CN104651324B - Cordyceps sinensis CTP synzyme, encoding gene and its application - Google Patents
Cordyceps sinensis CTP synzyme, encoding gene and its application Download PDFInfo
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- CN104651324B CN104651324B CN201510023806.7A CN201510023806A CN104651324B CN 104651324 B CN104651324 B CN 104651324B CN 201510023806 A CN201510023806 A CN 201510023806A CN 104651324 B CN104651324 B CN 104651324B
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Abstract
The present invention relates to a CTP synzyme for the anabolism cytidine that set out from " hundred make " production bacterium Cordyceps sinensis Hirsutella sinensis participation uridine triphosphate, gene and its application of this enzyme are encoded；The CTP synzyme, with the homology of amino acid sequence more than 90% shown in SEQ ID No.2, its encoding gene has the homology of nucleotide sequence more than 90% shown in SEQ ID No.4；Metabolic pathway of the present invention to uridine triphosphate synthesis cytidine from principle is studied in detail, clone DNA comprising nucleotide sequence provided by the present invention can be used to be transferred in engineering bacteria by the method for transduction, conversion, Conjugative tiansfer, by the expression for adjusting cytidine biosynthesis gene, assign the expressivity high of host's cytidine, for the yield for expanding cytidine provides effective way, with major application prospect.
Description
(1) technical field
Set out the present invention relates to a participation uridine triphosphate from " hundred make " production bacterium Cordyceps sinensis Hirsutella sinensis
The CTP synzyme (CTP synthase) of anabolism cytidine, encodes gene and its application of this enzyme.
(2) background technology
Cordyceps sinensis (Cordyceps sinensis (Berk.) Sacc.) is that aweto colonizes in Lepidoptera
(Lepidoptera) stroma and larva corpse on Hepialidae insect (Hepialus armoricanus Oberthur) larva
Complex (including stroma and polypide) on body.Cordyceps sinensis is traditional fungi herb resource that a class is treasured, and is produced with metabolism
The characteristics of thing and diverse biological activities, huge application and development prospect is shown in biomedicine field.Cordyceps sinensis is with it
Various medicinal efficacies extensively, substantially receive much concern, and worldwide enjoy high praise.The traditional Chinese medical science thinks that Cordyceps sinensis enters lung kidney
Two warps, can tonifying lung it is cloudy, and can kidney-replenishing, cure mainly kidney deficiency, impotence and seminal emission, soreness of waist and knee joint, eak after being ill, chronic cough is weak, phthisical cough
Phlegm blood, spontaneous sweating etc., be it is unique it is a kind of can be while balancing, adjusting the Chinese medicine of negative and positive.Modern pharmacology is it has proven convenient that the worm summer in winter
Grass has immunological regulation, antibacterial, antitumor, anti-oxidant, anti-aging, reducing blood sugar and blood lipid, gonadotropic Effect etc. extensive biological
Activity.
Aweto is a kind of sac fungus, has Conidial Stage (phorozoon) and ascospore in its history of life
Stage (epigamous).And the aweto in imperfect stage is used in the actual productions such as artificial culture, liquid fermentation, because
And the identification of Anamorph of Cordyceps Sinensis is extremely important.Domestic and foreign scholars Cordyceps Resources investigate, phorozoon confirmation, activity into
Separate analysis and the mechanism of action, development and application aspect are done a lot of work.Cordyceps sinensis Hirsutella sinensis has proved to be the winter
The worm summer grass phorozoon existence form, with natural cordyceps identical active component and drug effect.
Natural cses have strict parasitics and special ecological environment, therefore its yield is very low, and price is high.It is wild
Cordyceps sinensis is due to by the restriction of the factors such as growing environment, scarcity of resources.It is wild due to being made little progress on artificial cultivation in recent years
The research of Cordyceps sinensis substitute focuses mostly on liquid fermentation.Using liquid deep layer fermenting culture aweto mycelium, extract
Thing or zymotic fluid, are a kind of effective ways for solving Cordyceps sinensis medicine source.Chinese caterpillar fungus fermentation produces Chinese caterpillar fungus substitute, both can effectively protect
Shield Chinese caterpillar fungus this precious resources, and not climate, geographical environment and the strict limitation of Chinese caterpillar fungus parasitic conditions, are suitable for industrialization big
Large-scale production.Its composition of the substitute produced such as mycelium and drug effect are also similar to natural cses, thus cause always both at home and abroad
Power is in the fermented and cultured of cordyceps mycelia.The mycelia that aweto cultured by artificial fermentation Hirsutella sinensis is obtained, through toxicity, medicine
Reason, plant research, it was demonstrated that basically identical with natural cses chemical composition, pharmacological action, can replace natural cses to produce Chinese caterpillar fungus system
Product, to make up the shortage of natural resources, by the optimization to fermentation condition, the amount of mycelial biomass and metabolite has bright
It is aobvious to improve.
In recent years, developing rapidly with natural product chemistry and modern chromatographic techniques, in being researched and developed to worm grass product
Deeper functional metabolic Study on product is progressively turned to by direct utilization of Chinese caterpillar fungus raw material or crude extract.Both at home and abroad to worm
Careless metabolite has done substantial amounts of research, the main a few major class compounds such as including nucleosides, polysaccharide, polypeptide, sterol of metabolite, its
The representative functional metabolic product such as middle purines nucleosides, Cordyceps sinensis polysaccharide, mannitol is in biosynthesis, pharmacological action etc.
