CN104130985B - Cordyceps sinensis China pilose spore malate dehydrogenase (malic acid dehydrogenase) A, encoding gene and application thereof - Google Patents
Cordyceps sinensis China pilose spore malate dehydrogenase (malic acid dehydrogenase) A, encoding gene and application thereof Download PDFInfo
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- CN104130985B CN104130985B CN201410308610.8A CN201410308610A CN104130985B CN 104130985 B CN104130985 B CN 104130985B CN 201410308610 A CN201410308610 A CN 201410308610A CN 104130985 B CN104130985 B CN 104130985B
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- 230000007226 seed germination Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000035900 sweating Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 235000015099 wheat brans Nutrition 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The present invention relates to one and produce bacterium Cordyceps sinensis China pilose spore participation tricarboxylic acid cycle for the malate dehydrogenase (malic acid dehydrogenase) A in citric acid synthesis from " hundred make ", the gene of this enzyme of encoding and application thereof.The aminoacid sequence of described malate dehydrogenase (malic acid dehydrogenase) A and SEQ? ID? does albumen shown in No.1 have more than 90% homology, its encoding gene nucleotide sequence and SEQ? ID? sequence shown in No.2 has more than 90% homology.The cloned DNA of nucleotide sequence provided by the present invention can be used for proceeding in engineering bacteria by the method for transduction, conversion, Conjugative tiansfer, by the expression regulating catalysis oxysuccinic acid to prepare the corresponding encoding gene of enzyme of corresponding oxaloacetic acid, give the high expression level of host's malate dehydrogenase (malic acid dehydrogenase) A, for the biologic applications expanding malate dehydrogenase (malic acid dehydrogenase) A provides effective way, there is major application prospect.
Description
(1) technical field
The present invention relates to one and produce bacterium Cordyceps sinensis China pilose spore participation tricarboxylic acid cycle for the malate dehydrogenase (malic acid dehydrogenase) A (Malicdehydrogenase) in citric acid synthesis from " hundred make ", the gene of this enzyme of encoding and application thereof.
(2) background technology
Cordyceps sinensis (Cordycepssinensis (Berk.) Sacc.) is that Cordyceps fungus colonizes in the stroma on lepidopteran (Lepidoptera) Hepialidae insect (HepialusarmoricanusOberthur) 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.
But, blank is almost to the research of malate dehydrogenase (malic acid dehydrogenase) in Cordyceps sinensis China pilose spore, especially in the research participating in the malate dehydrogenase (malic acid dehydrogenase) of tricarboxylic acid cycle in citric acid synthesis.
Malate dehydrogenase (malic acid dehydrogenase) (EC:1.1.1.37) is the enzyme that the dehydrogenation of catalysis L MALIC ACID becomes oxaloacetic acid.It is with NAD
+as electron acceptor(EA).Broadly also comprise with NAD
+or NADP
+the malic enzyme (EC:1.1.1.38-40) of pyruvic acid and carbonic acid is generated as acceptor.With NADP
+also weak reaction is had, also can by other hydroxy acid dehydrogenation.Extensively being present on plastosome, bacterial cell membrane, is a kind of enzyme in tricarboxylic acid cycle.Because the source of enzyme is different, its some character is also different.Oxysuccinic acid dehydrogenation under malate dehydrogenase enzyme catalysis generates oxaloacetic acid, and the hydrogen taken off is by NAD
+accept to generate NADH
+h
+.The oxaloacetic acid of regeneration can enter the synthesis of tricarboxylic acid cycle for citric acid again.
Malate dehydrogenase (malic acid dehydrogenase) is prevalent in animal, plant, in the various organism of bacterium, has the conservative property of height.Malate dehydrogenase (malic acid dehydrogenase) not only participates in numerous physiological activity, comprises C4 circulation, respiration, the oxidation of lipid acid, TCA circulation etc., and at seed germination, plant-growth, pollen development, fruit development, the aspects such as plant stress-resistance play a significant role.
1993, the people such as FCendrin were separated and the gene of enzyme malate dehydrogenase (malic acid dehydrogenase) (MDH) of the coding that checked order from extreme halotolerant archeobacteria Haloarculamarismortui.This enzyme is made up of 303 amino acid, and its molecular weight is 32638 dalton.
