CN108239658A - Application of the recombinant plasmid of height expression DNMT2 genes in weaning stress of piglet is prevented - Google Patents
Application of the recombinant plasmid of height expression DNMT2 genes in weaning stress of piglet is prevented Download PDFInfo
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Abstract
The invention belongs to animal gene engineering technology fields, and in particular to application of the recombinant plasmid of height expression DNMT2 genes in weaning stress of piglet is prevented.It is a feature of the present invention that using pcDNA3.1 carriers as initial vector, structure obtains the eukaryotic expression recombinant plasmid pcDNA3.1 DNMT2 of high expression DNMT2 genes, which contains SEQ ID NO：Coded sequence described in 2.The method that the present invention uses for reference genomic medicine treatment, the recombinant plasmid is prepared into the acceptable pharmaceutical composition of animal to be injected into weanling pig or animal body, make DNMT2 albumen of its expression with natural structure and activity, so as to achieve the purpose that prevent weaning stress of piglet and Heat stress.
Description
Technical field
The invention belongs to animal gene engineering technology fields, and in particular to one kind high in animal body can express DNMT2 bases
The recombinant plasmid of cause and its application in prevention weaning stress of piglet and Heat stress.
Technical background
In pig production, early weaning piglet is one of key technology for improving sow productivity.But wean is young
An Important Change in pig life.The piglet digestive function of early weaning is not yet sound, in addition the change of environment and cause of disease are micro-
The influence of many factors such as biology, wean will certainly cause weaning stress of piglet.Weaning stress of piglet often show as loss of appetite,
Weight loss, seriously have loose bowels, premunition is weak, growth retardation even causes death, cause heavy economic losses to pig breeding industry.Cause
This, reduce weanling pig stress influence for improve pig breeding industry productivity effect it is most important.
In order to enhance the premunition of weanling pig, prevent the conventional method of weaning stress of piglet generally from weaning time, feeding
It supports and sets about in environment, feed and method.Antibiotic or the injection of antibiotics after weaned piglet are added in feed in many pig farms.
But low dosage antibiotic leads to antibiotic resistance getting worse in the use of aquaculture, disabling antibiotic in pig breeding industry exists
Common recognition is had become in world wide.Live pig grows up healthy and sound, and there is an urgent need for new means to prevent weaning stress of piglet.
DNMT2 gene code DNMT2 albumen.DNMT2 albumen is a member of dnmt rna (DNMT) family, mainly
Methylate 38 cytimidines (Goll, 2006) of eukaryon aspartic acid, glycine and valine tRNA, and generation 5- methyl born of the same parents are phonetic
Pyridine.This modification helps to enhance the stability of tRNA, promotes albumen synthesis (Tuorto, 2012), can also prevent tRNA from answering
Swash and be sheared (Schaefer, 2010) under state.Document report, DNMT2 participate in the RNA processing of stress reaction process
(Thiagarajan, 2011), and during effectively antiviral immune response essential (Durdevic, 2013).
DNMT2 can also improve amoeba histolytica to NO stress tolerance (Hertz et al, 2014).In vivo study shows
The murine protein synthesis that DNMT2 is knocked out is reduced, impaired cellular differentiation, and the phenomenon that early lethality (Tuorto, 2012) occurs.And
Life span of drosophila melanogaster (Lin, 2005) can then be extended by being overexpressed DNMT2.It can be seen that DNMT2 may play an important roll resisting stress.
Studies in China shows that in the different pig kind tissue of anti-stress ability the mRNA level in-site of DNMT2 genes exists significantly
Difference (Wu Yingyan, 2011).We early-stage study have found that DNMT2 expression significantly reduces in piglet gastrointestinal tract after wean.This and text
It is consistent to offer report weanling pig Intestinal Morphology variation (including intestinal wall atrophy, crypts intensification, villi damage, shortening).We are therefore
Speculate, DNMT2 is in the molecular basis that the reduction of weanling pig enteron aisle expression may be the variation of its Intestinal Morphology, increase
DNMT2 gene expressions may alleviate the symptom of weaning stress of piglet.
Non-virus carrier of the eukaryotic expression vector pcDNA3.1 for high copy has the advantages that unconformity and easy to operate,
It can ensure the effective transcription and translation of exogenous DNA, and daughter cell can be entailed with division of host cells.Gene therapy refers to
The gene with normal function or therapeutic gene are imported by gene transfer mode in diseased organism body in gene level, make trouble
It is not present in the normally functioning gene of diease occurrence object representation or protozoa body or the gene of low expression, assigns its new disease-resistant work(
Can, reach therapeutic purposes.
Invention content
It can the high expression in weanling pig body it is an object of the invention to overcome the deficiencies of the prior art and provide one kind
The eukaryotic expression recombinant plasmid of DNMT2.Using plasmid height copy, unconformable advantage, and it is injected into weanling pig body
In interior or other animal bodies, to prevent weaning stress of piglet and other Heat stress.
It is described that technical scheme is as follows
A kind of eukaryon expression plasmid pcDNA3.1-DNMT2 of the recombination of high expression DNMT2 genes, the recombinant plasmid contain
Such as SEQ ID NO：Coded sequence shown in 2, the plasmid can be replicated and be survived in host.
A kind of eukaryon expression plasmid pcDNA3.1-DNMT2 of recombinant expression DNMT2 genes of the present invention can prevent preparing
It is applied in weaning stress of piglet pharmaceutical composition.
A kind of eukaryon expression plasmid pcDNA3.1-DNMT2 of recombinant expression DNMT2 genes of the present invention can prevent preparing
It is applied in Heat stress pharmaceutical composition.
A kind of eukaryon expression plasmid pcDNA3.1-DNMT2 of recombinant expression DNMT2 genes of the present invention can prepare feed
It is applied in additive.