The research of aspect wins initial success.
Ucleosides is one of topmost active material of Chinese caterpillar fungus, and wherein miazines nucleosides includes cytidine and uracil
Nucleosides.Uridine (uracil riboside) has another name called by snow riboside, dihydro-pyrimidin core also known as uridine (uIidine)
Glycosides, 1- β-D- mutter ribosyl uracil.Cytidine (cytosine riboside) also known as cytidine (cytidine), cytidine,
1- β-D- ribofuranosyl cytidines.Pyrimidine nucleoside is multiduty nucleosides, can be used as health products and the additive of food.Its conduct
Beauty and anti-ultraviolet radiation cosmetics are increasingly becoming the necessity of people's life, and used as medicine, clinical practice is in maincenter god
Through many diseases such as, uropoiesis, metabolism and angiocarpy, in the U.S., nucleic acid similar drug is in recent years in antiviral, anti-tumor aspect
Show irreplaceable effect.
Due to the important physiological action of pyrimidine nucleoside and the like, both at home and abroad about microbial production of pyrimidine class nucleosides
There are many researchs.But as the aweto of important anabolism miazines nucleosides, also only rest on metabolite composition
In analysis and the research of effect, to the research of related gene and albumen in aweto miazines nucleosides metabolic pathway of synthesizing also
It is rarely found.
(3) content of the invention
Present invention aim at for the not enough and technical issues that need to address present on, is produced to " hundred make " the bacterium winter
The enzyme and its encoding gene of worm summer grass Hirsutella sinensis anabolism cytidine are furtherd investigate, there is provided " hundred make " is raw
Produce bacterium Cordyceps sinensis Hirsutella sinensis and participate in uridine triphosphate and set out the enzyme of anabolism cytidine, encoding gene and its should
With.
The technical solution adopted by the present invention is：
Set out conjunction from " hundred makes " production bacterium Cordyceps sinensis Hirsutella sinensis participation uridine triphosphate the present invention relates to one kind
Into the CTP synzyme of metabolism cytidine, the enzyme has amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2
More than 90% homology, preferred sequence is the pynK2 eggs of SEQ ID No.2 for the pynK1 albumen or sequence of SEQ ID No.1
In vain；The enzyme can be catalyzed uridine triphosphate and prepare cytidine.It is any to contain SEQ ID due to the particularity of amino acid sequence
The fragment or its variant of the peptide albumen of amino acid sequence shown in No.1 or SEQ ID No.2, such as its examples of conservative variations, bioactivity
Fragment or derivative, as long as the fragment of the peptide albumen or peptide protein variant and aforementioned amino acid sequences homology are more than 90%,
Belong to the row of the scope of the present invention.It is specific it is described change may include the missing of amino acid in amino acid sequence, insertion or
Replace；Wherein, sexually revised for the conservative of variant, the amino acid replaced has the structure similar to original acid or chemically
Matter, such as replaces isoleucine with leucine, and variant can also change with non-conservation, such as replaces glycine with tryptophan.
The path for obtaining cytidine by uridine triphosphate anabolism is as follows：
The invention further relates to described Cordyceps sinensis CTP synzyme cytidine is prepared in living things catalysis uridine triphosphate
In application, specific described application is preferably：Passed through with the wet thallus that the synzyme thalline of CTP containing Cordyceps sinensis fermented and cultured is obtained
Broken mixed liquor after clasmatosis is catalyst, with uridine triphosphate as substrate, in the cushioning liquid structure that pH is 6.5~8.5
Into transformation system in, 2~3h of conversion reaction under the conditions of 30 DEG C, 150rpm, reaction terminate after, by reacting liquid filtering, take filter
Liquid is the crude product containing cytidine, and the crude product isolates and purifies acquisition cytidine；The initial concentration of the substrate is
10g/L, the volumetric usage of the catalyst is 100mL/g substrates, and the dry mycelium concentration of the catalyst is 6.7mg/mL.
The preparation method of the catalyst is：By Cordyceps sinensis CTP synzyme recombinant bacterium E.coli BL21/pET-28a/
PynK1 or E.coli BL21/pET-28a/pynK2 are inoculated in the LB fluid nutrient mediums that 5mL contains Kan resistances (50mg/L),
37 DEG C, 250r/min overnight incubations.1mL cultures are taken, is transferred in the 50mL fresh LB Liquid Cultures containing Kan resistances
37 DEG C in base, 250r/min cultivate to cell concentration OD600 and be about 0.6~0.8 or so, to adding finite concentration in culture
The IPTG Fiber differentiation 8h of (240mg/ml), take induction broth filtering, collect wet thallus, and the wet thallus 0.5g for weighing collection is used
Phosphate buffer (50mM, pH8.0) 15mL suspends, ultrasonication (power 350W, broken 2s, interval 2s, ultrasonication altogether
The thalline mixed liquor for 5min) obtaining afterwards is as catalysis enzyme.