2008, based on grass carp (Ctenopharyngodonidellus) enteron aisle cDNA library malate dehydrogenase (malic acid dehydrogenase) (cMDH) est sequence that the people such as red legend drench build by laboratory, adopt end rapid amplification (RACE), from Healthy female grass carp (Ctenopharyngodonidellus) intestinal cell RNA, obtain the cDNA sequence (NCBI accession number: EU569765) of the grass carp intestinal cMDH of 1391bp.Result shows: this sequence comprises the 5' non-coding region (5'-UTR) of 62bp, the 3' non-coding region (3'-UTR) of 330bp, and allusion quotation plough tailing signal AATAA is positioned at polyA starting point upstream 16bp.The long 999bp of open reading frame ORF, 333 amino acid of encoding altogether, predicted protein iso-electric point is 6.67, and size is 36kDa.
2008, the people such as Yao Yuxin were separated the full length cDNA sequence of cyMDH from Apple, and this sequence contains the open reading frame (ORF) of a 996bp.Sequence homology analysis shows that Mal-cyMDH and other species cyMDH has higher homology.Prokaryotic expression obtains the fusion rotein of an about 37kD, and enzyme activity determination shows this enzyme major catalytic synthesizing apple acid.
2011, the people such as Li Qian were with genome of E.coli DNA for template, and amplification obtains malate dehydrogenase (malic acid dehydrogenase) (mdh) encoding gene mdh, constructed recombinant bacterium pET-28a-mdh/BL21 and successful expression malate dehydrogenase (malic acid dehydrogenase), size about 36000.Select the activated malate dehydrogenase (malic acid dehydrogenase) of Ni post affinity chromatography purifying tool, living than enzyme after purifying reaches 112.5U/mg, and purification reaches 2.62 times, and the rate of recovery is 59%.
2011, the people such as GNava were cloned into the complete encoding sequence of a malate dehydrogenase (malic acid dehydrogenase) from taeniasis suis endochylema.The cDNA sequence of this malate dehydrogenase (malic acid dehydrogenase) finds from taeniasis suis Genome Project database, and the protein of coding 332 amino-acid residues, molecular weight is approximately 36.5kDa.
2012, the people such as Li Wenshuan utilize the malate dehydrogenase gene cDNA sequence in electronic cloning technology acquisition soybean, and bioinformatic analysis and prediction are carried out to gene and proteins encoded thereof, provide foundation for cloning the problem such as this gene, solution plant phosphorus high efficiency gene quantity shortage further.Result shows, malate dehydrogenase gene (GmMDH1) the cDNA sequence length in soybean is 1574bp, open reading frame 1230bp, 409 amino-acid residues of encoding.
2013, the people such as YYChang increased a malate dehydrogenase gene from the genome of Thermusthermophilus, and cloned on the pET21b carrier of island and express.This albumen exists with soluble form in coli strain BL21 (DE3).
But, in current ncbi database, also retrieve the gene-correlation information less than malate dehydrogenase (malic acid dehydrogenase) in China pilose spore at present.
(3) summary of the invention
The object of the invention is for the above deficiency that exists and the technical issues that need to address, bacterium Cordyceps sinensis China pilose spore is produced participating in tricarboxylic acid cycle to " hundred make " and is used for enzyme in citric acid synthesis and encoding gene is furtherd investigate, provide " hundred makes " produce bacterium Cordyceps sinensis China pilose spore participate in tricarboxylic acid cycle be used for citric acid synthesize in a kind of malate dehydrogenase (malic acid dehydrogenase) A and encoding gene and application thereof.
The technical solution used in the present invention is:
A kind of participate in China pilose spore tricarboxylic acid cycle for citric acid synthesis in malate dehydrogenase (malic acid dehydrogenase) A (Malicdehydrogenase), with aminoacid sequence shown in SEQIDNo.1, there is more than 90% homology.Due to the singularity of aminoacid sequence; the fragment of any peptide protein containing aminoacid sequence shown in SEQIDNO.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.
Preferably, described malate dehydrogenase (malic acid dehydrogenase) A aminoacid sequence (is designated as mdhA albumen) as shown in SEQIDNo.1; This enzyme can prepare corresponding oxaloacetic acid by catalysis oxysuccinic acid.
The present invention states malate dehydrogenase (malic acid dehydrogenase) A and produces bacterium Cordyceps sinensis China pilose spore from " hundred make ".
