CN102676527B - Skeletal muscle specific MYHC-IIx promoter and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology, relates to a skeletal muscle specific MYHC-IIx promoter and application thereof, and in particular provides a skeletal muscle specific expression gene 5' control region specific promoter and application thereof in transgenic pigs. The promoter has a promoting characteristic in skeletal muscle only, and does not have any promoting activity in various cells except the skeletal muscle. Therefore, the gene promoter specifically promote the expression of downstream gene in the skeletal muscle only, can meet the requirement that target gene is specifically expressed in the skeletal muscle and is an important element for improving the quality of pork through a transgenic technology.

Description

A kind of skeletal muscle specificity MYHC-IIx 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 MYHC-IIx promotor 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 of some specific gene expression pattern can cause that transcription factor is combined with the specificity of related elements, becomes the committed step of transcription initiation, and finally causes time and the space expression of gene.The series family 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 transgenic animal, the specificity promoter that obtains the energy widespread use seems 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.

Myosin (myosin) is the protein in one group of muscle and the nonmuscle cells constriction device, has atpase activity.The major part of heavy chain is the α spiral, and its head has atpase activity, and is combined with Actin muscle, and light chain has kinase activity.Myosin in the muscle is the composition unit of sarcostyle crin.Head has atpase activity, belong to can with the interactional motor protein of actin filament; Myosin also is the main constitutive protein matter of muscle, accounts for 60% of sarcostyle gross protein.The skeletal muscle myoglobulin heavy chain gene M YHC-I of pig, MYHC-IIa, MYHC-IIb, MYHC-IIx are positioned at karyomit(e) No. 12.Wherein MYHC-IIx is made up of 27 exons and 26 introns, 1881 amino acid of encoding.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 cultivation and the quality raising of transgenic pig.

Summary of the invention

Based on above-mentioned reason, the present inventor is through discovering that pig skeletal muscle myoglobulin heavy chain gene M YHC-IIx gene 5 ' control region has skeletal muscle specificity starting characteristic, and a kind of skeletal muscle specificity MYHC-IIx promoter vector 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 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 ID No:1;

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

" variant " refers to pig skeletal muscle MYHC-IIx promotor Seq ID No:1 sequence is carried out the sequence that any replacement, variation, modification, replacement, disappearance or the interpolation of one or more base produce, and this sequence still shows the activity that is similar to Seq ID No:1DNA sequence.

" fragment " refers to one or more zone of basic pig skeletal muscle MYHC-IIx promotor Seq ID No:1 sequence, and it still is similar to the activity of basic dna sequence dna.

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 promotor, the general recombinant nucleic acid sequence that adopts contains above-mentioned promotor or its variant, perhaps fragment in this sequence, so just can make the goal gene in promotor downstream specific expressed in skeletal muscle, as analyzing, develop, transform 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 by gene leading-in techniques such as microinjections, be used for setting up transgenic animal.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, 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, improve the meat nutritive value.

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 goal gene to the influence of animal, improve genetically modified effect.

The present invention mainly determines the startup feature of this promotor by the reporter gene analysis.At first obtain no promotor red fluorescent protein carrier.Obtain specific expressed pig skeletal muscle myoglobulin heavy chain IIx type 5 ' control region in the skeletal muscle by technology such as PCR or gene traps then, length is 2.9kb, insert the red fluorescent protein upstream, 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, transfection H9c2 rat myocardial cell 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 red fluorescent protein expression with inverted fluorescence microscope behind the 48h, analyze the promotor characteristic.Wherein why select in the empty carrier that CMV is connected into as positive control, mainly be because CMV is known generally acknowledged strong promoter, strong promoter is connected into the expression that promoterless carrier upstream can start reporter gene RFP, this carrier transfection such as cell are as positive control, can prove that cell used in the experiment and transfection reagent, rotaring dyeing technology etc. do not have problems, it is owing to the existence that is not activated son that the proof negative control does not have the RFP expression, rather than other factors cause.And then the stronger proof carrier that is connected into the experiment promotor to change the expression that starts RFP behind the cell over to be because the effect of this promotor.