A kind of preparation method of the recombinant plasmid pcDNA3.1-DNMT2 of high expression DNMT2 genes is applicant provided, it should
The specific steps of method include：
(1) it is cDNA from pig spleen extraction total serum IgE and reverse transcription, is FJ384441's by the template amplification number of logging in of cDNA
The coded sequence of DNMT2, the code sequence such as SEQ ID NO compiled：Shown in 2；
(2) using agarose gel electrophoresis purified pcr product, purpose piece is recycled using Ago-Gel QIAquick Gel Extraction Kit
Section；
(3) with the appropriate target fragment of Nhe1 and XhoI digestions and pcDNA3.1 carriers, digestion products are recycled；
(4) target fragment and carrier DNA are connected with T4 ligases；
(5) connection product is converted and to TG1 competent cells, is coated on the agarose containing 100 μ g/mL ampicillins
Tablet, 37 DEG C are incubated overnight；
(6) picking single bacterium colony expands culture, plasmid is extracted, by the plasmid double digestion of gained, screening and cloning；
(7) sequence verification is carried out to Plasmid DNA, chooses the clone of correct sequence, obtain Eukaryotic expression recombinant plasmid
PcDNA3.1-DNMT2 (its sequence such as SEQ ID NO:Shown in 2).
The present invention is using pcDNA3.1 as carrier (carrier is purchased from Invitrogen companies, and plasmid map is shown in Fig. 2) structure DNMT2
The eukaryotic expression recombinant plasmid pcDNA3.1-DNMT2 of gene, and the method for using for reference gene therapy, by the recombinant plasmid obtained by this
It is injected into weanling pig body, allows its expression with natural structure and active DNMT2 albumen, it should to reach prevention weaned piglet
Swash the purpose with other Heat stress
Eukaryotic expression recombinant plasmid of the present invention containing DNMT2 has following feature：
1) eukaryotic expression recombinant plasmid of the DNMT2 genes of the high expression of the present invention is included just like sequence table SEQ ID NO：1
In nucleotide sequence shown in 1-1227 bit bases, there is amicillin resistance；
2) high expression DNMT2 recombinant plasmids of the present invention can replicate simultaneously high efficient expression DNMT2 in eukaryocyte
Activated protein, protein sequence such as SEQ ID NO：Shown in 2；
3) eukaryon expression plasmid of DNMT2 genetic recombination of the present invention has the advantages that high copy and unconformable.
The eukaryotic expression recombination plasmid pcDNA3.1-DNMT2 of the present invention, (is purchased from pcDNA3.1 carriers
Invitrogen companies) it is starting plasmids, eukaryotic expression recombination plasmid is prepared by following step：
(1) it is cDNA from healthy Large White spleen extraction total serum IgE and reverse transcription, using cDNA as template amplification such as SEQ ID
NO：Coded sequence (the gene number of the logging in FJ384441 of DNMT2 described in 2；
(2) using agarose gel electrophoresis purified pcr product, and using Ago-Gel QIAquick Gel Extraction Kit recycling purpose piece
Section；
(3) with the appropriate target fragment of Nhe1 and XhoI digestions and pcDNA3.1 carriers, digestion products are recycled；
(4) target fragment and carrier DNA after digestion are connected with T4 ligases；
(5) connection product is converted and to TG1 competent cells, is coated on the agarose containing 100 μ g/mL of ampicillin
Tablet, 37 DEG C are incubated overnight；
(6) picking single bacterium colony expands culture, extracts plasmid, and gained plasmid is carried out double digestion screening and cloning；
(7) to gained Plasmid DNA sequence verification, the clone of correct sequence is chosen, finally obtains Eukaryotic expression recombinant plasmid
pcDNA3.1-DNMT2。
In order to verify that pcDNA3.1-DNMT2 recombinant plasmids can be such that DNMT2 genes overexpress in animal body, 6 bodies are chosen
The consistent male mouse of kunming of weight age in days is randomly divided into two groups, and every group 3, (PBS is purchased one group of intraperitoneal injection phosphate buffer
From Fisher companies), pcDNA3.1-DNMT2 recombinant plasmids prepared by another group of intraperitoneal injection present invention.After feeding 3 days, take small
Mouse nephridial tissue, and therefrom extracted total RNA and albumen compare two groups of mouse DNMT2 expression using RT-PCR and Western blot
It is horizontal.The result shows that plasmid group mouse DNMT2 expressions of the invention are significantly higher than and PBS group mouse.
In order to verify that pcDNA3.1-DNMT2 recombinant plasmids can effectively antagonize weaning stress of piglet, it is big to choose 12 wean
White piglet is randomly divided into 2 groups, and every group 6, one group of jugular vein injects PBS, and another group of jugular vein injects the present invention's
PcDNA3.1-DNMT2 recombinant plasmids.Wean in one week, daily observation grice diarrhoea situation in real time simultaneously weighs pig weight.As a result
Show compared with PBS group piglets, recombinant plasmid group grice diarrhoea rate of the invention is relatively low, and body weight increase is very fast.
PcDNA3.1-DNMT2 recombinant plasmids provided by the invention have the following advantages：
(1) recombinant plasmid prepared by the present invention can make the high expression in piglet body of DNMT2 genes.
(2) recombinant plasmid prepared by the present invention can alleviate the symptom of diarrhea of weanling pig, and pig weight is promoted to increase；
(3) recombinant plasmid of the invention is prepared easy, is had no toxic side effect, and has the potentiality of large-scale production, young to solve
Pig weaning stress and Heat stress provide new technical solution.
Description of the drawings
Sequence table SEQ ID NO：1 be the present invention clone DNMT2 genes nucleotide sequence, sequence length 1227bp
(1-1227bp),
Its corresponding code area is described in 1-1227bp), 408 amino acid sequences are encoded altogether.
Sequence table SEQ ID NO：2 be the sequence of the protein of DNMT2 gene codes.
Fig. 1：The coding nucleotide sequence schematic diagram for the DNMT2 genes that the present invention clones.
Fig. 2：The structure figure of the initial vector pcDNA3.1 of the present invention (picture is originated from the specification of Invitrogen companies).