The path obtaining corresponding oxaloacetic acid through the catalysis of malate dehydrogenase (malic acid dehydrogenase) A by oxysuccinic acid is as follows:
The invention still further relates to described malate dehydrogenase (malic acid dehydrogenase) A and prepare application in corresponding oxaloacetic acid at biocatalysis oxysuccinic acid, described in be applied as: prepare corresponding oxaloacetic acid with malate dehydrogenase (malic acid dehydrogenase) A catalysis oxysuccinic acid of the present invention.Concrete, described is applied as: with the wet thallus phosphate buffered saline buffer (50mM obtained through inducing culture containing the recombinant bacterial strain of malate dehydrogenase (malic acid dehydrogenase) A gene, pH8.0) suspend, after ultrasonic cell disintegration, centrifugal, getting supernatant liquor is catalyzer, with 0.1M aqueous solution of malic acid for substrate, with the NADH aqueous solution of 3.75mM for coenzyme, be in the phosphoric acid buffer of 7.0 in pH value, water-bath at 37 DEG C, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and namely obtain mixed solution containing oxaloacetic acid, separation and purification after mixing, namely (described separation purification method is known in this field to obtain oxaloacetic acid, the method of usual employing affinity chromatography).
The consumption of described substrate is in oxysuccinic acid quality, the starting point concentration of described oxysuccinic acid is 0.05M, the volumetric usage of described catalyzer is in the quality of broken front wet thallus, final concentration is 4.2 ~ 5g/L (preferred 4.2g/L), and in described reaction system, the final concentration of NADH is 3.75 μm of ol/L.
Described catalyzer is prepared as follows: be inoculated in the LB liquid nutrient medium containing the Kan resistance of final concentration 50 μ g/ml by the recombinant bacterial strain containing malate dehydrogenase (malic acid dehydrogenase) A gene, 37 DEG C, 250r/min overnight incubation, get 1mL culture, transferred (inoculum size is volumetric concentration 2%) contain in 50mL in the LB liquid nutrient medium of final concentration 50 μ g/mlKan resistance, 37 DEG C, it is 0.6 ~ 0.8 that 250r/min is cultured to cell concentration OD600, the IPTG inducing culture 8h of final concentration 0.05mmol/L is added in culture, collect wet thallus, by wet thallus at power 40%, broken 1s to stop under 1s condition ultrasonication (preferably 3 times, each 5min), centrifugal, get supernatant liquor and be catalyzer.
The invention still further relates to described malate dehydrogenase (malic acid dehydrogenase) A encoding gene, the nucleotide sequence of described encoding gene (is designated as mdhA gene as shown in SEQIDNo.2; MdhA genes encoding mdhA albumen).
Due to the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQIDNO.2, 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 malate dehydrogenase (malic acid dehydrogenase) A gene can be used for building can prepare the genetic engineering bacterium of corresponding oxaloacetic acid by biocatalysis oxysuccinic acid, to expand the application of malate dehydrogenase (malic acid dehydrogenase) A, be specially: build the recombinant vectors containing described malate dehydrogenase (malic acid dehydrogenase) A gene, described recombinant vectors is converted in intestinal bacteria (preferred E.coliBL21 (DE3)), the recombination engineering bacteria obtained carries out inducing culture, and nutrient solution separation and purification obtains the somatic cells containing malate dehydrogenase (malic acid dehydrogenase) A gene.
Main points of the present invention there are provided the nucleotide sequence shown in the aminoacid sequence shown in SEQIDNO.1 and SEQIDNO.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 malate dehydrogenase (malic acid dehydrogenase) A of the present invention and encoding gene thereof can be provided to be China pilose spore (Hirsutellasinensis) L0106, this culture presevation is in China typical culture collection center, deposit number is CCTCCNo:M2011278, discloses in the patent CN102373190A of previously application.