Compared with prior art, the present invention has selected unique carrier DsRed carrier (commercial carrier full name is pDsRed-Express2-1vector) for use, to adapt to reporter gene RFP of the present invention (red fluorescent protein), the present invention simultaneously uses for research for MYHC-IIx has designed primer, promoter sequence and restriction enzyme site etc. especially, all has unique representativeness, therefore compare with existing skeletal muscle specificity promoter, technology of the present invention has tangible difference and obvious improvement and specific aim.

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, for the research in this field good reference value be arranged.

Description of drawings

Fig. 1 is MYHC-II xF and MYHC-II xR primer PCR amplification MYHC-II x promoter fragment electrophorogram, and swimming lane 1 is the PCR product among the figure, and M is MassRuler Maker;

Fig. 2 is promoterless DsRed carrier collection of illustrative plates;

Fig. 3 is transfection MYHC-II x promoter DsRed 48h mouse C2C12 cell photo gray-scale map,

The left side is photo under 40 times of light microscopics among the figure, and cell state is good; The right side is that green glow excites down photo, and visible RFP expresses, and MYHC-H x promoter has the activity of startup;

Fig. 4 is transfection MYHC-II x promoter DsRed 48h rat heart muscle H9c2 cell photo gray-scale map,

The left side is photo under 40 times of light microscopics among the figure, and cell state is good; The right side is that green glow excites down photo, and no RFP expresses, and MYHC-II x promoter does not have the activity of startup;

Fig. 5 is transfection CMV Promoter DsRed 48h Pigs Hearts cell photo gray-scale map,

The left side is photo under 40 times of light microscopics among the figure, and cell state is good; The right side is that green glow excites down photo, and visible RFP expresses, and rotaring dyeing technology is effective;

Fig. 6 is transfection MYHC-II x promoter DsRed 48hPK15 porcine kidney cell photo gray-scale map,

The left side is photo under 40 times of light microscopics among the figure, and cell state is good; The right side is that green glow excites down photo, and no RFP expresses, and MYHC-II x promoter does not have the activity of startup;

Fig. 7 is transfection CMV Promoter DsRed 48hPK15 porcine kidney cell photo gray-scale map,

The left side is photo under 40 times of light microscopics among the figure, and cell state is good; The right side is that green glow excites down photo, and visible RFP 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 advises.

The structure of embodiment 1 promotor reporter gene carrier

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

Promoterless DsRed carrier is as negative control; Be that skeleton is cut with Sma I (Fermentas) enzyme with DsRed, reclaim the 4107bp fragment, obtain not having promoter fragment.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 100 μ g/ml LB substratum, and 200rpm acutely rocks 14h.4 ℃ of 6000g collect thalline to the 50ml centrifuge tube, are used for no 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.

Design following primer according to NCBI (Gene ID:100125538), MYHC-II xF gene order is shown in Seq ID No:2, MYHC-II xR gene order is shown in Seq ID No:3, and it has comprised the scope from transcription initiation site downstream 36bp to upstream 2885bp.

According to following system preparation PCR reaction solution

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.8%TAE sepharose with 6 * Loading buffer, cutting glue behind the 5V/cm electrophoresis 20min reclaims, obtain purified product by the operation of Promega sepharose purification kit purifying specification sheets, using T4Polynucleotide Kinase (Fermentas) to carry out phosphorylation simultaneously handles, promoterless DsRed carrier is cut with Sma I (Fermentas) enzyme, adds FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, MYHC-II x promotor is connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse and order-checking that the bacterium checking is inserted behind the coated plate, check order errorless back obtains MYHC-IIx promotor DsRed plasmid, and promoter sequence is shown in Seq ID No:1.To comprise the correct bacterium liquid of order-checking and be seeded to according to 1: 500 ratio and contain in the kantlex 100 μ g/ml LB liquid nutrient mediums, 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used for no 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 MYHC-II x Promoter DsRed carrier, and utilize the method for transient transfection cell at the start-up characteristic of cell levels checking MYHC-II x promotor.