Fig. 3：The structure figure of the recombined pronucleus expression plasmid pcDNA3.1-DNMT2 of the present invention.DNMT2 gene coded sequences
It is inserted between NheI the and XhoI restriction enzyme sites of pcDNA3.1 plasmids.
Fig. 4：PcDNA3.1-DNMT2 clone's digestion agarose gel electrophoresis figures of the present invention.Reference sign：In Fig. 4
M be DNA molecular amount standard, 1-5 is the electrophoresis knot after plasmid NheI and the XhoI double digestion that 5 different bacterium colonies obtain respectively
Fruit.The band of corresponding 6000bp is carrier DNA after double digestion, and the band of corresponding 1176bp is the coding DNA of DNMT2.
Fig. 5：The RT-PCR testing results that the RNA of DNMT2 genes is expressed in mouse kidney.Reference sign：In Fig. 5
PBS represents that injection has the control mice kidney samples A of PBS, and DNMT2 represents that injection has the mouse kidney sample of high expression DNMT2 plasmids
Product.
Fig. 6：Expression of the DNMT2 albumen in mouse kidney.Reference sign：A figures in Fig. 6 are in mouse kidney
The Western blot analysis results of DNMT2 gene expressions.Reference sign：The injection of DNMT2 gene representations has high table in Fig. 6
Up to the mouse kidney sample of DNMT2 plasmids, PBS represents that injection has the control mice kidney samples A of PBS；Serial number (1), (2) and (3)
DNMT2 plasmids and three mouse kidney samples of PBS control group are represented respectively.B figures in Fig. 6 are western blot in A figures
Quantitative analysis result.
Fig. 7：Using the influence of recombinant plasmid pcDNA3.1-DNMT2 per day weightenings to mouse.
Fig. 8：Expression of the DNMT2 albumen in Large White blood.Reference sign：A figures in Fig. 8 are weanling pig blood
The Western blot analysis results that DNMT2 is expressed in liquid.In A figures in fig. 8：The control that PBS represents injection and has PBS is weaned
Piglet blood sample；PcDNA3.1 is the control weanling pig blood sample that injection has empty plasmid pcDNA3.1；pcDNA3.1+
DNMT2 represents that injection has the weanling pig blood sample of high expression DNMT2 plasmids；DNMT2 and GAPDH shows both eggs respectively
White band.B figures in Fig. 8 are the gray value analysis of the A corresponding protein bands shown in figure in Fig. 8.
Fig. 9：The measure of the per day weightening of weanling pig.Reference sign：A figures in Fig. 9 are after weaned piglet in 7 days
Average weight gain；B figures in Fig. 9 are piglet 1 after wean, the average weight growth rate of 3,5 and 7 days.PBS is marked to represent note in figure
Penetrate the control group piglet of PBS；DNMT2 represents the experimental group piglet of the high expression DNMT2 recombinant plasmids of injection.
Specific embodiment
The structure of 1 recombinant plasmid pcDNA3.1-DNMT2 of embodiment and verification
(1) from pig spleen extraction total serum IgE and reverse transcription cDNA, using cDNA as the coded sequence of template amplification DNMT2.Amplification
The primer sequence of gene DNMT2 is as follows：
Forward primer F：5 ' CTA CTA GCT AGC ATG GAG CCC CTG CGG GTC 3 ',
Reverse primer R：5′CGA CTG CTC GAG TTT GGA TAT GAT GGA ATA CTG GA 3′；
PCR reaction systems are shown in Table 1
1 PCR reaction systems of table
Reaction condition is 95 DEG C of 5min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 1min, 30 cycles；72℃10min.
(2) target fragment is recycled using Ago-Gel QIAquick Gel Extraction Kit, 1 μ L is taken to measure purpose with Nano Drop 2000
The purity and concentration of segment.
(3) digestion products are recycled first with the appropriate target fragment of Nhe1 digestions and pcDNA3.1 carriers, then with Xho digestions,
Secondly recycling digestion products take 1 μ L to recycle segment, its purity and concentration are measured with Nano Drop 2000.Digestion system is shown in Table
2。
2 digestion with restriction enzyme system of table
(4) according to carrier：Target fragment molar ratio is 1:10, with T4DNA ligases 1h is connected in PCR instrument for 16 DEG C.It carries
Body is shown in Table 3 with target fragment linked system.
3 carrier of table and target fragment linked system
(5) connection product is converted to the agar for TG1 competent cells, being coated on the ampicillin containing 100 μ g/mL
Sugared tablet, 37 DEG C of overnight incubations.
(6) picking single bacterium colony expands culture, and plasmid is extracted with the small extraction reagent kit of plasmid.Double digestion screening and cloning, as a result such as
Shown in Fig. 2, choose double digestion and correctly clone carry out DNA sequencing, sequencing result is as shown in Figure 3.
(7) it chooses the correct plasmid of sequencing and is named as eukaryotic expression recombination plasmid (or recombinant plasmid) pcDNA3.1-
DNMT2。
A large amount of preparations of 2 recombinant plasmid pcDNA3.1-DNMT2 of embodiment
1st, the preparation of competent escherichia coli cell
(1) take -80 DEG C of bacillus coli DH 5 alpha strains frozen, streak inoculation on the LB agar plates without antibiotic,
After 37 DEG C of incubator culture 12-16h, the well-grown single bacterium colony of picking is inoculated in 5mL LB culture solutions, 37 DEG C of culture 5-6h,
Shaking table concussion speed 250-300rpm；
(2) Escherichia coli bacteria liquid after activation is aseptically transferred in the conical flask of dress 50mL LB, continues to shake
Culture about 2.5-3h is swung, until when the bacterium solution OD values at 600nm reach 0.5-0.6, bacterium is aseptically transferred to one
In ice-cold 50mL sterile centrifugation tubes, 30min is placed on ice；
(3) 10min is centrifuged under 4 DEG C of 4000rpm, supernatant is abandoned under aseptic condition, trains residual centrifuge tube inversion 1min
After nutrient solution is flow to end, (formula is seen below the 0.1mol/L TSS buffer solutions of addition 2mL (depending on amount of bacteria) ice precooling in precipitation
Face), thalline is resuspended, ice bath 30min is to get to the competent cell prepared.