Beneficial effect of the present invention is mainly reflected in: the present invention prepares corresponding oxaloacetic acid to catalysis oxysuccinic acid and studies in detail principle, provide " hundred make " and produce bacterium Cordyceps sinensis China pilose spore participation tricarboxylic acid cycle for the malate dehydrogenase (malic acid dehydrogenase) A in citric acid synthesis and encoding gene thereof, the cloned DNA of nucleotide sequence provided by the present invention can be used for by transduction, transform, the method of Conjugative tiansfer proceeds in engineering bacteria, by the expression regulating catalysis oxysuccinic acid to prepare the corresponding encoding gene of enzyme of corresponding oxaloacetic acid, give the high expression level of host's malate dehydrogenase (malic acid dehydrogenase) A, for the biologic applications expanding malate dehydrogenase (malic acid dehydrogenase) A 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 that China pilose spore malate dehydrogenase (malic acid dehydrogenase) participates in tricarboxylic acid cycle process annotated map;
Fig. 3 is malate dehydrogenase (malic acid dehydrogenase) A gene PCR amplified production gel electrophoresis figure;
Fig. 4 is cloning vector pMD18-TVector and expression vector pET-28a physical map;
Fig. 5 is restructuring cloned plasmids pMD18-T/mdhA physical map;
Fig. 6 is recombinant expression plasmid pET-28a/mdhA building process schematic diagram;
Fig. 7 is recombinant expression plasmid pET-28a/mdhA physical map;
Fig. 8 is the SDS-PAGE figure of malate dehydrogenase (malic acid dehydrogenase) A albumen.
(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 (Hirsutellasinensis) through this bacterial strain of strain identification, this culture presevation is in China typical culture collection center, deposit number is CCTCCNo:M2011278, 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, lower with), 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; Cultivate 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%, and 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
Extract total serum IgE with TRIzol reagent, step is specially:
1) liquid nitrogen grinding: get the new fresh thalli of 1g and put into mortar, repeatedly add liquid nitrogen and be fully ground to Powdered, be dispensed in the 1.5mL centrifuge tube of precooling, add 1mLTRIzol 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 1mL75% (v/v) ethanol, precipitation hanged, 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 the enrichment with magnetic bead mRNA with Oligo (dT).Add fragmentationbuffer 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 (randomhexamers), then Article 2 cDNA chain is synthesized, do end reparation after adding EB buffer solution elution through QiaQuickPCR kits, 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 IlluminaGAIIx built up checks order.The raw image data obtained that checks order is converted into sequence data through basecalling, i.e. rawdata or rawreads.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, Zhuetal.Denovoassemblyofhumangenomeswithmassivelyparalle lshortreadsequencing [J] .GenomeRes, 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-endreads, these Contig connect together by SOAPdenovo, and middle unknown nucleotide sequence N represents, so just obtains Scaffold.Utilize paired-endreads 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, Jongeneeletal.ESTScan:aprogramfordetecting, evaluating, andreconstructingpotentialcodingregionsinESTsequences [J] .InProceedingsof9thInternationalConferenceonIntelligentS ystemsforMolecularBiology.AAAIPress, MenloPark, 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 GeneOntology (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, Gotzetal.Blast2GO:auniversaltoolforannotation, visualizationandanalysisinfunctionalgenomicsresearch [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, Fangetal.WEGO:awebtoolforplottingGOannotations [J] .NucleicAcidsResearch, 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 the analysis of the working cycle of bacterium Cordyceps sinensis China pilose spore malate dehydrogenase (malic acid dehydrogenase) A catalysis
Fig. 2 is the procedure chart that China pilose spore malate dehydrogenase (malic acid dehydrogenase) A participates in tricarboxylic acid cycle.Tricarboxylic acid cycle is the recycle system of the enzymatic reaction for the ethanoyl in acetyl-CoA being oxidized to carbonic acid gas and reducing equivalent, and the first step of this circulation forms citric acid by acetyl-CoA and oxaloacetic acid condensation.Reactant acetyl-CoA is the common intermediate product of carbohydrate, lipid, amino acid metabolism, and can be decomposed after entering circulation and finally generate product carbon dioxide and produce H, H will pass to coenzyme--nicotinamide adenine dinucleotide (NAD
+) and flavin adenine dinucleotide (FAD), make it to become NADH
+h
+and FADH
2.NADH
+h
+and FADH
2carry H and enter respiratory chain, respiratory chain by electron transmission to O
2produce water, the phosphorylation of coupled oxidation simultaneously produces ATP, provides energy.The Unigene of malate dehydrogenase (malic acid dehydrogenase) A is detected from the order-checking of China pilose spore transcript profile and annotation information.By the ORFFinder software on-line checkingi in NCBI, have found the open reading frame (SEQIDNo.2) of this gene and obtain corresponding protein sequence (SEQIDNo.1).