The invention further relates to the encoding gene of above-mentioned CTP synzyme, i.e. Cordyceps sinensis CTP synthase genes, the gene
With the homology of nucleotide sequence more than 90% shown in SEQ ID No.3 or SEQ ID No.4, preferred sequence is SEQ ID
The pynK1 genes or sequence of No.3 are the pynK2 genes of SEQ ID No.4.Due to the particularity of nucleotide sequence, any SEQ
ID NO：3 or SEQ ID NO：The variant of polynucleotides shown in 4, as long as it has more than 90% homology with the polynucleotides,
Belong to the row of the scope of the present invention.The variant of the polynucleotides refers to a kind of with the change of one or more nucleotides
Polynucleotide sequence.The variant of this polynucleotides can make the displacement variant of life or the variant of non-life, including substitution variation
Body, Deletion variants and insert variation.As known in the art, allelic variant is an alternative forms for polynucleotides,
It is probably a substitution for polynucleotides, missing or inserts, but will not be from the function of the peptide albumen for substantially changing its coding.
Described gene can be used to building can living things catalysis uridine triphosphate prepare the genetic engineering bacterium of cytidine,
To expand the yield of cytidine or derivatives thereof；Described application is：Build and contain the Cordyceps sinensis CTP synzyme bases
The recombinant vector of cause, the recombinant vector is converted into Escherichia coli, and the recombination engineering bacteria of acquisition carries out Fiber differentiation,
Nutrient solution isolates and purifies somatic cells of the acquisition containing Cordyceps sinensis CTP synzyme.
The bacterial strain that Cordyceps sinensis CTP synzyme of the present invention and its encoding gene can be provided is Hirsutella sinensis
(Hirsutella Sinensis) L0106, in China typical culture collection center, deposit number is CCTCC for the culture presevation
No：M 2011278, discloses in the patent CN102373190A of previous application.
It is of the invention to be characterized by there is provided SEQ ID NO：1 or SEQ ID NO：Amino acid sequence and SEQ shown in 2
ID NO：3 or SEQ ID NO：Nucleotide sequence shown in 4, in the case of the known amino acid sequence and nucleotide sequence,
The acquisition of the amino acid sequence and nucleotide sequence, and relevant carriers, host cell acquisition, for those skilled in the art
For be obvious.
The beneficial effects are mainly as follows：The present invention synthesizes cytidine generation from principle to uridine triphosphate
The approach of thanking is studied in detail, and the clone DNA comprising nucleotide sequence provided by the present invention can be used to by transduction, turn
Change, the method for Conjugative tiansfer is transferred in engineering bacteria, by adjusting the expression of cytidine biosynthesis gene, assign host three
The expressivity high of cytidine phosphate, for the yield for expanding cytidine or derivatives thereof provides effective way, answers with great
Use prospect.
(4) illustrate
Fig. 1 is the denaturing formaldehyde gel electrophoretogram that " hundred make " produces bacterium Cordyceps sinensis Hirsutella sinensis total serum IgE；
Fig. 2 is purine metabolism approach annotated map；
Fig. 3 is CTP synthase gene pcr amplification product gel electrophoresis figures；
Fig. 4 is cloning vector pMD18-T Vector and expression vector pET-28a physical maps；
Fig. 5 is restructuring cloned plasmids pMD18-T/pynK physical maps；
Fig. 6 is recombinant expression plasmid pET-28a/pynK building process schematic diagrames；
Fig. 7 is recombinant expression plasmid pET-28a/pynK physical maps；
Fig. 8 schemes for CTP synthetase albumens SDS-PAGE.
(5) specific embodiment
With reference to 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 culture of bacterium Cordyceps sinensis Hirsutella sinensis
Bacterium source：Gathering natural cordyceps from Qinghai first, and bring it back into Hangzhou carries out separation screening, obtains
L0106 bacterial strains, and the bacterial strain is Hirsutella sinensis (Hirsutella Sinensis) through strain idenfication, the culture presevation is in
State's Type Tissue Collection, deposit number is CCTCC No：M 2011278, in the patent of previous application
Disclosed in CN102373190A.
The strain is inoculated in inclined-plane, (this is the liquid formulations before solidification to culture medium prescription, good by following proportions
Bevel again afterwards) it is glucose 2.0% (w/v contains 1g, similarly hereinafter in 1% expression 100mL culture mediums), corn flour
1.0%th, murphy juice 0.5%, dextrin 0.5%, dusty yeast 0.5%, wheat bran 1.0%, dried silkworm chrysalis meal 2.0%, peptone 1.0%, sulphur
Sour magnesium 0.05%, potassium dihydrogen phosphate 0.05%, agar powder 1.0%, balance of water are cultivated 25 days at 12~16 DEG C；Then by bacterium
Plant and be inoculated in fermentation medium, culture medium prescription is glucose 1.0%, molasses 1.0%, dried silkworm chrysalis meal 0.5%, soybean cake powder
1.0%th, yeast extract 0.5%, magnesium sulfate 0.01%, potassium dihydrogen phosphate 0.02%, balance of water, are placed on shaking table, and temperature 12~
16 DEG C are cultivated 25 days, are cultivated after terminating aseptically, carry out separation of solid and liquid, and solid is placed in into sterilized equipment, standby.
