CN102127545A - Skeletal muscle specificity CKM (Creatine Kinase Muscle) promoter and applications thereof - Google Patents

Abstract

The invention belongs to the biotechnology field and relates to a skeletal muscle specificity CKM (Creatine Kinase Muscle) promoter and the applications thereof, in particular to a skeletal muscle specificity expression gene 5' control region specificity promoter and the applications thereof to transgenic pigs. The promoter has promotion specificity only in skeletal muscle and has no promotion activity in various cells except for skeletal muscle so that the gene promoter only specifically promotes the expression of a downstream gene in the skeletal muscle. Accordingly, the invention can meet the need for the specific expression of a target gene in the skeletal muscle and provides an important element for improving pork quality through a transgene technology.

Description

A kind of skeletal muscle specific C KM promotor and application thereof

Technical field

The present invention relates to the molecular genetics field, be specifically related to a kind of skeletal muscle different expression gene 5 ' control region specificity promoter and the application in transgenic pig thereof.

Background technology

Time and level that eukaryotic gene is expressed are carried out in proper order in strict accordance with growth.The adjusting that some specific gene is expressed is incorporated into the control region related elements by the transcription factor specificity, becomes the committed step of transcription initiation, and finally causes the time and the space expression of gene.The gene that coding is organized differential protein is in strict accordance with tissue specificity and the expression of etap property, for gene expression regulation mechanism provides the excellent research pattern.In the transgenic animal of specific high-efficiency expression, the specificity promoter that obtains the energy widespread use is extremely important.But the specificity of promotor is controlled by a plurality of elements, and the controlling element that plays a crucial role at different sorts and different development stage is also inequality, and the interaction between controlling element and the trans-acting factor is also very complicated.Foreign gene can be expressed on differential high efficient ground in animal tissues, must structure one can specific high-efficiency expression the animal expression carrier, wherein the selection of promotor is one of important condition.

Creatine kinase (Creatine Kinase; CK) be present in usually in the cytoplasm and plastosome of tissue such as heart, muscle and brain of animal; be the important kinases that and intracellular energy running, Muscle contraction, ATP regeneration have direct relation, it is the commentaries on classics phosphoryl reaction between catalysis creatine and the ATP reversibly.Creatine kinase has four kinds of isozyme forms: muscularity, brain type, hydridization type and plastosome type.Wherein (Creatine Kinase Muscle CKM) mainly is present in the Skeletal Muscle Cell muscularity, and pig CKM gene comprises eight exons and seven introns, 381 amino acid of encoding.This gene 5 ' control region comprises a plurality of MyoD binding sites, and transcription initiation site upstream-60bp comprises a CAAT Box, and-33bp comprises a TATA Box.By cloning this gene promoter area and in its starting characteristic of analyzed in vitro, utilizing correlated series to make up the skeletal muscle specific expression vector is a kind of effective means.But above-mentioned research all also is in the starting stage, does not all play promoter action preferably for the cultivation and the raising of raising meat quality of transgenic pig.

Summary of the invention

Based on above-mentioned reason, the present inventor is through discovering that pig creatine kinase gene 5 ' control region has skeletal muscle specificity starting characteristic, and a kind of skeletal muscle specific expressing promoter carrier that utilized this characteristics design, this promotor can be used in the cultivation of transgenic pig.This promoter vector has skeletal muscle specificity starting characteristic, and it is active all not have startup in the various kinds of cell except that skeletal muscle, as seen this promotor starts the expression of downstream gene specifically in skeletal muscle, can satisfy goal gene specific expressed requirement in skeletal muscle, for transgene carrier provides a kind of important element.

Skeletal muscle different expression gene 5 ' control region specificity promoter provided by the present invention, its gene order is shown in Seq IDNo:1;

Except above-mentioned promotor, can also be the variant or the fragment of this promotor, all have the gene order shown in Seq ID No:1.

" variant " is meant pig CKM promotor Seq ID No:1 sequence carried out any replacement, variation, modification, replacement, the disappearance of one or more base or the sequence that interpolation produced, and this sequence still shows the activity that is similar to Seq ID No:1 sequence.

" fragment " is meant one or more zone of basic dna sequence dna, and it still is similar to the activity of pig CKM promotor Seq IDNo:1.

Promotor or its variant with above-mentioned sequence, perhaps fragment, has skeletal muscle specificity starting characteristic, and it is active all not have startup in the various kinds of cell outside skeletal muscle, as seen this gene promoter starts the expression of downstream gene specifically in skeletal muscle, can satisfy goal gene specific expressed requirement in skeletal muscle, and can be applied in the cultivation of transgenic pig.