Embodiment 7: " hundred make " produces the design of bacterium Cordyceps sinensis China pilose spore malate dehydrogenase (malic acid dehydrogenase) A gene primer
Use GENERUNNER primer-design software according to predicting each gene open proofreading dna primers obtained, produce the degraded of bacterium China pilose spore for clone's " hundred make " and participate in tricarboxylic acid cycle for the malate dehydrogenase (malic acid dehydrogenase) A gene in citric acid synthesis, primer by Sani bio tech ltd, Shanghai synthesize, primer sequence as follows listed by:
MdhA gene: forward primer 5 ' AGAGAATTCATGACCTCGGCCGGCCCCCG3 '
Reverse primer 5 ' ATACTCGAGTCACGATACGGTGGCATACAT3 '
MdhA mrna length is 1299bp.
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 PrimeScript1stStrandcDNASynthesisKit test kit (TaKaRa) reverse transcription synthesis cDNA first chain from TotalRNA, 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℃，5min
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℃15～30min
70℃15min
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 (providing in PrimeScript1stStrandcDNASynthesisKit) in the dT region developed alone by TaKaRa 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 malate dehydrogenase (malic acid dehydrogenase) A gene, expression and protein vigor
1, the pcr amplification of malate dehydrogenase (malic acid dehydrogenase) A gene
With cDNA first chain obtained in embodiment 8 for template, malate dehydrogenase (malic acid dehydrogenase) A gene primer with synthesis in embodiment 7: 5 ' AGAGAATTCATGACCTCGGCCGGCCCCCG3 ' and 5 ' ATACTCGAGTCACGATACGGTGGCATACAT3 ' carries out PfuDNA polysaccharase pcr amplification reaction, and condition setting is as follows:
PfuPCR amplification reaction system:
PfuDNAPloymerasePCR amplification condition:
2, malate dehydrogenase (malic acid dehydrogenase) A gene PCR product detected through gel electrophoresis
Concrete detection method is:
1) it is made to be uniformly dissolved the sepharose microwave-oven-heating of prepare 0.9%;
2) get 15mL sepharose, when sepharose is cooled to about 50 DEG C, add 1 μ L staining fluid Goldview, pour into after mixing on electrophoresis agarose gel plate, after removing bubble, insert point sample comb;
3), after on agarose gel plate, sepharose solidifies, 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 × LoadingBuffer1.5 μ L and ddH
2o4 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 5V/cm;
7) electrophoresis can be stopped when sample ran 2/3 of agarose gel plate;
8), after cutting off the electricity supply, gel on agarose gel plate taken out and puts into the observation of gel imaging instrument, take pictures.
The size of transcript profile order-checking prediction malate dehydrogenase (malic acid dehydrogenase) A gene is 1299bp, and agarose gel electrophoresis result shows that Successful amplification has gone out malate dehydrogenase (malic acid dehydrogenase) A gene, and size is about 1300bp.Fig. 3 is that " hundred make " produces bacterium China pilose spore malate dehydrogenase (malic acid dehydrogenase) A functional gene PCR primer gel electrophoresis figure.
3, malate dehydrogenase (malic acid dehydrogenase) A gene PCR product add base A process and purifying
Because PfuDNA polysaccharase 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 20min, finally purify with AxyPrepPCR cleaning agents box.