Embodiment 2：" hundred make " produces the extraction of bacterium Cordyceps sinensis Hirsutella sinensis total serum IgE
Total serum IgE is extracted with TRIzol reagents, step is specially：1) liquid nitrogen grinding：Take the new fresh thallis of 1g to be put into mortar, instead
It is added with and is fully ground to powdered into liquid nitrogen, be dispensed into the 1.5mL centrifuge tubes of precooling, add 1mL TRIzol reagents, mixes,
5min is stood on ice, is kept completely separate nucleic acid-protein compound.2) RNA is separated：0.2mL chloroforms are added, firmly concussion is mixed
15s, stands 2~3min on ice, and 4 DEG C, 12000rpm centrifugation 15min, layering take upper strata aqueous phase, about 600 μ L.3) RNA precipitate：
500 μ L isopropanols are added, 10min, 4 DEG C, 12000rpm centrifugation 10min are stood on ice, abandon supernatant.4) RNA washings：Add
1mL 75% (v/v) ethanol, precipitation is hanged, and 10min, 4 DEG C, 7500rpm centrifugations 15min are stood on ice；Repeat washing above
Step, then wash one time.5) RNA is dissolved：Centrifuge tube to be placed in open wide on ice and dries 5~10min, plus appropriate DEPC water dissolves.
Embodiment 3：The RNA sample sequencing of " hundred make " production bacterium Cordyceps sinensis Hirsutella sinensis
After extracting sample total serum IgE, with the enrichment with magnetic bead mRNA with Oligo (dT).Add fragmentation
MRNA is broken into short-movie section (200~700bp) by buffer, with mRNA as template, with hexabasic base random primer (random
Hexamers) synthesize first cDNA chain, then synthesize Article 2 cDNA chains, then by the purifying of QiaQuick PCR kits simultaneously
Plus end reparation is done after EB buffer solution elutions, is added polyA and is connected sequence measuring joints, then carry out piece with agarose gel electrophoresis
Duan great little is selected, and finally enters performing PCR amplification, and the sequencing library built up is sequenced with Illumina GA IIx.What sequencing was obtained
Raw image data is converted into sequence data, i.e. raw data or raw reads through base calling.Remove primitive sequencer
It is standby with subsequent analysis containing only the reads of adaptor sequences in reads.
Embodiment 4：" hundred make " produces the bacterium Cordyceps sinensis Hirsutella sinensis short reading sequence assemblings of RNA
Use short reads composite softwares SOAPdenovo (Li, Zhu et al.De novo assembly of human
genomes with massively parallel short read sequencing[J].Genome Res,2010,20:
265-272.) transcript profile is done from the beginning to assemble.Reads with certain length overlap is linked to be longer by SOAPdenovo first
The Contig fragments without N.Then reads is compared into back Contig, is determined from same by paired-end reads
The distance between different Contig of transcript and these Contig, SOAPdenovo connect together these Contig, in
Between unknown nucleotide sequence represented with N, thus obtain Scaffold.Scaffold is mended further with paired-end reads
Hole is processed, and finally obtains, Unigene sequence that two ends can not again extend minimum containing N.Finally, by Unigene sequences and albumen number
Blastx is according to storehouse nr, Swiss-Prot, KEGG and COG compare (evalue<0.00001) the best albumen of comparison result, is taken
Determine the sequence direction of Unigene.If the comparison result between different storehouses is contradictory, by nr, Swiss-Prot, KEGG and
The priority of COG determines the sequence direction of Unigene, the Unigene software ESTScan with four storehouses of the above all to being less than
(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 code area and determine the side of sequence
To.Unigene for can determine that sequence direction provides the sequence in its direction from 5' to 3', for that cannot determine sequence direction
Unigene provides the sequence that composite software is obtained.
Embodiment 5：" hundred make " produces bacterium Cordyceps sinensis Hirsutella sinensis Unigene functional annotations
Functional annotation information provides protein function annotation, Pathway annotations, COG functional annotations and the Gene of Unigene
Ontology (GO) functional annotation.First, by blastx by Unigene sequence alignments to albumen database nr, Swiss-
Prot, KEGG and COG (evalue<0.00001), obtain that there is the albumen of highest serial similitude with giving Unigene, so that
Obtain the protein function annotation information of the Unigene.The Pathway of Unigene can be further obtained according to KEGG annotation informations
Annotation.Unigene and COG databases are compared, the possible functions of Unigene is predicted and function statistic of classification is done to it.
According to nr annotation informations, Blast2GO softwares (Conesa, Gotz et al.Blast2GO are used:a universal tool
for annotation,visualization and analysis in functional genomics research[J]
.Bioinformatics,2005,21(18):3674-3676.) obtain the GO annotation informations of Unigene.Obtain each
After the GO annotations of Unigene, with WEGO softwares (Ye, Fang et al.WEGO:a web tool for plotting GO
annotations[J].Nucleic Acids Research,2006,34:GO functions 293-297.) are done to all Unigene
Statistic of classification, from the gene function distribution characteristics for macroscopically recognizing the species.
