CN103305517B - Lung tissue specific SP-D promoter and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, relates to a lung tissue specific SP-D promoter and an application of the promoter, and particularly provides a specific promoter within a lung tissue specific expression gene 5' control region and an application of the promoter to a transgenic pig. The promoter only has starting characteristics in a lung tissue and does not have starting activity in a plurality of cells except for the lung tissue; the gene promoter only specifically starts the expression of downstream gene in the lung tissue, and therefore can meet the requirements for specific expression of the target gene in the lung tissue; the promoter provides an important element for improving the disease resistance of the pig by means of transgenic technology.

Description

A kind of lung tissue specificity SP-D promotor and application thereof

Technical field

The present invention relates to molecular genetics field, be specifically related to a kind of lung tissue different expression gene 5 ' control region specificity SP-D promotor and the application in transgenic pig thereof.

Background technology

Time and level that eukaryotic gene is expressed are sequentially carried out in strict accordance with growth.The adjusting of some specific gene expression pattern can cause the specific binding of transcription factor and related elements, becomes the committed step of transcription initiation, and finally causes the spatial-temporal expression of gene.Coding organizes the series family gene of differential protein to express in strict accordance with tissue specificity and etap property, for gene expression regulation mechanism provides good research mode.In transgenic animal, the specificity promoter that obtains energy widespread use seems extremely important.But the specificity of promotor is controlled by a plurality of elements, the controlling element playing a crucial role at different sorts and different development stage is not identical, and the interaction between controlling element and trans-acting factor is also very complicated.Therefore to make foreign gene in animal tissues, express differential high efficient, must build one can specific high-efficiency expression animal expression carrier, wherein the selection of promotor is one of important condition.

Pulmonary surfactant (Pulmonary Surfactant, PS) is a kind of lipoprotein complex body, main active ingredient be DPPC (DPPC) and with the protein-specific protein of its combination.Be present in PS and in the interactional specific proteins of phospholipid molecule and be called pulmonary surfactant in conjunction with albumen, be called for short surfactant albumen (Surfactant Protein, SP).SP only accounts for below 5% of PS total amount, but has physiological function widely, is the indispensable important component of PS.The at present existing four kinds of surfactant albumen of pig surfactant protein gene SP are determined, are divided into two large classes: (1) small molecules hydrophobicity SP, comprises SP-B and SP-C by its biochemical characteristic; (2) macromole wetting ability SP, comprises SP-A and SP-D.Wherein the SP-D of pig is positioned at karyomit(e) No. 14.By 8 exons and 7 introns, formed 378 amino acid of encoding.By cloning this gene promoter area and analyzing in vitro its starting characteristic, utilizing correlated series to build lung tissue specific expression vector is a kind of effective means, but above-mentioned research is all also in the starting stage, for cultivation and the disease resistance raising of transgenic pig, all do not play good promoter action.

Summary of the invention

Reason based on above-mentioned, the present inventor finds that through research pig surfactant protein gene SP-D gene 5 ' control region has lung tissue specificity starting characteristic, and a kind of lung tissue specificity SP-D promoter vector that utilized this characteristics design, this promotor can be applied in the cultivation of transgenic pig.This promoter vector has lung tissue specificity starting characteristic, and it is all active without starting in the various kinds of cell except lung tissue, visible this promotor starts specifically the expression of downstream gene in lung tissue, can meet goal gene specific expressed requirement in lung tissue, for transgene carrier provides a kind of important element.

Lung tissue different expression gene provided by the present invention 5 ' control region specificity promoter, its gene order is as shown in Seq ID No:1;

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

" variant " refers to pig lung tissue SP-D promotor Seq ID No:1 sequence carried out to 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 region of basic pig lung tissue SP-D promotor Seq ID No:1 sequence, and it is still similar to the activity of basic DNA sequence dna.

Promotor or its variant with above-mentioned sequence, or fragment, there is lung tissue specificity starting characteristic, and it is all active without starting in various kinds of cell outside lung tissue, visible this gene promoter starts specifically the expression of downstream gene in lung tissue, can meet goal gene specific expressed requirement in lung tissue, and can be applied in the cultivation of transgenic pig.

While applying above-mentioned promotor, the general recombinant nucleic acid sequence that adopts, contains above-mentioned promotor or its variant in this sequence, or fragment, so just can make in the goal gene in promotor downstream specific expressed in lung tissue, as analyzing, the research model of exploitation, transformation lung tissue characteristic.Can also utilize existing molecular biology operative technique to obtain the recombinant nucleic acid that comprises goal gene, by gene Transfection Technologies such as microinjections, obtain transgenic embryos, for setting up transgenic animal.Goal gene in recombinant nucleic acid sequence, contain at least one following functional gene, mainly comprise disease-resistant gene, accelerate the gene of speed of growth gene and production certain drug, so just can obtain outside lung tissue specificity starting characteristic, improve the performance of this recombinant nucleic acid sequence, for the cultivation of transgenic pig provides more functional direction, increase its additional output value, as Toll sample acceptor gene is expressed under this promotor instructs, can specificity improve interleukin and Interferon, rabbit in lung tissue, improve the disease-resistant performance of pig.