2nd, it converts and expands culture
(1) 100 μ L competent cells are taken, after slowly melting on ice, add in 2 μ L recombinant plasmids, gently mixing；Ice bath
30min, 42 DEG C of heat shock 90s, then ice bath 2min；0.9mL LB culture mediums are added in, while separately 100 μ L competent cells are taken to add in
0.9mL LB culture mediums are as blank control group, and in 37 DEG C of shaking table, 220rpm shakes 60min；
(2) two tissue culture nutrient solutions are centrifuged into 5min with 3000rpm, draws supernatant, leave 300 μ L culture solutions and be coated on 100 μ g/
On mL amicillin resistance tablets, 37 DEG C of insulating box overnight incubations (17-20h) are put in after drying；
(3) picking single bacterium colony is added in 4mL LB culture solutions, in shaking table 37 DEG C of cultures 8h, rotating speed 180rpm；
(4) bacterium solution prepared by 3mL steps (3) is added in 1L culture solutions and expands culture 12h.
3rd, a large amount of Prepare restructuring plasmids
The configuration of large-scale/a large amount of plasmid extraction kit of table 4
The explanation of table 4：The kit of a large amount of Prepare restructuring plasmids of the present embodiment application is purchased from Beijing Ai Delai biologies section
The a large amount of plasmid extraction kits (article No. PL1401) of large size of skill Co., Ltd.
Concrete application step is as described below：
(1) it takes converted eukaryotic expression recombination plasmid pcDNA3.1-DNMT2 to be incubated overnight bacterium solution 150mL or so, is packed into
In suitable centrifugal bottle, 10000 × g centrifuges 2min precipitation thalline, complete reject supernatant in 4 DEG C.
(2) 5mL solution P1 (kit carries) is added in, be fully suspended concussion bacterial sediment, is completely dispersed it, until nothing
Block of wadding a quilt with cotton exists.Bacterial suspension is moved into 50mL centrifuge tubes, is placed at room temperature for 3-5min.
(3) 5mL solution P2 (kit carries) is added in, gently overturns centrifuge tube 6-8 times, is placed at room temperature for 4-5min, is made thin
Bacterium cracks completely, and solution is transparent.
(4) add 5mL solution P3 (kit carries), overturn centrifuge tube immediately 6-8 times, abundant mixing, until White Flocculus
It generates.Above-mentioned lysate centrifuges 10-15min in 4 DEG C of 12000-16000 × g, and supernatant is carefully sucked out, and moves into new 50mL centrifugations
Guan Zhong.
(5) 10mL isopropanols are added in, overturn centrifuge tube, abundant mixing.
(6) 10min are centrifuged in 4 DEG C of 12000-16000 × g, carefully discarded supernatant, be inverted on blotting paper gently drain it is residual
Extraction raffinate body adds in 3-5mL70% ethyl alcohol and rinses one time, and most high speed centrifugation 5min abandons supernatant, dries precipitation.
(7) it adds in 1.4mL solution P1 and is completely dissolved pellet, plasmid solution then is transferred to 2 new 1.5mL centrifuges
In pipe (each 700 μ L).
(8) often pipe adds in 55 μ L impurity and removes liquid A (kit carries), and about 10% volume is added in after overturning abundant mixing
The Endotoxin removal agent of (about 80 μ L) ice precooling, overturns rotation 7-10 times (30s or so), and abundant mixing or is put ice bath on ice
5min is put, it is intermediate to overturn mixing several times once in a while.
(9) 42 DEG C of water-baths, solution can become muddy, overturn 42 DEG C of warm bath 5min after mixing again.
(10) 14000 × g of room temperature centrifuges 5min split-phases.Upper strata aqueous phase contains DNA, and lower floor's blue oily is mutually containing endotoxin and its
His impurity.Upper strata aqueous phase containing DNA is transferred to new pipe, abandons oily layer.
(11) isometric impurity will be added in upper strata aqueous phase obtained by previous step and removes liquid B (kit carries), about 750 μ L),
Soft mixing, 4 DEG C of 14000 × g centrifuge 10min, abandon supernatant, gently add in the washing of 70% ethyl alcohol of 1mL, and supernatant is abandoned in centrifugation, and totally 2
Secondary, 5-10min is dried in room temperature inversion makes ethyl alcohol volatilize completely.
(12) each centrifuge tube add appropriate TE or pure water (100-200 μ L) dissolve precipitation (can 37 DEG C of water-baths concussions with
Auxiliary dissolving).
(13) concentration of detection gained plasmid.
The recombinant plasmid of 3 present invention of embodiment makes its DNMT2 overexpressions verification in animal body
1. feed mouse
The identical SPF male mouse of kunming of 6 weight ages in days (being purchased from Disease Prevention Control Center, Hubei Prov) is chosen, at random
It is divided into two groups, every group 3, another group of intraperitoneal injection of one group of intraperitoneal injection phosphate buffer (PBS, purchased from Hyclone companies)
Prepared by the present invention, the 25 μ L of Eukaryotic expression recombinant plasmid pcDNA3.1-DNMT2 of a concentration of 1 μ g/ μ L.By SPF males Kunming
Mouse is fed to being suffocated with carbon dioxide after 3 days and puts to death, and takes kidney.