4, the connection of malate dehydrogenase (malic acid dehydrogenase) A gene and cloning vector
Cloning vector pMD18-TVector is purchased from TaKaRa company (TaKaRacodeD101A), its physical map is shown in Fig. 4, the malate dehydrogenase (malic acid dehydrogenase) A gene of step 3 purifying is connected construction recombination plasmid pMD18-T/mdhA with cloning vector, 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 malate dehydrogenase (malic acid dehydrogenase) A recombinant plasmid pMD18-T/mdhA
Recombinant plasmid pMD18-T/mdhA is proceeded in E. coli JM109, build the recombinant bacterium E.coliJM109/pMD18-T/mdhA carrying malate dehydrogenase (malic acid dehydrogenase) A gene, concrete steps are: 1) go in competent cell E.coliJM109 by 10 μ L reaction systems (i.e. the connection product of step 4), 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 malate dehydrogenase (malic acid dehydrogenase) AE.coliJM109/pMD18-T/mdhA
Bacterium colony PCR can extract genomic dna, and directly with the DNA exposed after thalline pyrolysis for template carries out pcr amplification, the method is easy and simple to handle, quick, can Rapid identification bacterium colony be whether positive bacterium colony containing object plasmid, comparatively common in conversion qualification.In experiment, carrying out bacterium colony PCR by being inoculated into single bacterium colony corresponding in liquid nutrient medium, whether proceeding to goal gene to verify.First, 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 malate dehydrogenase (malic acid dehydrogenase) A recombinant plasmid pMD18-T/mdhA
LB liquid medium is seeded to the positive recombinant bacterium that bacterium colony PCR detects, 37 DEG C, after 150rpm overnight incubation, 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 SEQIDNo.2 has recombinated in pMD18-T/mdhA.
8, the structure of malate dehydrogenase (malic acid dehydrogenase) A recombinant expression plasmid pET-28a/mdhA
Experimental basis foreign gene is in the principle of expression in escherichia coli, and expression vector pET-28a and malate dehydrogenase (malic acid dehydrogenase) A gene restriction enzyme site comparison situation, determine mdhA EcoR I/Xhol double enzyme site, and the cultivation of liquid LB test tube shaker, recombinant plasmid extraction are carried out to restructuring E. coli JM109/pMD18-T/mdhA.
The recombinant plasmid pMD18-T/mdhA of malate dehydrogenase (malic acid dehydrogenase) A gene and expression vector pET-28a use respectively EcoR I/Xhol restriction enzyme 37 DEG C respectively enzyme cut process 6h, it is as follows that enzyme cuts system:
EcoR I/Xhol double digestion system:
After enzyme cuts end, 65 DEG C of deactivation 15min, then reclaim that test kit carries out reclaiming, purifying respectively with AxygenDNA gel.
Malate dehydrogenase (malic acid dehydrogenase) A gene and expression vector pET-28a are after double digestion, purifying, connect with T4 ligase enzyme 16 DEG C again and spend the night, build recombinant expression plasmid pET-28a/mdhA, its building process is shown in Fig. 6, builds the recombinant expression plasmid pET-28a/mdhA collection of illustrative plates obtained and sees Fig. 7.Linked system is composed as follows:
Linked system:
9, the conversion of malate dehydrogenase (malic acid dehydrogenase) A recombinant expression plasmid and the screening of positive monoclonal
By heat-shock transformed in E.coliBL21 (DE3) Host Strains for the expression plasmid built, be then applied on the LB agar plate containing kantlex (Kan) resistance (final concentration 50 μ g/ml), 37 DEG C of overnight incubation.Random choose list bacterium colony from flat board, carries out pcr amplification with the malate dehydrogenase (malic acid dehydrogenase) A gene primer of synthesis in step 7, selects positive colony.
10, the abduction delivering of malate dehydrogenase (malic acid dehydrogenase) A recombinant bacterium
The mono-clonal being accredited as the positive is inoculated in 5mL to be contained in the LB liquid nutrient medium of Kan resistance (final concentration 50 μ g/ml), 37 DEG C, 250r/min overnight incubation.Get 1mL culture, transferred in 50mL to contain in the LB liquid nutrient medium of Kan resistance (final concentration 50 μ g/ml) 37 DEG C, 250r/min is cultured to cell concentration OD600 and is about about 0.6 ~ 0.8.Certain density IPTG (final concentration 0.05mmol/L) inducing culture 8h is added respectively in culture.Collect thalline for electrophoretic analysis and Enzyme activity assay.
11, malate dehydrogenase (malic acid dehydrogenase) A recombinant bacterium expression product SDS-PAGE analyzes
With E.coliBL21 (DE3) 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 7h, get 0.5mL Induced cultures, collected by centrifugation thalline, be resuspended in 50 μ L distilled water, add 50 μ L sample-loading buffers, 10min is boiled after mixing, carry out SDS-PAGE electrophoretic analysis, " A " swimming lane in Fig. 8 is the SDS-PAGE figure of e. coli bl21 ghost, " B " swimming lane is the SDS-PAGE figure after recombinant bacterium E.coliBL21 (DE3)/pET-28a adds IPTG induction, " C " swimming lane is that the contrast SDS-PAGE that recombinant bacterium E.coliBL21 (DE3)/pET-28a/mdhA does not add IPTG schemes, " D " swimming lane is the SDS-PAGE figure of recombinant bacterium E.coliBL21 (DE3)/pET-28a/mdhA abduction delivering.Show that recombinant bacterium E.coliBL21 (DE3)/pET-28a/mdhA is containing malate dehydrogenase (malic acid dehydrogenase) A (through its aminoacid sequence of sequence verification as shown in SEQIDNo.1).