Embodiment 6：The metabolic pathway analysis of " hundred make " production bacterium Cordyceps sinensis Hirsutella sinensis cytidine
Fig. 2 is the pyrimidine metabolic (map00240) in KEGG metabolic pathways annotation, and the enzyme for having annotated is " hundred for having detected that
Make " production bacterium Cordyceps sinensis Hirsutella sinensis pyrimidine metabolic pathway relevant enzymes, it can be seen that detecting from three phosphorus
Uraturia glycosides sets out and synthesizes 2 Unigene of CTP synzyme of cytidine.It is online by the ORF Finder softwares in NCBI
Detection, have found the ORFs (SEQ ID No.3 and SEQ ID No.4) of this gene and has obtained corresponding protein
Sequence (SEQ ID No.1 and SEQ ID No.2).
Embodiment 7：" hundred make " produces bacterium Cordyceps sinensis Hirsutella sinensis CTP synthase gene design of primers
Drawn according to the gene open proofreading dna sequences Design that prediction is obtained with GENE RUNNER primer-design softwares
Thing, the CTP synthase genes of bacterium Hirsutella sinensis anabolism cytidine are produced for cloning " hundred make ", and primer is by Shanghai
Sheng Gong bioengineering Co., Ltd synthesizes, and primer sequence is listed below：
PynK1 genes：The ATAGAATTCATGCGCTGCGGTCGGAAACTG3 ' of forward primer 5 '
The ACGAAGCTTCTAAAAGCAAGAATTATCAGG of reverse primer 5 ' 3 '
PynK1 mrna lengths are 741bp
PynK2 genes：The ACAGAATTCATGAATCCGAAGGAGCACGGG of forward primer 5 ' 3 '
The ACGAAGCTTTCAGTGTTGCTCGAGCAAGATGG of reverse primer 5 ' 3 '
PynK2 mrna lengths are 627bp
Embodiment 8：" hundred make " produces the preparation of bacterium Cordyceps sinensis Hirsutella sinensis the first chains of cDNA
After the method for first being provided according to embodiment 1 turns out sutella sinensis fermented mycelium, carried according still further to embodiment 2
The method of confession carries out the extraction of total serum IgE to Hirsutella sinensis, and " hundred make " production bacterium Cordyceps sinensis is carried out by following after obtaining total serum IgE
The synthesis of the chains of Hirsutella sinensis cDNA first, tests for follow-up each gene cloning.
Using PrimeScript 1st Strand cDNA Synthesis Kit kits (TaKaRa) from Total
The reverse transcription synthesis chains of cDNA first in RNA, experimental procedure is as follows：
1) following mixed liquor is prepared in Microtube pipes.
2) denaturation, annealing operation are conducive to the denaturation of template ribonucleic acid and the specificity annealing of reverse transcription primer and template, can
Improve reverse transcription reaction efficiency, thus be denatured in PCR instrument, annealing reaction, condition setting is as follows：
65 DEG C, 5min
3) several seconds is centrifuged after annealing terminates makes the mixed liquor of template ribonucleic acid/primer etc. be gathered in Microtube bottom of the tube.
4) following inverse transcription reaction liquid is prepared in above-mentioned Microtube pipes.
5) reverse transcription reaction is carried out by following condition in PCR instrument.
42 DEG C of 15~30min
70℃ 15min
Ordinary circumstance, has a PolyA structure in the end of eukaryote mRNA 3 ', and the quantity of A bases is ten to hundreds of
It is individual, Oligo (dT) primer can be utilized using this structure, in the presence of reverse transcriptase, with mRNA as templated synthesis
The chains of cDNA first, the present invention uses sequence (the PrimeScript 1st Strand in the dT regions developed alone by TaKaRa
There is provided in cDNA Synthesis Kit) it is primer, if the mRNA integralities for obtaining are preferable, then can by process of reverse-transcription
To obtain the chains of cDNA first of all zymoprotein encoding genes in species.
Embodiment 9：" hundred make " production bacterium Cordyceps sinensis Hirsutella sinensis anabolism miazines nucleosides functional gene CTP is closed
Clone, expression and the detection of protein vigor into enzyme pynK1 and pynK2 gene
1st, the PCR amplifications of CTP synzyme pynK1 and pynK2 gene
The chains of cDNA first obtained with embodiment 8 as template, with embodiment 7 synthesize pynK1 gene primers：5’
The ACG AAG CTT CTA AAA GCA AGA ATT of ATA GAA TTC ATG CGC TGC GGT CGG AAA CTG 3 ' and 5 '
ATC AGG 3 ' and pynK2 gene primers：The ACG of 5 ' ACA GAA TTC ATG AAT CCG AAG GAG CAC GGG 3 ' and 5 '
AAGCTTTCAGTGTTGCTCGAGCAAGATGG 3 ' carries out Pfu archaeal dna polymerase pcr amplification reactions respectively, and condition setting is such as
Under：
Pfu pcr amplification reaction systems：
Pfu DNA Ploymerase PCR amplification conditions：
2nd, CTP synzyme pynK1 and pynK2 gene PCR product detected through gel electrophoresis
Specifically detection method is：1) the Ago-Gel microwave stove heat of 0.9% for preparing is uniformly dissolved it；
2) 15mL gels are taken, when gel is cooled to 50 DEG C or so, 1 μ L dyeing liquor Gold view is added, electrophoresis is poured into after being well mixed
On gel slab, point sample comb is inserted after removing bubble；3) it is careful to take out point sample comb after gel sets, offset plate is put into electrophoresis tank
(negative pole of the loading wells one end near electrophoresis tank), adds TAE electrophoretic buffers in electrophoresis tank；4) 5 μ L samples are taken and is subsequently adding 6
× Loading Buffer 1.5 μ L and ddH2Liquid-transfering gun loading is used after the μ L of O 4 mixing, applied sample amount is 10 μ L；5) electrophoresis tank is connected
Power line between electrophoresis apparatus, just extremely red, negative pole is black；6) power-on, starts electrophoresis, and ceiling voltage is no more than
5V/cm；7) electrophoresis can be terminated when sample ran the 2/3 of offset plate；8) after cutting off the electricity supply, gel is taken out and is put into gel imaging instrument
Middle observation, take pictures.