2. utilize DNMT2 expression quantity in real-time fluorescence quantitative PCR detection kidney
(1) total tissue RNA is extracted
It is operated in superclean bench：The above-mentioned kidney of mouse samples of 100mg are taken, shred the mill for being placed on precooling
In, 1mL PBS are added in, is ground and is homogenized using homogenizer, take 200 μ L treated tissue fluid to add in 1mL in 1.5mL EP pipes
'sReagent (is purchased from precious bioengineering Dalian Co., Ltd), and 5~10min is stored at room temperature after the mixing that turns upside down；
200 μ L chloroforms are added in, vortex 15s (or acutely shaking 15s) stands 10min, 4 DEG C of 13000rpm, centrifuges 15min；It sucts clearly extremely
In new 1.5mL EP pipes, 500 μ L isopropanols are added in, are gently overturned several times up and down, stand 10min, 4 DEG C of 13000rpm, centrifugation
15min；Supernatant is abandoned, adds in 75% ethyl alcohol of 1mL, 4 DEG C of 7500rpm after gently overturning several times up and down centrifuge 5min；Abandon supernatant,
Pipe abandon bottom liquid is inhaled, air-dries 5min, 30 μ L pyrocarbonic acid diethyl esters (DEPC) processed water is added in and fully dissolves, using being divided light
Degree meter measures the content and purity of RNA, and putting -80 DEG C of preservation, (RNA extractions institute is using EP pipes and the consumptive materials such as pipette tips by going RNA enzyme
It is processed).
(2) RNA purity and concentration mensuration
Measure the purity and concentration of RNA with Nano Drop 2000, and using dissolve RNA by the use of DEPC water as blank control
Calibration.
(3) reverse transcription of RNA
The synthesis of cDNA：Take RNA PCR Kit (AMV) the reverse transcription reagent box specification of 1 μ g total serum IgEs by Toyobo companies
Prepare cDNA.Reverse transcription reaction system is shown in Table 5.
5 reverse transcription reaction system of table
The response procedures of RT are：42℃60min；94℃5min；4℃5min.Synthetic cDNA is placed in -80 DEG C of preservations
It is spare.
(4) RT-PCR is detected
Real time fluorescent quantitative reaction system is shown in Table 6.
6 real time fluorescent quantitative reaction system of table
The explanation of table 6：The full name of referred to as " Shanghai life work " is Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Reacting amplification condition is：95℃30s；95 DEG C of 5s, 52 DEG C of 15s, 72 DEG C of 30s, 40 cycles.Fluorescence in reaction process
The variation of signal is detected by 480 real-time fluorescence quantitative PCR instrument of Roche Light Cycler.
3rd, Western Blot are detected
(1) tissue sample is cracked, measures protein concentration, sample size is adjusted according to protein concentration, makes its Tot Prot consistent,
Sample-loading buffer is added in, 95 DEG C of 10min place cooled on ice, 5000rpm centrifugations 5min.It is ready for electrophoresis.
(2) SDS-PAGE electrophoresis
The glass plate for recording polyacrylamide gel is placed, prepares 12% SDS-PAGE separation gels, after rapid mixing
It pours into the gap of glass plate (every piece of plank about 5mL), and adds in 400 μ L isopropanols and flatten the liquid level of separation gel, standing is treated
After it is solidified, ddH is used2O cleans the isopropanol on separation gel upper strata, and 5% SDS-PAGE concentration glue is prepared after the residual liquid that exhausts,
It is poured into after rapid mixing in the gap of glass plate (every piece of plank about 2mL), and is rapidly inserted into comb, standing treats its solidification.Colloid
Comb is extracted after solidification, fixed electrophoretic apparatus adds in electrophoretic buffer, and the broken glue in gel glue hole is sucked out using liquid-transfering gun,
By the sample handled well and albumen Marker per 20 μ L of hole loading.Electrophoretic apparatus is connected, setting voltage is 80V, proceeds by electricity
Swimming.When bromophenol blue is run to separation gel, adjustment voltage to 120V when bromophenol blues are run to separation gel bottom, stops electrophoresis.
(3) electrotransfer：After SDS-PAGE electrophoresis, take out gel and be placed in transferring film buffer solution (seeing below surface compositions).With
The length and width of the good gel of scale dipstick metering, an equal amount of pvdf membrane one of clip are opened, while prepare thick filter paper and each two of thin filter paper, leaching
Bubble 5min in transferring film buffer solution, pvdf membrane are positioned in methanol and are taken out after effect 1min, be placed in transferring film buffer solution.It is cleaning
On the clean and graphite electrode plate that has dried, the lower thick filter paper of paving, thin filter paper, pvdf membrane, gel, thin filter paper, thickness filter paper one by one, and
Edge is aligned, one layer is often spread, is uniformly rolled using glass bar to drive bubble away, finally cover cathode graphite electrode plate, by electrode
Plate is put into transfer groove, is powered on, and adjusting voltage is 100V, and setting time is 1h 20min, carries out constant pressure transfer.
(4) it closes
After electrotransfer, instrument is closed, pvdf membrane is carefully taken out, and place it in distilled water, then switches into and fill
In the capsule of the confining liquid of TBST buffer solutions of the 20mL containing 10% skimmed milk, it is placed in 1~2h of shaking mild on shaking table at room temperature.
(5) it is incubated primary antibody
After closing, pvdf membrane is rinsed with TBST buffer solutions (formula is shown in annex), the TBST of about 15mL is added in, in shaking table
Upper shaking, each 5min are rinsed 3 times.After rinsing, pvdf membrane is transferred in another clean plate, is delayed with TBST
Fliud flushing is with suitable concentration dilution primary antibody (source) be added thereto, it is placed on shaking table and leniently shakes 1h.Box is then placed in 4
It is stored overnight in DEG C.
(6) HRP label secondary antibodies are incubated
After primary antibody is incubated, pvdf membrane is rinsed with TBST buffer solutions, the TBST of about 15mL is added in, is shaken on shaking table,
Each 5min is rinsed 3 times.
After rinsing, pvdf membrane is transferred in another clean plate, it is dilute with suitable concentration with TBST buffer solutions
It releases HRP label secondary antibodies (source) to be added thereto, is placed on shaking table and leniently shakes 1h and be incubated.Subsequent secondary antibody incubation terminates
Afterwards, pvdf membrane is rinsed with TBST buffer solutions, adds in the TBST of about 15mL, shaken on shaking table, each 5min, rinsed 5 times.