When using above-mentioned nucleic acid, the general recombinant nucleic acid sequence that adopts wherein contains above-mentioned promotor or its variant, perhaps fragment, so just can make in the goal gene in promotor downstream specific expressed in skeletal muscle, as analyzing, exploitation, transforming skeletal muscle The Characteristic Study model.Can also utilize existing molecular biology operative technique to obtain to comprise the recombinant nucleic acid of goal gene, obtain transgenic embryos, be used to set up transgenic animal by gene leading-in techniques such as microinjections.Goal gene in the recombinant nucleic acid sequence, contain following at least a functional gene, mainly comprise and improve the meat quality gene, accelerate speed of growth gene, disease-resistant gene, improve the gene of the price of deed and the gene of production certain drug, so just can obtain outside the skeletal muscle specificity starting characteristic, improve the performance of this recombinant nucleic acid sequence, for the cultivation of transgenic pig provides the more function direction, increase its additional output value,, improve the meat nutritive value as desaturation fatty acid enzyme gene is expressed the content that can specificity improves unsaturated fatty acids in the skeletal muscle under this promotor instructs.

After having obtained above-mentioned recombinant nucleic acid sequence, can also set up special expression system with it, so that better application is in the cultivating process of transgenic pig.Goal gene is recombinated to this promotor downstream, changing technology over to by gene makes control region and goal gene be integrated in the karyomit(e) jointly, obtain transgenic animal, goal gene is only expressed in skeletal muscle under this promotor effect, in other histoorgans, do not express, reduce the influence of goal gene, improve genetically modified effect animal.

The present invention mainly determines the startup feature of this promotor by the reporter gene analysis.At first make up no promotor green fluorescent protein carrier,, insert internal ribosome entry site (IRES) in the green fluorescence protein gene upstream for guaranteeing the reporter gene correct translation.Obtain specific expressed creatine kinase 5 ' control region in the skeletal muscle by PCR or fragment technology such as catch then, length is 1.4kb, insert upstream, internal ribosome site, obtain the promotor reporter gene and analyze carrier, CMV promotor (589bp) is connected in the empty carrier as positive control the negative contrast of empty carrier.After plasmid removes intracellular toxin, transfection C2C12 mouse muscle-forming cell system, former generation myocardial cell of transfection pig and pig renal epithelial cell are that PK15 is contrast simultaneously, change behind the transfection 24h and contain the inducing culture of horse serum to sarcoplast, the inducing cell differentiation is merged, observe the egfp expression situation with inverted fluorescence microscope behind the 48h, analyze the promotor characteristic.

When above-mentioned promotor, after perhaps recombinant nucleic acid sequence or expression system are applied in the cultivation of transgenic pig, can effectively improve the effect of cultivation, and by adding various functioning genes, make transgenic pig can obtain better meat quality, the higher speed of growth, better disease resistance and reduction feeding cost, can also in the production of certain drug, obtain using more widely, good reference value be arranged for the research in this field.

Description of drawings

Fig. 1 is CKMF and CKMR primer PCR amplification CKM 5 ' control region fragment electrophorogram, and swimming lane 1 is the PCR product, and M is DL2000 Maker;

Fig. 2 is no promotor CMV Enhancer IRES AcGFP carrier collection of illustrative plates;

Fig. 3 is a transfection CMV Enhancer CKM Promoter pIRES-AcGFP 48h mouse C2C12 cell photo,

The left side is a photo under 80 times of light microscopics, and cell state is good, and the right side is a photo under the blue excitation module, and visible GFP expresses, and CKM Promoter has the activity of startup;

Fig. 4 is a transfection CMV Enhancer CKM Promoter pIRES-AcGFP 48h Pigs Hearts cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is a photo under the blue excitation module, and no GFP expresses, and CKM Promoter does not have the activity of startup;

Fig. 5 is a transfection CMV Promoter pIRES-AcGFP 48h Pigs Hearts cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is a photo under the blue excitation module, and visible GFP expresses, and rotaring dyeing technology is effective;

Fig. 6 is a transfection CMV Enhancer CKM Promoter pIRES-AcGFP 48h porcine kidney cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is a photo under the blue excitation module, and no GFP expresses, and CKM Promoter does not have the activity of startup;

Fig. 7 is a transfection CMV Promoter pIRES-AcGFP 48h porcine kidney cell photo,

The left side is a photo under 40 times of light microscopics, and cell state is good, and the right side is a photo under the blue excitation module, and visible GFP expresses, and rotaring dyeing technology is effective;

Embodiment

In the context of the present specification, unless specialize otherwise the used any term of this specification sheets has those skilled in the art's implication of common sense in the art, and the experimental technique of unreceipted detailed conditions is to carry out according to the routine test method or according to the process specifications that supplier advised.