Adopt the MYHC-II x promoter fragment electrophorogram of MYHC-II xF and MYHC-II xR primer PCR amplification acquisition as shown in Figure 1.

Embodiment 2 cell cultures and transfection

Rat myocardial cell is that H9c2 cultivates in the DMEM that contains 10vt% foetal calf serum (Gibco) (Gibco) substratum, and culture condition is 37 ℃, 5%CO ₂, keep cell degree of converging 50%.

Ren sus domestica clone PK15 cultivates in the DMEM that contains 10vt% foetal calf serum (Gibco) (Gibco) substratum, and culture condition is 37 ℃, 5%CO ₂

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

To treat that cells transfected is with twice of 1 * PBS (Gibco) rinsing attached cell, 0.05% pancreatin (Gibco) that adds 37 ℃ of preheatings, digestion 5min, the perfect medium that adds antibiotic-free adopts DMEM (Gibco) the substratum re-suspended cell that contains 10vt% foetal calf serum (Gibco), 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 (Gibco) 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 12h, and this moment, cell degree of converging was 90%～95%.

With positive control support C MV Promoter DsRed, the promoterless DsRed of negative control carrier, experiment carrier MYHC-II x three kinds of plasmids of Promoter DsRed transfection simultaneously mouse muscle-forming cell is C2C12, rat myocardial cell is H9c2, porcine kidney cell line PK15, 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 3 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 RFP expression behind the transfection 48h with under the fluorescence inverted microscope: the transfection efficiency of positive control carrier is at 30% (as Fig. 5 and shown in Figure 7), redfree fluorescent protein expression after the transfection of negative control carrier.Behind the transfection MYHC-II x Promoter DsRed carrier, having only through the mouse muscle-forming cell of inducing differentiation is that C2C12 has red fluorescent protein to express (as shown in Figure 3), equal redfree fluorescent protein expression (as Fig. 4 and shown in Figure 6) in rat heart muscle and porcine kidney cell line.

Transfection results only show in the myocyte of maturation pig MYHC-H x promotor just have start active, in another kind of voluntary muscle myocardial cell, do not start active, in nephrocyte, there is not the activity of startup yet, show that pig MYHC-IIx promotor has the skeletal muscle specificity and starts active, can the driving purposes gene specific expressed at skeletal muscle.

Embodiment 3MYHC-II x 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 driving purposes expression of gene.

The application of embodiment 4 series connection MYHC-IIx promotors

Design following primer according to NCBI (Gene ID:100125538), the upstream and downstream primer is introduced XhoI, KpnI restriction enzyme site and protection base respectively.MYHC-II x2F gene order is shown in Seq ID No:4, and MYHC-II x2R gene order is shown in Seq ID No:5.

The PCR program setting is as follows

Product after the amplification is mixed sample to the 0.8%TAE sepharose with 6 * Loading buffer, cut glue behind the 5V/cm electrophoresis 20min and reclaim the 2.9kb fragment, obtain purified product by the operation of Promega sepharose purification kit purifying specification sheets.Cut the PCR purified product and reclaim enzyme and cut product with XhoI, KpnI (Fermentas) enzyme.

MYHC-II x Promoter DsRed carrier is cut with Xho I, KpnI (Fermentas) enzyme, enzyme is cut MYHC-II x control region that purifying crosses to be connected with carrier after enzyme is cut 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 100 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 for no 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 Double MYHC-II x Promoter DsRed carrier, and utilize the method for transient transfection cell at the start-up characteristic of the two MYHC-II x promotors of cell levels checking.