(7) it develops the color
It is protected from light and prepares developing solution 1mL, it is now with the current.Start chemiluminescent autography system, developing solution is added drop-wise to exposure
Camera lens center slowly covers upper pvdf membrane, makes its covering uniform.It exposure colour developing certain time, obtains and preserves picture.
As a result such as Fig. 4, shown in 5, compared with PBS control group, the RNA of the mouse DNMT2 of recombinant plasmid and albumen table are injected
Up to horizontal apparent rising, significant difference (p ＜ 0.05) illustrates that injecting Eukaryotic expression recombinant plasmid pcDNA3.1-DNMT2 can make
DNMT2 genes overexpress in object.
Embodiment 4 is overexpressed the influence of the anti-chronic heat stress of DNMT2 gene pairs mouse
(1) the identical SPF male mouse of kunming of 10 weight ages in days is chosen, is randomly divided into two groups, every group 5, one group of abdomen
Chamber injects PBS, the Eukaryotic expression recombinant plasmid pcDNA3.1-DNMT2 of another group of a concentration of 1 μ g/ μ L of 25 μ L of intraperitoneal injection.
(2) by this two groups of Mouse feeders three days, a few days ago raised under the conditions of chronic heat stress, every night 275W Europe
General board infrared heating light bulb, the control of mouse cage ambient temperature were normally raised in 37 DEG C or so, the 3rd day.Every mouse is recorded daily
Weight, compare each group mouse weight daily gain situation.
The results are shown in Figure 7, and under the conditions of chronic heat stress group, the 1st day PBS group and plasmid group weight are all notable after injection
It reduces, but the reduction of PBS groups becomes apparent from than plasmid group；The 2nd day PBS group weight is still remarkably decreased after injection, but plasmid group weight
Start to increase；The 3rd day environment PBS group and plasmid group weight due to being detached from chronic heat stress all increases after injection.This showed
Expression DNMT2 can effectively antagonize chronic heat stress.
Effect of the recombinant plasmid of 5 present invention of embodiment in weaning stress of piglet is prevented
(1) 12 wean of " great Bai " piglet of three 6~7kg of week old weight are selected from Hua Zhong Agriculture University's fine work pig farm, with
Machine is divided into two groups, every group each 6.On the wean same day, one group of piglet Jugular vessel injects 5mL PBS as blank control group, another
The pcDNA3.1-DNMT2 recombinant plasmids of a concentration of 100 μ g/mL of group Jugular vessel injection 5mL are as experimental group.
(2) normal to raise in 1 week, the daily grice diarrhoea situation of observation in real time simultaneously records weight.
The results are shown in Figure 8, and 7 days after wean, recombinant plasmid group piglet average weight increases dramatically increases (p than control group
＜ 0.05), moreover, weight is intended to increase always in recombinant plasmid group piglet one week, and PBS control group then has weight loss
Phenomenon.Recombinant plasmid group diarrhea of weaned piglets rate is 0%, and control group diarrhea of weaned piglets rate is 17%, illustrates system of the present invention
Standby recombinant plasmid can effectively prevent weaning stress of piglet reaction.
Annex：Culture solution or buffer formulation：
1) 1X TSS are prepared:1% tryptone, 0.5% yeast extract, 1% sodium chloride, 10% polyethylene glycol (molecule
Measure 3350-8000), 5% dimethyl sulfoxide (DMSO), 20-50mM magnesium chlorides.It is stirred to dissolve, with dilute hydrochloric acid or sodium hydroxide solution tune
PH is saved between 6.4-6.8,0.22 μm of membrane filtration degerming after constant volume, 4 DEG C are in store for.
2) transferring film buffer solution：2.9g glycine is weighed, 5.8g Tris, 0.37g SDS add 200mL methanol, add
ddH2O to 1000mL.
3) TBST buffers：10mM Tris-HCl, 150mM NaCl add in final concentration of 0.05% Tween-20,
Stir evenly, it is fixed it is molten after with 0.22 μm of membrane filtration degerming.
Sequence table
<110>Hua Zhong Agriculture University
<120>Application of the recombinant plasmid of height expression DNMT2 genes in weaning stress of piglet is prevented
<141> 2016-12-25
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1227
<212> DNA
<213>Pig (Sus scrofa)
<220>
<221> gene
<222> (1)..(1227)
<220>
<221> CDS
<222> (1)..(1227)
<400> 1
atg gag ccc ctg cgg gtc ctg gag ctg tac agc ggc att ggg ggc atg 48
Met Glu Pro Leu Arg Val Leu Glu Leu Tyr Ser Gly Ile Gly Gly Met
1 5 10 15