Embodiment 1 no promotor IRES-AcGFP vector construction

ATG is to the influence of reporter gene translation in the analytical sequence, guarantee the correct expression of downstream reporter gene GFP, the no promotor IRES-AcGFP carrier of special structure, this carrier comprises an internal ribosome and enters element, this method helps keeping the controlling element in transcription initiation site downstream, can obtain bigger control region analystal section.With pIRES-AcGFP is skeleton, cuts with AseI, Nhe I enzyme, reclaims the 4723bp fragment, obtains not having the fragment of CMV promotor; Pcr amplification CMV Enhancer element, downstream primer adds Xba I restriction enzyme site and protectiveness base, and overstriking is represented.CMV Enhancer Forward gene order is shown in Seq ID No:2, and CMV Enhancer Reverse gene order is shown in Seq ID No:3.

Prepare the PCR reaction solution according to following system:

2×PrimeSTAR ^TM?GC?Buffer 10μl

dNTP?Mixture(2.5mM?each) 2μl

CMV?Enhancer?Forward 0.3μM(final?conc.)

CMV?Enhancer?Reverse 0.3μM(final?conc.)

PIRES-AcGFP plasmid DNA 1ng

PrimeSTAR ^TM?HS?DNA?Polymerase(2.5U/μl) 0.2μl

H ₂O complements to 20 μ l

The PCR program setting is as follows:

Product after the amplification is mixed sample to the 1%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim the purpose fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets, cut purified product with Ase I (Fermentas) and Xba I (Fermentas) enzyme, agarose was cut glue and is reclaimed the CMV Enhancer that obtains sticky end after enzyme was cut, CMV Enhancer element is connected with Rapid DNA Ligation (Fermentas) with carrier after enzyme is cut, the clone transforms, and obtains to contain the promoterless pIRES-AcGFP of CMV Ehancer.Complete CMV promotor forward is connected in this carrier, obtains the positive control carrier of this carrier.The bacterium liquid that will comprise negative control and positive control carrier is seeded to respectively according to 1: 500 ratio and contains in the kantlex 50 μ g/ml LB substratum, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (QIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.

The structure of embodiment 2 promotor reporter gene carriers

Genome extracts: get the LargeYorkshire ear tissue, carry out extracting genome DNA according to TIANamp genomic DNA (day root) test kit specification sheets.

Follow according to NCBI (Gene ID:DQ153192.1) and design following primer, the CKMF gene order is shown in Seq ID No:4, and the CKMR gene order is shown in Seq ID No:5.

According to following system preparation PCR reaction solution

2×PrimeSTAR ^TM?GC?Buffer 10μl

dNTP?Mixture(2.5mM?each) 2μl

CKMF 0.3μM(final?conc.)

CKMF 0.3μM(final?conc.)

Pig genomic dna 100ng

PrimeSTAR ^TM?HS?DNA?Polymerase(2.5U/μl) 0.2μl

H ₂O complements to 20 μ l

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.5%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim big fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets, purified product is connected in the pJET1.2 cloning vector (Fermentas), the clone transforms DH5 α, the order-checking of picking list bacterium colony, errorless back acquisition CKM promotor-pJET1.2 plasmid checks order, cut this plasmid with Bgl II (Fermentas) enzyme, agarose is cut glue and is reclaimed CKM 5 ' control region shown in Seq ID No:1.

Promoterless pIRES-AcGFP carrier is cut with Bgl II (Fermentas) enzyme, adds FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, CKM 5 ' control region mover is connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse of bacterium checking insertion behind the coated plate, the bacterium liquid that will comprise correct direction of insertion is seeded to according to 1: 500 ratio and contains in the kantlex 50 μ g/mlLB liquid nutrient mediums, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (QIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid CMV Enhancer CKM Promoter pIRES-AcGFP carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of CKM promotor.