12, the protein vigor of malate dehydrogenase (malic acid dehydrogenase) A recombinant bacterium detects
The protein vigor of malate dehydrogenase (malic acid dehydrogenase) A detects:
1. the phosphoric acid buffer of 0.2M: take the Sodium phosphate dibasic of 3.561g and the SODIUM PHOSPHATE, MONOBASIC of 3.121g in the volumetric flask of 100mL, and add water to scale marks place, be stored in brown bottle for subsequent use, with the soft rubber ball of cleaning, bottleneck covered tightly.
2. the 3.75mMNADH aqueous solution: the NADH taking 2.86g in the volumetric flask of 100mL, and adds water to scale marks place with water-soluble, is stored in brown bottle for subsequent use, is covered tightly by bottleneck with the soft rubber ball of cleaning.
3. the standardized solution of 1mM oxaloacetic acid: the oxaloacetic acid taking 13.2mg in the volumetric flask of 100mL, and adds water to scale marks place with water-soluble, is stored in brown bottle for subsequent use, is covered tightly by bottleneck with the soft rubber ball of cleaning.Make the typical curve of oxaloacetic acid by table 1, equation is: y=2.5488x+0.0334, R
2=0.9963, wherein y is the light absorption value under 340nm, and x is the concentration of standard substance.
The typical curve of table 1 oxaloacetic acid
Prepared by enzyme liquid: take recombinant bacterium E.coliBL21 (DE3)/pET-28a/mdhA wet thallus 2g that step 10 is collected, suspend with phosphate buffered saline buffer (50mM, pH8.0) 100mL, high pressure cracker (model FS-600, Shanghai Sheng Xi ultrasonic instrument company limited) at power 40%, broken 1s broken 3 times (35Kpa) under stopping 1s condition, each 5min, centrifugal removing thalline, gets supernatant liquor and obtains crude enzyme liquid 79.3ml.
Malate dehydrogenase (malic acid dehydrogenase) A transformation system: transform in bottle the aqueous solution of malic acid 3mL of the NADH aqueous solution 1mL and 0.1M adding the crude enzyme liquid that E.coliBL21 (DE3)/fragmentation of pET-28a/madA high pressure is collected afterwards of 1mL, phosphoric acid buffer 1mL, 3.75mM of 0.2M at 50mL.After 37 DEG C of water-bath 10min, the centrifugal 5min of 12000r/min, termination reaction, gets supernatant liquor survey enzyme and lives.
Measure the amount of supernatant liquor mesoxalyl acetic acid, malate dehydrogenase (malic acid dehydrogenase) A decomposes oxysuccinic acid and generates oxaloacetic acid, along with the increase of oxaloacetic acid, and A
340nmalso become large gradually, oxaloacetic acid concentration corresponding to arbitrary absorbance can be obtained from oxaloacetic acid concentration one absorbance curve.
Malate dehydrogenase (malic acid dehydrogenase) A Mei Huo unit (U) is defined as: malate dehydrogenase (malic acid dehydrogenase) A per minute decomposes the enzyme amount that oxysuccinic acid produces 1 μm of ol oxaloacetic acid.
The blank detecting malate dehydrogenase (malic acid dehydrogenase) A enzyme alive is that after boiling 20min, the crude enzyme liquid of inactivation substitutes enzyme liquid.In addition, also have detected the vigor of the crude enzyme liquid after E.coliBL21 (DE3) and E.coliBL21 (DE3)/pET-28a induction under similarity condition, all do not detect that malate dehydrogenase (malic acid dehydrogenase) A enzyme is lived.