cac cag gcg ctg aga gaa agc tgt ata cct gcg caa gtg gtg gct gcc 96
His Gln Ala Leu Arg Glu Ser Cys Ile Pro Ala Gln Val Val Ala Ala
20 25 30
att gat gta aac acc gtc gct aac gaa gta tac aag tat aat ttt cct 144
Ile Asp Val Asn Thr Val Ala Asn Glu Val Tyr Lys Tyr Asn Phe Pro
35 40 45
cac aca cag tta ctg gcc aag aca att gaa ggt gtt aca cta gaa gaa 192
His Thr Gln Leu Leu Ala Lys Thr Ile Glu Gly Val Thr Leu Glu Glu
50 55 60
ttt gac aga tta tct ttc aat atg gtt tta atg agc cct ccc tgt cag 240
Phe Asp Arg Leu Ser Phe Asn Met Val Leu Met Ser Pro Pro Cys Gln
65 70 75 80
cca ttt aca aga att ggc ctg caa ggt gat gtg acg gat ccg agg aca 288
Pro Phe Thr Arg Ile Gly Leu Gln Gly Asp Val Thr Asp Pro Arg Thr
85 90 95
aat agc ttc tta tat att cta gac att ctc cca aga ttg caa aaa tta 336
Asn Ser Phe Leu Tyr Ile Leu Asp Ile Leu Pro Arg Leu Gln Lys Leu
100 105 110
cca aag tat att ctt cta gaa aat gtt aaa ggt ttt gaa gta tct tca 384
Pro Lys Tyr Ile Leu Leu Glu Asn Val Lys Gly Phe Glu Val Ser Ser
115 120 125
aca aga gac cta tta ata caa aca ata gaa aat tgt ggc ttt cag tac 432
Thr Arg Asp Leu Leu Ile Gln Thr Ile Glu Asn Cys Gly Phe Gln Tyr
130 135 140
caa gaa ttt ctc ttg tct cca acc tct ctt ggc att cca aat tca agg 480
Gln Glu Phe Leu Leu Ser Pro Thr Ser Leu Gly Ile Pro Asn Ser Arg
145 150 155 160
cta cgg tat ttc ctt att gca aag ctt cag tca gag cca ttc cct ttt 528
Leu Arg Tyr Phe Leu Ile Ala Lys Leu Gln Ser Glu Pro Phe Pro Phe
165 170 175
caa gcc cct ggt cag gtg cta atg gag ttc ccc caa atg gaa tcc gaa 576
Gln Ala Pro Gly Gln Val Leu Met Glu Phe Pro Gln Met Glu Ser Glu
180 185 190
cat cca caa aaa cac gca ata gat gca caa agt aaa atc gaa gaa aag 624
His Pro Gln Lys His Ala Ile Asp Ala Gln Ser Lys Ile Glu Glu Lys
195 200 205
aaa att gaa cga aat att tgc ttg gat agc agt gca caa tgt tct gga 672
Lys Ile Glu Arg Asn Ile Cys Leu Asp Ser Ser Ala Gln Cys Ser Gly
210 215 220
aaa gag gcc att ctt ttc aag ctt gaa act gca gga gaa att gac agg 720
Lys Glu Ala Ile Leu Phe Lys Leu Glu Thr Ala Gly Glu Ile Asp Arg
225 230 235 240
aaa cat caa cag gac agt gat ctc tct gtg caa atg cta aaa ggt ttt 768
Lys His Gln Gln Asp Ser Asp Leu Ser Val Gln Met Leu Lys Gly Phe
245 250 255
ctt gaa gat gac att gac atg aac tcg tac ttt tta ccg cca aag tcg 816
Leu Glu Asp Asp Ile Asp Met Asn Ser Tyr Phe Leu Pro Pro Lys Ser
260 265 270
ttg ctc cga tat gct ctt ttg tta gac att gtt aag ccc act tcc aga 864
Leu Leu Arg Tyr Ala Leu Leu Leu Asp Ile Val Lys Pro Thr Ser Arg
275 280 285
aga tcc atg tgc ttt acg aaa ggt tat gga cgc tac ata gaa ggg aca 912
Arg Ser Met Cys Phe Thr Lys Gly Tyr Gly Arg Tyr Ile Glu Gly Thr
290 295 300
gga tct gtg tta cag acc tca gaa gat gtg cag att gag aat atc tac 960
Gly Ser Val Leu Gln Thr Ser Glu Asp Val Gln Ile Glu Asn Ile Tyr
305 310 315 320
aaa tcc ctt acc agt ttg tca cca gaa gaa aag ata atg aaa ttg tta 1008
Lys Ser Leu Thr Ser Leu Ser Pro Glu Glu Lys Ile Met Lys Leu Leu
325 330 335
atg ctt aaa ctt cga ttt ttc act cct aaa gaa ata gca aat ctc ctt 1056
Met Leu Lys Leu Arg Phe Phe Thr Pro Lys Glu Ile Ala Asn Leu Leu
340 345 350
gga ttt cct cca gag ttc gga ttt cct gag aag ata aca gtc aaa cag 1104
Gly Phe Pro Pro Glu Phe Gly Phe Pro Glu Lys Ile Thr Val Lys Gln
355 360 365
cgt tat cgt cta ctt gga aat agc ctc aac gtg cat gtt gta gct aaa 1152
Arg Tyr Arg Leu Leu Gly Asn Ser Leu Asn Val His Val Val Ala Lys
370 375 380
cta atc aaa atc ctg tat gcg tcg gtt ttg agt agc tct gcg agg tgg 1200
Leu Ile Lys Ile Leu Tyr Ala Ser Val Leu Ser Ser Ser Ala Arg Trp
385 390 395 400
ttc cag tat tcc atc ata tcc aaa tag 1227
Phe Gln Tyr Ser Ile Ile Ser Lys
405
<210> 2
<211> 408
<212> PRT
<213>Pig (Sus scrofa)
<400> 2
Met Glu Pro Leu Arg Val Leu Glu Leu Tyr Ser Gly Ile Gly Gly Met
1 5 10 15
His Gln Ala Leu Arg Glu Ser Cys Ile Pro Ala Gln Val Val Ala Ala
20 25 30
Ile Asp Val Asn Thr Val Ala Asn Glu Val Tyr Lys Tyr Asn Phe Pro
35 40 45
His Thr Gln Leu Leu Ala Lys Thr Ile Glu Gly Val Thr Leu Glu Glu
50 55 60
Phe Asp Arg Leu Ser Phe Asn Met Val Leu Met Ser Pro Pro Cys Gln
65 70 75 80
Pro Phe Thr Arg Ile Gly Leu Gln Gly Asp Val Thr Asp Pro Arg Thr
85 90 95
Asn Ser Phe Leu Tyr Ile Leu Asp Ile Leu Pro Arg Leu Gln Lys Leu
100 105 110
Pro Lys Tyr Ile Leu Leu Glu Asn Val Lys Gly Phe Glu Val Ser Ser
115 120 125
Thr Arg Asp Leu Leu Ile Gln Thr Ile Glu Asn Cys Gly Phe Gln Tyr
130 135 140
Gln Glu Phe Leu Leu Ser Pro Thr Ser Leu Gly Ile Pro Asn Ser Arg
145 150 155 160
Leu Arg Tyr Phe Leu Ile Ala Lys Leu Gln Ser Glu Pro Phe Pro Phe
165 170 175
Gln Ala Pro Gly Gln Val Leu Met Glu Phe Pro Gln Met Glu Ser Glu
180 185 190
His Pro Gln Lys His Ala Ile Asp Ala Gln Ser Lys Ile Glu Glu Lys
195 200 205