Embodiment 3 cell cultures and transfection

The two age in days two-way cross piggys that will be born are put to death, aseptic condition takes out heart, kidney down to the physiological saline of room temperature, heart is transferred among the 1 * PBS (Hyclone) of 37 ℃ of preheatings after the rinsing in physiological saline gently, blood stains are removed in flushing, tear pericardium off, cut off the atrium, ventricle is shredded into 1mm ³Fritter, add the resuspended tissue block of 1 * PBS (Hyclone), leave standstill 3min, remove supernatant, add 4 ℃ of 0.05% pancreatin (Hyclone), place 4 ℃ of refrigerators to hatch, turn upside down behind the 3h 1 time, remove pancreatin behind the 6h, place 37 ℃ to hatch 20min tissue block, add 37 ℃ of 10ml perfect medium DMEM/F12 and (contain 20%FBS, penicillin 100U/ml, Streptomycin sulphate 100 μ g/ml), blow and beat tissue block gently with suction pipe, disappear, leave standstill 30sec until tissue block, get the upper strata cell suspension and measure cell viability and counting, cell is pressed 1 * 10 ⁴Individual/ml is seeded in the perfect medium, places 37 ℃, 5%CO ₂Cultivate.

With the kidney of aseptic taking-up, in physiological saline, be transferred among 1 * PBS of 37 ℃ of preheatings after the rinsing gently, blood stains are removed in flushing, and fat tears surperficial manadesma off, cuts off kidney, cuts off renal plevis and only stays the about 1g of renal cortex, and it is shredded into 1mm ³Fritter, add the resuspended tissue block of 40ml 1 * PBS (Hyclone), leave standstill 3min, remove supernatant, add 37 ℃ of 0.1% collagenase of 30ml, turn upside down 10 times, place 37 ℃ to hatch 60min, add 2ml foetal calf serum (Gibco), blow and beat tissue block gently with suction pipe, disappear, leave standstill 1min until tissue block, get the upper strata cell suspension and measure cell viability and counting, cell is pressed 1 * 10 ⁴Individual/ml is seeded in the perfect medium, places 37 ℃, 5%CO ₂Cultivate.

Mouse muscle-forming cell is that C2C12 cultivates in the DMEM that contains 10% foetal calf serum (Gibco) (Hyclone) substratum, and culture condition is 37 ℃, 5%CO ₂, had digestive transfer culture keeps cell degree of converging 50%, and inducing culture is for containing the DMEM substratum of 2% horse serum (Hyclone).

To treat cells transfected with 1 * PBS (Hyclone) rinsing attached cell twice, and add 0.05% pancreatin (Hyclone) of 37 ℃ of preheatings, digestion 7min adds the perfect medium re-suspended cell of antibiotic-free, and every hole is by 1 * 10 after the cell counting ⁴Be seeded to 24 orifice plates, with 37 ℃ of serum-free antibiotic-free DMEM (Hyclone) substratum washed cell once, every hole adds the perfect medium of the fresh antibiotic-free of 500 μ l, carries out transfection behind the 12h behind the 24h, and this moment, cell degree of converging was 90%～95%.

With positive control support C MV Promoter pIRES-AcGFP, the negative control carrier does not have promotor CMV Enhancer pIRES-AcGFP, experiment support C MV Enhancer CKM three kinds of plasmids of Promoter pIRES-AcGFP transfection simultaneously mouse muscle-forming cell is C2C12, the pig myocardium cell, porcine kidney cell, every hole is diluted to the 750ng plasmid among the 100 μ l opti-MEM, adds 0.75 μ l Lipofectamine ^TMPLUS (Invitrogen) is mixing thoroughly, adds 4 μ l Lipofectamine behind the 5min ^TMLTX (Invitrogen) transfection reagent is put upside down mixing, hatches for 25 ℃, adds in each hole mixing behind the 30min gently.Change inducing culture behind the transfection 12h to C2C12 clone, the inducing cell differentiation is merged.Observe the GFP expression down with the fluorescence inverted microscope behind the transfection 48h.Positive control carrier reflection transfection conditions is (Fig. 5, Fig. 7) effectively, negative control redgreen fluorescent protein expression.The C2C12 clone that the experimental group of transfection CKM Promoter pIRES-AcGFP carrier was only induced has egfp expression (Fig. 3), equal redgreen fluorescent protein expression (Fig. 4, Fig. 6) in cardiac muscle and nephrocyte.

Transfection results only show in sophisticated myocyte pig CKM promotor just have start active, in another kind of voluntary muscle myocardial cell, do not start active, in renal epithelial cell, there is not the activity of startup yet, show that pig CKM promotor has the skeletal muscle specificity and starts active, can the driving purposes gene specific expressed at skeletal muscle.