The size of transcript profile sequencing prediction CTP synzyme pynK1 and pynK2 genes is respectively 741bp and 627bp, agar
Sugared Gel electrophoresis results (Fig. 3) show that Successful amplification has gone out CTP synzyme pynK1 and pynK2 genes.
3rd, the base A that adds of CTP synzyme pynK1 and pynK2 gene PCR product is processed and purifying
Due to Pfu archaeal dna polymerase PCR primers end be flush end, so also need to carry out after glue reclaim plus base A treatment,
Just can be used for carrier T connection after purification.Glue reclaim product adds base A systems as follows：
72 DEG C plus A base 20min in PCR instrument, finally purified with AxyPrep PCR cleaning agents boxes.
4th, the connection of CTP synzyme pynK1 and pynK2 gene and cloning vector
Cloning vector pMD18-T Vector are purchased from TaKaRa companies (TaKaRa code D101A), and its physical map is shown in
Fig. 4, respectively by CTP synzyme pynK1 and pynK2 genes be connected with cloning vector construction recombination plasmid pMD18-T/pynK1 and
PMD18-T/pynK2, physical map is shown in Fig. 5, and linked system and condition of contact are as follows.
Linked system：
Condition of contact：16 DEG C, 16h；Inactivation：65 DEG C, 15min.
5th, the conversion of CTP synzyme recombinant plasmid pMD18-T/pynK1 and pMD18-T/pynK2
Recombinant plasmid pMD18-T/pynK1 and pMD18-T/pynK2 are transferred in E. coli JM109 respectively
Build recombinant bacterium the E.coli JM109/pMD18-T/pynK1 and E.coli for carrying CTP synzyme pynK1 and pynK2 genes
JM109/pMD18-T/pynK2.Concretely comprise the following steps：1) in 10 μ L reaction systems being gone into competent cell E.coli JM109,
Ice bath 30min；2) thermal shock：42 DEG C, 90s；3) ice bath：2-3min；4) 800 μ L liquid LB, 37 DEG C, 250rpm, 1h are added；5) apply
Cloth flat board (resistance containing Amp)；6) 37 DEG C of incubator overnight incubations.
6th, CTP synzyme E.coli JM109/pMD18-T/pynK1 and E.coli JM109/pMD18-T/pynK2 are positive
The screening of recombinant bacterium
Bacterium colony PCR can extract genomic DNA, and directly enter performing PCR as template with exposed DNA after thalline pyrolysis and expand
Increase, whether the method is easy to operate, quick, can be the positive bacterium colony containing purposeful plasmid with Rapid identification bacterium colony.In experiment, will
Corresponding single bacterium colony carries out bacterium colony PCR in being inoculated into fluid nutrient medium, to verify whether to be transferred to genes of interest.First, toothpick is used
Picking single bacterium colony is added in the 1.5mL centrifuge tubes containing 50 μ L sterilized waters, and boiling water bath 30min is then centrifuged for using supernatant as template,
Enter performing PCR amplification, PCR program settings are that Taq enzyme expands general procedure.Finally detected using 0.9% agarose gel electrophoresis
Bacterium colony PCR primer.
7th, the sequencing of CTP synzyme recombinant plasmid pMD18-T/pynK1 and pMD18-T/pynK2
The positive restructuring bacteria liquid LB medium cultures detected to bacterium colony PCR overnight after, take 4mL bacterium solutions extract plasmid,
Method presses the operating instruction of AxyPrep DNA small volume of reagent box offer.Sequencing is complete by Shanghai Sani bio tech ltd
Into.Through sequence verification, sequence SEQ ID No.3 and SEQ ID No.4 have been recombinated to pMD18-T/pynK1 and pMD18- respectively
In T/pynK2.
8th, the structure of CTP synzyme recombinant expression plasmid pET-28a/pynK1 and pET-28a/pynK2
Test the principle in expression in escherichia coli according to foreign gene, and expression vector pET-28a and CTP synzyme
PynK1 and pynK2 genes restriction enzyme site compares situation, it is determined that pynK1 genes EcoR I and the double enzyme sites of Hind III,
PynK2 genes EcoR I and the double enzyme sites of Hind III, and to restructuring E. coli JM109/pMD18-T/pynK1
The culture of liquid LB test tube shakers, recombinant plasmid are carried out with E.coli JM109/pMD18-T/pynK2 to extract.