With positive control support C MV Promoter DsRed, the promoterless DsRed of negative control carrier, above-mentioned recombinant nucleic acid experiment carrier Double MYHC-II x three kinds of plasmids of Promoter DsRed transfection simultaneously mouse muscle-forming cell is C2C12, rat myocardial cell is H9c2, porcine kidney cell line PK15, 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 3 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 RFP expression behind the transfection 48h with under the fluorescence inverted microscope: the transfection efficiency of positive control carrier is 30%, redfree fluorescent protein expression after the transfection of negative control carrier.Behind the transfection Double MYHC-II x Promoter DsRed carrier, having only through the mouse muscle-forming cell of inducing differentiation is that C2C12 has red fluorescent protein to express, equal redfree fluorescent protein expression in rat heart muscle and porcine kidney cell line.

Transfection results only show in the myocyte of maturation pig Double MYHC-II x promotor just have start active, in another kind of voluntary muscle myocardial cell, do not start active, in nephrocyte, there is not the activity of startup yet, show that pig Double MYHC-II x promotor has the skeletal muscle specificity and starts active, can the driving purposes gene specific expressed at skeletal muscle.

Embodiment 5MYHC-IIx promoter fragment is used

Design following primer according to NCBI (Gene ID:100125538), the upstream and downstream primer is introduced XhoI, KpnI restriction enzyme site and protection base respectively.MYHC-II x3F gene order is shown in Seq ID No:6, and MYHC-II x3R 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 1.2%TAE sepharose with 6 * Loading buffer, cutting glue behind the 5V/cm electrophoresis 15min reclaims, obtain purified product by the operation of Promega sepharose purification kit purifying specification sheets, using T4Polynucleotide Kinase (Fermentas) to carry out phosphorylation simultaneously handles, promoterless DsRed carrier is cut with Sma I (Fermentas) enzyme, adds FastAP simultaneously ^TMThermosensitive Alkaline Phosphatase (Fermentas) carries out dephosphorylation to be handled, MYHC-II x promotor is connected with dephosphorylized carrier with Rapid DNA Ligation (Fermentas), and conversion DH5 α, choose the forward and reverse and order-checking that the bacterium checking is inserted behind the coated plate, check order errorless back obtains MYHC-II x promotor DsRed plasmid, and promoter sequence is shown in Seq ID No:8.To comprise the correct bacterium liquid of order-checking and be seeded to according to 1: 500 ratio and contain in the kantlex 100 μ g/ml LB liquid nutrient mediums, 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to the 50ml centrifuge tube, are used for no 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 Segment MYHC-II x Promoter DsRed carrier, and utilize the method for transient transfection cell at the start-up characteristic of cell levels checking MYHC-II x promotor.

With positive control support C MV Promoter DsRed, the promoterless DsRed of negative control carrier and above-mentioned recombinant nucleic acid experiment carrier S egment MYHC-II x three kinds of plasmids of Promoter DsRed transfection simultaneously mouse muscle-forming cell are C2C12, rat myocardial cell is H9c2, porcine kidney cell line PK15, 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 3 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 RFP expression behind the transfection 48h with under the fluorescence inverted microscope: the transfection efficiency of positive control carrier is 30%, redfree fluorescent protein expression after the transfection of negative control carrier.Behind the transfection Segment MYHC-II xPromoter DsRed carrier, having only through the mouse muscle-forming cell of inducing differentiation is that C2C12 has red fluorescent protein to express, equal redfree fluorescent protein expression in rat heart muscle and porcine kidney cell line.

Transfection results only show in the myocyte of maturation pig Segment MYHC-II x promotor just have start active, in another kind of voluntary muscle myocardial cell, do not start active, in nephrocyte, there is not the activity of startup yet, show that pig Segment MYHC-II x promotor has the skeletal muscle specificity and starts active, can the driving purposes gene specific expressed at skeletal muscle.

Claims (1)

1. skeletal muscle specificity MYHC-II x promotor, it is characterized in that: the gene order of this promotor is shown in Seq ID No:1.

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