber (loading wells one end is near the negative pole of electrophoresis chamber), adds TAE electrophoretic buffer in electrophoresis chamber; 4) get 5 μ L samples and then add 6 × Loading Buffer 1.5 μ L and ddH ₂o 4 μ L uses liquid-transfering gun loading after mixing, and applied sample amount is 10 μ L; 5) connect the supply lead between electrophoresis chamber and electrophoresis apparatus, just very red, negative pole is black; 6) power-on, start electrophoresis, maximum voltage is no more than 5 V/cm; 7) electrophoresis can be stopped when sample ran 2/3 of offset plate; 8), after cutting off the electricity supply, gel taken out and puts into the observation of gel imaging instrument, take pictures.

The size of transcript profile order-checking prediction Isocytosine deaminase pynL gene is 1971bp, and agarose gel electrophoresis result (Fig. 3) shows that Successful amplification has gone out Isocytosine deaminase pynL gene, and actual size is in the same size with prediction, sees Fig. 3.

3, Isocytosine deaminase pynL gene PCR product add base A process and purifying

Because Pfu archaeal dna polymerase PCR primer end is flush end, connect so just can be used for carrier T also need to carry out adding base A process, purifying after glue reclaims after.It is as follows that glue recovery product adds base A system:

In PCR instrument, 72 DEG C add A base 20 min, finally purify with AxyPrep PCR cleaning agents box.

4, the connection of Isocytosine deaminase pynL gene and cloning vector

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

Linked system:

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

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

Recombinant plasmid pMD18-T/pynL is proceeded in intestinal bacteria E. coli JM109 the recombinant bacterium E. coli JM109/pMD18-T/pynL building and carry Isocytosine deaminase pynL gene respectively.Concrete steps are: 1) go in competent cell E. coli JM109 by 10 μ L reaction systems, ice bath 30min; 2) thermal shock: 42 DEG C, 90s; 3) ice bath: 2-3min; 4) 800 μ L liquid LB are added, 37 DEG C, 250rpm, 1h; 5) spread plate (containing Amp resistance); 6) 37 DEG C of incubator overnight incubation.

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

Bacterium colony PCR is carried out to single bacterium colony, whether proceeds to goal gene to verify.Bacterium colony PCR experiment flow process: add in the 1.5mL centrifuge tube containing 50 μ L sterilized waters with toothpick picking list bacterium colony, boiling water bath 30min, then centrifugal using supernatant as template, carry out pcr amplification, PCR program setting is Taq enzyme amplification general procedure.The agarose gel electrophoresis of 0.9% is finally adopted to detect bacterium colony PCR primer.

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

After the positive recombinant bacterium LB liquid medium overnight incubation that bacterium colony PCR is detected, 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 completed by Sani bio tech ltd, Shanghai.Through sequence verification, sequence SEQ ID No.2 has recombinated in pMD18-T/pynL.

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

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

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

EcoR I/Hind III double digestion system:

Enzyme is cut and is terminated rear 65 DEG C of deactivation 15min, then respectively with Axygen DNA gel recovery test kit carry out reclaiming, purifying.

Isocytosine deaminase pynL gene and expression vector pET-28a connect with T4 ligase enzyme 16 DEG C again and spend the night after double digestion, purifying, build recombinant expression plasmid pET-28a/pynL, its building process is shown in Fig. 6, builds the Isocytosine deaminase recombinant expression plasmid pET-28a/pynL collection of illustrative plates obtained and sees Fig. 7.Linked system is composed as follows:

Linked system:

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

Respectively by heat-shock transformed in E. coli BL21 Host Strains for two the Isocytosine deaminase recombinant expression plasmid pET-28a/pynL built, then be applied on the LB agar plate containing kantlex (Kan) resistance (50mg/L), 37 DEG C of overnight incubation.From flat board, random choose list bacterium colony, 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

The mono-clonal being accredited as the positive is inoculated in 5mL to be contained in the LB liquid nutrient medium of Kan resistance (50mg/L), 37 DEG C, 250r/min overnight incubation.Get 1mL culture, transferred in 50mL fresh containing the LB liquid nutrient medium of Kan resistance (50mg/L) in 37 DEG C, 250r/min is cultured to cell concentration OD600 and is about about 0.6 ~ 0.8.The IPTG inducing culture 8h of finite concentration (240mg/L) is added respectively in culture.Collect thalline for electrophoretic analysis and Enzyme activity assay.

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

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

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

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

Isocytosine deaminase pynL transformation system: transform in bottle at 50mL and add E. coli BL21/pET-28a/pynL ultrasonication thalline 10mL, 0.1g cytosine(Cyt) or 0.1g thymus pyrimidine, 30 DEG C, 150r/min conversion reaction 2 ~ 3h, after conversion terminates, 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 is centrifugal, supernatant liquor is crossed 0.22 μm of microporous membrane and is detected for high performance liquid chromatography.Condition: chromatographic column: Agilent C18 post (4.6mm × 250mm i.d.5 μm); Column temperature: 35 DEG C; Sampling volume: 20 μ L; Flow velocity 1mL/min; Determined 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 uridylic (or cytosine(Cyt)) standard substance, about 1.00mg-2.00mg, ultrapure water constant volume.6 gradients (1 μ g/mL, 30 μ g/mL, 60 μ g/mL, 90 μ g/mL, 120 μ g/mL, 150 μ g/mL) prepared by each standard substance.Then loading successively, each concentration repeats for 5 times.After the reference liquid getting certain volume maximum concentration mixes, constant volume, then HPLC analyzes the optimal conditions that uridylic (cytosine(Cyt)) detects.

Detect through above-mentioned chromatographic condition and calculate, we draw to draw a conclusion: high specific enzyme work (Specific Activity) of the Isocytosine deaminase expressed by Isocytosine deaminase recombinant bacterium E. coli BL21/pET-28a/pynL is ratio enzyme maximum in 144mol/min/mg(cytosine(Cyt) and thymus pyrimidine work), 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

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

2. Cordyceps sinensis Isocytosine deaminase prepares the application in uridylic or 5-methylcytosine at biocatalysis cytosine(Cyt) or thymus pyrimidine 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 Isocytosine deaminase thalline fermentation culture for catalyzer, with cytosine(Cyt) or thymus pyrimidine for 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 uridylic or 5-methylcytosine, described crude isolate purified acquisition uridylic or 5-methylcytosine.

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 biocatalysis cytosine(Cyt) or thymus pyrimidine can prepare application in the genetic engineering bacterium of uridylic or 5-methylcytosine.

8. apply as claimed in claim 7, it is characterized in that described being applied as: build the recombinant vectors containing described Cordyceps sinensis cytosine 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 Isocytosine deaminase.