Respectively by the recombinant plasmid pMD18-T/pynK1 and pMD18-T/pynK2 of CTP synzyme pynK1 and pynK2 genes
And corresponding expression vector pET-28a processes 6h, digestion body with the restriction enzymes of I/Hind of EcoR III in 37 DEG C of digestions
It is as follows：
The double digestion systems of I/Hind of EcoR III：
Digestion terminate after 65 DEG C of inactivation 15min, then reclaimed with Axygen DNA gel QIAquick Gel Extraction Kits respectively, pure
Change.
CTP synzyme pynK1 and pynK2 genes and expression vector pET-28a through double digestion, use T4 ligases again after purification
16 DEG C of connections overnight, build recombinant expression plasmid pET-28a/pynK1 and pET-28a/pynK2, and its building process is shown in Fig. 6, structure
Build recombinant expression plasmid pET-28a/pynK1 and the pET-28a/pynK2 collection of illustrative plates for obtaining and see Fig. 7.Linked system composition is as follows：
Linked system：
9th, the conversion of CTP synzyme recombinant expression plasmid pET-28a/pynK1 and pET-28a/pynK2 and positive Dan Ke
Grand screening
In the heat-shock transformed BL21 Host Strains to E.coli of expression plasmid that will be built, it is then applied to containing card that is mould
On the LB agar plates of plain (Kan) resistance (50mg/L), 37 DEG C of overnight incubations.The random picking individual colonies from flat board, with each work(
The primer of energy gene enters performing PCR amplification, selects positive colony.
10th, CTP synzyme recombinant bacterium E.coli BL21/pET-28a/pynK1 and E.coli BL21/pET-28a/
The induced expression of pynK2
The monoclonal that the positive will be accredited as is inoculated in the LB fluid nutrient mediums that 5mL contains Kan resistances (50mg/L), and 37
DEG C, 250r/min overnight incubations.1mL cultures are taken, the LB Liquid Cultures for containing Kan resistances (50mg/L) in 50mL of being transferred
37 DEG C in base, 250r/min cultivates to cell concentration OD600 and is about 0.6~0.8 or so.It is certain dense to being separately added into culture
Spend the IPTG Fiber differentiations 8h of (240mg/L).Collects thalline supplies electrophoretic analysis and Enzyme activity assay.
11st, CTP synzyme recombinant bacterium E.coli BL21/pET-28a/pynK1 and E.coli BL21/pET-28a/
PynK2 expression products SDS-PAGE is analyzed
Using the E.coli BL21 bacterium for being transferred to empty carrier and the recombinant bacterium for not adding derivant IPTG as control.It is accredited as
After positive recombinant bacterium is through IPTG Fiber differentiations certain hour (8h), 0.5mL Induced cultures are taken, thalline is collected by centrifugation, it is resuspended
In 50 μ L distilled water, 50 μ L sample-loading buffers are added, 10min is boiled after mixing, SDS-PAGE electrophoretic analysis is carried out, in Fig. 8
" K1 " swimming lane be recombinant bacterium E.coli BL21/pET-28a/pynK1 expression CTP synthetase albumens pynK1 (through sequencing
Verify its amino acid sequence for shown in SEQ ID No.1) SDS-PAGE figures, " K2 " swimming lane is recombinant bacterium E.coli BL21/
The CTP synthetase albumens pynK2 of pET-28a/pynK2 expression (is SEQ ID No.2 institutes through its amino acid sequence of sequence verification
Show) SDS-PAGE figure.
12nd, CTP synzyme recombinant bacterium E.coli BL21/pET-28a/pynK1 and E.coli BL21/pET-28a/
The protein active detection of pynK2
(1) the protein active detection of CTP synzyme recombinant bacterium E.coli BL21/pET-28a/pynK1
It is prepared by enzyme liquid：Weigh the recombinant bacterium E.coli BL21/pET-28a/pynK1 wet thallus 0.5g (dry weights of collection
0.1g) suspended with phosphate buffer (50mM, pH8.0) 15mL, ultrasonication is (power 350W, broken 2s, interval 2s, super altogether
Sound crushes 5min) as catalysis enzyme.
CTP synzyme pynK1 transformation systems：E.coli BL21/pET-28a/pynK1 are added to surpass in 50mL conversion bottles
Broken thalline 10mL, 0.1g uridine triphosphate of sound, 30 DEG C, 150r/min 2~3h of conversion reaction, after conversion terminates, in centrifuging and taking
It is clear standby with subsequent detection.
Detection method：High performance liquid chromatography quantitative determination cytidine.Pre-treatment：After conversion fluid centrifugation, supernatant
0.22 μm of microporous barrier is crossed for high performance liquid chromatography detection.Condition：Chromatographic column：Agilent C18 posts (4.6mm × 250mm i.d.5
μm)；Column temperature：35℃；Sampling volume：20μL；Flow velocity 1mL/min；Detection wavelength 260nm；Mobile phase：A：Ultra-pure water；B：Methyl alcohol.