Embodiment 4CKM promoter variants function

According to the explanation of manufacturers, utilize Stratagene QuikChange ^TMRite-directed mutagenesis test kit (Stratagene), the design mutant primer makes up this promoter variants, comprises the disappearance of nucleic acid, replaces and insertion.The promotor of change is by sequencing checking sudden change.Promotor after the sudden change is recombinated to the goal gene upstream, obtain recombinant nucleic acid, this recombinant nucleic acid imports among zooblast or the embryo by transgenic technology, can realize goal gene specific expressed in skeletal muscle.

The application of embodiment 5 series connection CKM promotors

Follow according to NCBI (Gene ID:DQ153192.1) and design following primer, the upstream and downstream primer all comprises Xho I restriction enzyme site and protection base.The CKM3F gene order is shown in Seq ID No:6, and the CKM3R gene order is shown in Seq ID No:7.

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.5%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim the 1.4kb fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets.Cut the PCR purified product and reclaim enzyme and cut product with XhoI (Fermentas) enzyme.

CKM Promoter pIRES-AcGFP carrier is cut with Xho I (Fermentas) enzyme, added FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, Xho I enzyme is cut the CKM control region that purifying crosses to be connected with dephosphorylized carrier with Rapid DNAL igation (Fermentas), and conversion DH5 α, choose the forward and reverse of bacterium checking insertion behind the coated plate, the bacterium liquid that will comprise correct direction of insertion is seeded to according to 1: 500 ratio and contains in the kantlex 50 μ g/ml LB liquid nutrient mediums, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (QIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid CMV Enhancer double CKM Promoter pIRES-AcGFP carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of two CKM promotors.

Embodiment 6CKM promoter fragment is used

Follow according to NCBI (Gene ID:DQ153192.1) and design following primer, the CKM2F gene order is shown in Seq ID No:8, and the CKM2R gene order is shown in Seq ID No:9.

According to following system preparation PCR reaction solution

2×PrimeSTAR ^TM?GC?Buffer 10μl

dNTP?Mixture(2.5mM?each) 2μl

CKMF 0.3μM(final?conc.)

CKMF 0.3μM(final?conc.)

Pig genomic dna 100ng

PrimeSTAR ^TM?HS?DNA?Polymerase(2.5U/μl) 0.2μl

H ₂O complements to 20 μ l

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.5%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim big fragment, obtain purified product by the operation of Axygen sepharose purification kit purifying specification sheets, purified product is connected in the pJET1.2 cloning vector (Fermentas), the clone transforms DH5 α, the order-checking of picking list bacterium colony, errorless back acquisition CKM promoter fragment-pJET1.2 plasmid checks order, cut this plasmid with Bgl II (Fermentas) enzyme, agarose is cut glue and is reclaimed CKM5 ' control region shown in Seq ID No:10.

Promoterless pIRES-AcGFP carrier is cut with Bgl II (Fermentas) enzyme, adds FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, CKM 5 ' control region mover is connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse of bacterium checking insertion behind the coated plate, the bacterium liquid that will comprise correct direction of insertion is seeded to according to 1: 500 ratio and contains in the kantlex 50 μ g/mlLB liquid nutrient mediums, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used to not have the intracellular toxin plasmid and extract, and concrete operations are carried out according to EndoFree Plasmid Maxi (QIAGEN) specification sheets, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid CMV Enhancer CKM Promoter pIRES-AcGFP carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of CKM promoter fragment.

＜110〉Shandong Agricultural University

＜120〉a kind of skeletal muscle specific C KM promotor and application thereof

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acacggccat?gcggctcgtg?gcaaggcaag?cctggtttca?cggagggcaa?agtgcccacg 300

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Claims (4)

1. skeletal muscle specific C KM promotor, it is characterized in that: the gene order of this promotor is shown in Seq ID No:1.

2. a recombinant nucleic acid sequence is characterized in that: include promotor or the variant of this promotor, the perhaps fragment of gene order shown in Seq ID No:1 in the sequence.

3. according to the recombinant nucleic acid sequence under the claim 2, it is characterized in that: also include at least a functional gene in the sequence, described functional gene comprises raising meat quality gene or accelerates speed of growth gene or disease-resistant gene or the gene of the raising price of deed or gene or its combination of production certain drug.

4. an expression system is characterized in that: include as claim 2 or 3 described recombinant nucleic acid sequences.

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