Lys Ile Glu Arg Asn Ile Cys Leu Asp Ser Ser Ala Gln Cys Ser Gly
210 215 220
Lys Glu Ala Ile Leu Phe Lys Leu Glu Thr Ala Gly Glu Ile Asp Arg
225 230 235 240
Lys His Gln Gln Asp Ser Asp Leu Ser Val Gln Met Leu Lys Gly Phe
245 250 255
Leu Glu Asp Asp Ile Asp Met Asn Ser Tyr Phe Leu Pro Pro Lys Ser
260 265 270
Leu Leu Arg Tyr Ala Leu Leu Leu Asp Ile Val Lys Pro Thr Ser Arg
275 280 285
Arg Ser Met Cys Phe Thr Lys Gly Tyr Gly Arg Tyr Ile Glu Gly Thr
290 295 300
Gly Ser Val Leu Gln Thr Ser Glu Asp Val Gln Ile Glu Asn Ile Tyr
305 310 315 320
Lys Ser Leu Thr Ser Leu Ser Pro Glu Glu Lys Ile Met Lys Leu Leu
325 330 335
Met Leu Lys Leu Arg Phe Phe Thr Pro Lys Glu Ile Ala Asn Leu Leu
340 345 350
Gly Phe Pro Pro Glu Phe Gly Phe Pro Glu Lys Ile Thr Val Lys Gln
355 360 365
Arg Tyr Arg Leu Leu Gly Asn Ser Leu Asn Val His Val Val Ala Lys
370 375 380
Leu Ile Lys Ile Leu Tyr Ala Ser Val Leu Ser Ser Ser Ala Arg Trp
385 390 395 400
Phe Gln Tyr Ser Ile Ile Ser Lys
405
Claims (6)
1. it is a kind of it is high expression DNMT2 genes recombinant plasmid pcDNA3.1-DNMT2, which is characterized in that the recombinant plasmid contain as
SEQ ID NO：Coded sequence described in 2.
A kind of 2. recombinant plasmid pcDNA3.1-DNMT2 of high expression DNMT2 genes, which is characterized in that the recombinant plasmid energy
It is enough to replicate and survive in host.
3. a kind of recombinant plasmid pcDNA3.1-DNMT2 of high expression DNMT2 genes described in claim 1 is preparing prevention son
Application in pig weaning stress pharmaceutical composition.
4. a kind of recombinant plasmid pcDNA3.1-DNMT2 of high expression DNMT2 genes described in claim 1 is dynamic in preparation prevention
Application in object heat stress pharmaceutical composition.
5. a kind of recombinant plasmid pcDNA3.1-DNMT2 of high expression DNMT2 genes described in claim 1 adds preparing feed
Add the application in agent.
6. a kind of preparation method for the eukaryon expression plasmid pcDNA3.1-DNMT2 for recombinantly expressing DNMT2 genes, which is characterized in that
Include the following steps：
(1) it is cDNA from pig spleen extraction total serum IgE and reverse transcription, is FJ384441's by the template amplification number of logging in of cDNA
DNMT2 coded sequences, the coded sequence such as SEQ ID NO：Shown in 2；
(2) using agarose gel electrophoresis purified pcr product, target fragment is recycled with Ago-Gel QIAquick Gel Extraction Kit；
(3) with the appropriate target fragment of Nhe1 and XhoI digestions and pcDNA3.1 carriers, digestion products are recycled；
(4) target fragment and carrier DNA are connected with T4 ligases；
(5) connection product is converted and to TG1 competent cells, is coated on the agarose containing 100 μ g/mL ampicillins and puts down
Plate, 37 DEG C are incubated overnight；
(6) picking single bacterium colony expands culture, extracts plasmid, the plasmid of gained is carried out double digestion, screening is cloned；
(7) sequence verification is carried out to Plasmid DNA, chooses the clone of correct sequence, obtain high recombinant expression pcDNA3.1-
DNMT2, the plasmid contain SEQ ID NO:Coded sequence shown in 2.
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| US20070287676A1 (en) * | 2006-05-16 | 2007-12-13 | Sun-Wei Guo | Diagnosis and treatment of endometriosis |
| CN105596352A (en) * | 2016-01-22 | 2016-05-25 | 徐毅 | Novel function of karounitriol on reducing expression level of DNA transmethylase |
| CN108103094A (en) * | 2016-11-25 | 2018-06-01 | 复旦大学 | Expression vector for the expression of targeted inhibition HIV-1 provirus and its preparation method and application |
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2017
- 2017-12-04 CN CN201711262843.9A patent/CN108239658B/en not_active Expired - Fee Related
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|---|---|---|---|---|
| US20070287676A1 (en) * | 2006-05-16 | 2007-12-13 | Sun-Wei Guo | Diagnosis and treatment of endometriosis |
| CN105596352A (en) * | 2016-01-22 | 2016-05-25 | 徐毅 | Novel function of karounitriol on reducing expression level of DNA transmethylase |
| CN108103094A (en) * | 2016-11-25 | 2018-06-01 | 复旦大学 | Expression vector for the expression of targeted inhibition HIV-1 provirus and its preparation method and application |
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| DEVI THIAGARAJAN等: ""The DNA methyltranferase Dnmt2 participates in RNA processing during cellular stress"", 《EPIGENETICS》 * |
| 刘玲娟: ""DNA 甲基转移酶 1 在同型半胱氨酸致人脐静脉平滑肌细胞增殖中的作用研究"", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》 * |
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