Gradient elution：0min, 15%B, keep 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 standard curve：Accurately weigh cytidine, about 1.00mg-2.00mg, ultra-pure water constant volume.Each standard items
Prepare 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).Then loading successively, often
5 repetitions of individual concentration.Take certain volume maximum concentration titer mix after, constant volume, then HPLC analysis triphosphoric acid
The optimal conditions of cytidine detection.
By above-mentioned chromatographic condition detection and calculating, we draw to draw a conclusion：CTP synzyme recombinant bacteriums E.coli
The maximum specific enzyme activity (Specific Activity) of the CTP synzyme expressed by BL21/pET-28a/pynK1 is 2.51mol/
min/mg.Substrate conversion efficiency is 74%.
(2) the protein active detection of CTP synzyme recombinant bacterium E.coli BL21/pET-28a/pynK2
It is prepared by enzyme liquid：Weigh the recombinant bacterium E.coli BL21/pET-28a/pynK2 wet thallus 0.5g (dry weights of collection
0.1g) suspended with phosphate buffer (50mM, pH8.0) 15mL, ultrasonication is (power 350W, broken 2s, interval 2s, super altogether
Sound crushes 5min) as catalysis enzyme.
CTP synzyme pynK2 transformation systems：E.coli BL21/pET-28a/pynK2 are added to surpass in 50mL conversion bottles
Broken thalline 10mL, 0.1g uridine triphosphate of sound, 30 DEG C, 150r/min 2~3h of conversion reaction, after conversion terminates, in centrifuging and taking
It is clear standby with subsequent detection.
The making of detection method and standard curve is with CTP synzyme recombinant bacterium E.coli BL21/pET-28a/pynK1's
Protein active is detected.
By above-mentioned chromatographic condition detection and calculating, we draw to draw a conclusion：CTP synzyme recombinant bacteriums E.coli
The maximum specific enzyme activity (Specific Activity) of the CTP synzyme expressed by BL21/pET-28a/pynK2 is 1.79mol/
min/mg.Substrate conversion efficiency is respectively 71%.
Claims (8)
1. a kind of Cordyceps sinensis CTP synzyme, it is characterised in that the amino acid sequence of the enzyme is shown in SEQ ID No.2.
2. Cordyceps sinensis CTP synzyme as claimed in claim 1 is in living things catalysis uridine triphosphate prepares cytidine
Using.
3. application as claimed in claim 2, it is characterised in that described application is：With the synzyme thalline of CTP containing Cordyceps sinensis
The wet thallus that fermented and cultured is obtained are catalyst through the broken mixed liquor after clasmatosis, with uridine triphosphate as substrate, in pH
In for the transformation system of 6.5~8.5 cushioning liquid composition, 2~3h of conversion reaction under the conditions of 30 DEG C, 150rpm, reaction is tied
Shu Hou, by reacting liquid filtering, takes filtrate and is the crude product containing cytidine, and the crude product isolates and purifies acquisition triphosphoric acid born of the same parents
Glycosides.
4. application as claimed in claim 3, it is characterised in that the initial concentration of the substrate is 10g/L, the catalyst
Volumetric usage is 100mL/g substrates, and the dry mycelium concentration of the catalyst is 6.7mg/mL.
5. it is a kind of encode claim 1 described in enzyme gene.
6. gene as claimed in claim 5, it is characterised in that the nucleotides sequence of the gene is classified as shown in SEQ ID No.4.
7. as described in one of claim 5~6 gene build can living things catalysis uridine triphosphate prepare cytidine
Application in genetic engineering bacterium.
8. application as claimed in claim 7, it is characterised in that described application is：Build and closed containing the Cordyceps sinensis CTP
Into the recombinant vector of enzyme gene, the recombinant vector is converted into Escherichia coli, the recombination engineering bacteria of acquisition is lured
Culture is led, nutrient solution isolates and purifies somatic cells of the acquisition containing Cordyceps sinensis CTP synzyme.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420172A (en) * | 2001-11-19 | 2003-05-28 | 杭州华大基因研发中心 | High-temp resistant CTP synthetase gene, polypeptide coded therewith and preparing method thereof |
| CN102690796A (en) * | 2012-05-28 | 2012-09-26 | 浙江工业大学 | Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme |
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2012
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1420172A (en) * | 2001-11-19 | 2003-05-28 | 杭州华大基因研发中心 | High-temp resistant CTP synthetase gene, polypeptide coded therewith and preparing method thereof |
| CN102690796A (en) * | 2012-05-28 | 2012-09-26 | 浙江工业大学 | Enzyme synthesized by Chinese caterpillar fungus hirsutella sinensis to metabolize guanine nucleotide and genes and application of enzyme |
Non-Patent Citations (2)
| Title |
|---|
| Cloning,sequencing and characterization of the Saccharomyces cerevisiae URA7 gene encoding CTP synthetase;Odile Ozier-Kalogeropoulos et al.;《Mol Gen Genet》;19911231;第231卷;全文 * |
| 冬虫夏草的水溶性成分-核苷类化合物的研究;徐文豪等;《中药通报》;19880430;第13卷(第4期);第226页左栏第3段 * |
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