The Coomassie Brilliant Blue protein content recorded in malate dehydrogenase (malic acid dehydrogenase) A crude enzyme liquid is utilized to be 0.214mg/mL, by Bandscan software, crude enzyme liquid band content in SDS-PAGE is analyzed, malate dehydrogenase (malic acid dehydrogenase) A accounts for 16.4% of total protein, therefore the malate dehydrogenase (malic acid dehydrogenase) A participating in catalyzed reaction is 0.214mg/mL × 1mL × 0.164=0.035mg.Live according to enzyme and define, the high specific enzyme work of malate dehydrogenase (malic acid dehydrogenase) A is: 45.6 μm of ol/ (0.035mg × 10min)=130.3 μm ol/mg/min=130.3U/mg.Therefore, the high specific enzyme of the malate dehydrogenase (malic acid dehydrogenase) A expressed by malate dehydrogenase (malic acid dehydrogenase) A recombinant bacterium is lived as 130.3U/mg, and the transformation efficiency of above-mentioned reaction is 15.2%.Than enzyme work calculation formula be: than the Tot Prot of enzyme work=enzyme work/enzyme.Transformation efficiency calculation formula is: transformation efficiency=(starting point concentration-equilibrium concentration)/starting point concentration.
Claims (9)
1. a Cordyceps sinensis China pilose spore malate dehydrogenase (malic acid dehydrogenase) A, is characterized in that the aminoacid sequence of described dehydrogenase A is as shown in SEQIDNo.1.
2. malate dehydrogenase (malic acid dehydrogenase) A as claimed in claim 1 prepares the application in oxaloacetic acid at biocatalysis oxysuccinic acid.
3. apply as claimed in claim 2, it is characterized in that described being applied as: suspend with the wet thallus pH8.0 phosphate buffered saline buffer obtained through inducing culture containing recombinant bacterial strain E.coliBL21 (the DE3)/pET-28a/mdhA of malate dehydrogenase (malic acid dehydrogenase) A gene, after ultrasonication, centrifugal, getting supernatant liquor is catalyzer, with 0.1M aqueous solution of malic acid for substrate, with the NADH aqueous solution of 3.75mM for coenzyme, be in the phosphoric acid buffer of 7.0 in pH value, react at 37 DEG C, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and namely obtain mixed solution containing oxaloacetic acid.
4. apply as claimed in claim 3, it is characterized in that: the volumetric usage of described substrate is in oxysuccinic acid quality, the starting point concentration of described oxysuccinic acid is 0.05M, the volumetric usage of described catalyzer counts 4.2g/L with the quality of broken front wet thallus, and in described reaction system, the consumption of NADH is 3.75 μm of ol/L.
5. apply as claimed in claim 3, it is characterized in that described catalyzer is prepared as follows: be inoculated in the LB liquid nutrient medium containing the Kan resistance of final concentration 50 μ g/ml by the recombinant bacterial strain containing malate dehydrogenase (malic acid dehydrogenase) A gene, 37 DEG C, 250r/min overnight incubation, get culture, transfer in the LB liquid nutrient medium containing final concentration 50 μ g/mlKan resistance with volumetric concentration 2% inoculum size, 37 DEG C, it is 0.6 ~ 0.8 that 250r/min is cultured to cell concentration OD600, the IPTG inducing culture 8h of final concentration 0.05mmol/L is added in culture, collect wet thallus, by wet thallus at power 40%, broken 1s stops ultrasonication under 1s condition, centrifugal, get supernatant liquor and be catalyzer.
6. the gene of malate dehydrogenase (malic acid dehydrogenase) A described in claim 1 of encoding.
7. encoding gene as claimed in claim 6, is characterized in that the nucleotide sequence of described encoding gene is as shown in SEQIDNo.2.
8. encoding gene is as claimed in claims 6 or 7 building and can prepare application in the genetic engineering bacterium of oxaloacetic acid by biocatalysis oxysuccinic acid.
9. apply as claimed in claim 8, it is characterized in that described being applied as: build the recombinant vectors pET-28a/mdhA containing described malate dehydrogenase (malic acid dehydrogenase) A gene, described recombinant vectors is converted in E. coli BL21 (DE3), recombination engineering bacteria E.coliBL21 (the DE3)/pET-28a/mdhA obtained carries out inducing culture, and nutrient solution separation and purification obtains the somatic cells containing malate dehydrogenase (malic acid dehydrogenase) A gene.
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