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

The present invention is mainly by the startup feature of this promotor of reporter gene Analysis deterrmination.First obtain without promotor red fluorescent protein carrier.Then by technology such as PCR or gene traps, obtain specific expressed pig surfactant protein gene SP-D type 5 ' control region in lung tissue, length is 2.97kb, insert red fluorescent protein upstream, obtain promotor reporter gene and analyze carrier, CMV promotor (589bp) is connected in empty carrier DsRed carrier as positive control to the negative contrast of empty carrier DsRed carrier.Plasmid goes after intracellular toxin, transfection V79 hamster pneumonocyte system, the 3T3-L1 of transfection simultaneously mouse embryo fibroblasts system, C2C12 mice skeletal clone and Marc145 African green monkey kidney cell line are contrast, after 48h, with inverted fluorescence microscope, observe red fluorescent protein expression, analyze promotor characteristic.Wherein why select in empty carrier that CMV is connected into as positive control, mainly 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 is if cell is as positive control, can prove that cell used in experiment and transfection reagent, rotaring dyeing technology etc. do not have problems, it is owing to being not activated sub existence that proof negative control is expressed without RFP, rather than other factors cause.And then the stronger proof carrier that is connected into experiment promotor to proceed to the expression that starts RFP after cell be because the effect of this promotor.

Compared with prior art, the present invention has selected unique carrier DsRed carrier (business carrier full name is pDsRed-Express2-1vector), to adapt to reporter gene RFP(red fluorescent protein of the present invention), simultaneously the present invention is SP-D special design primer, promoter sequence and restriction enzyme sites etc. are for studying it, all there is unique representativeness, therefore compare with existing lung tissue specificity promoter, technology of the present invention has obvious difference and significant progressive and specific aim.

When above-mentioned promotor, or after 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 disease-resistant quality, the higher speed of growth, better disease resistance and reduction feeding cost, can also in the production of certain drug, obtain applying more widely, for the research in this field, have good reference value.

Accompanying drawing explanation

Fig. 1 is SP-D (F) and SP-D (R) primer PCR amplification SP-D promoter fragment electrophorogram, and in figure, swimming lane M is MassRuler Maker, and 1 is PCR product;

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

Fig. 3 is transfection SP-D promoter DsRed48h hamster V79 cell photo gray-scale map,

In figure, left side is photo under 40 times of light microscopics, and cell state is good; Right side is that green glow excites lower photo, and visible RFP expresses, and SP-D promoter has the activity of startup;

Fig. 4 is transfection SP-D promoter DsRed48h mouse embryo fibroblast 3T3-L1 cell photo gray-scale map,

In figure, left side is photo under 40 times of light microscopics, and cell state is good; Right side is that green glow excites lower photo, without RFP, expresses, and SP-D promoter is active without starting;

Fig. 5 is transfection SP-D promoter DsRed48h mice skeletal C2C12 cell photo gray-scale map,

In figure, left side is photo under 40 times of light microscopics, and cell state is good; Right side is that green glow excites lower photo, without RFP, expresses, and SP-D promoter is active without starting;

Fig. 6 is transfection SP-D promoter DsRed48h African green monkey kidney Marc145 cell photo gray-scale map,

In figure, left side is photo under 40 times of light microscopics, and cell state is good; Right side is that green glow excites lower photo, without RFP, expresses, and SP-D promoter is active without starting;

Fig. 7 is transfection CMV Promoter DsRed48h hamster V79 cell photo gray-scale map,

In figure, left side is photo under 40 times of light microscopics, and cell state is good; Right side is that green glow excites lower photo, and visible RFP expresses, and rotaring dyeing technology is effective;

Fig. 8 is transfection promoter less DsRed48h hamster V79 cell photo gray-scale map,

In figure, left side is photo under 40 times of light microscopics, and cell state is good; Right side is that green glow excites lower photo, without RFP, expresses, and transfection reagent is effective.

Embodiment

In the context of the present specification, unless specialize otherwise this specification sheets any term used has the implication that those skilled in the art understand in the art conventionally, and the experimental technique of unreceipted detailed conditions is to carry out according to conventional methods or the process specifications of advising according to supplier.

The structure of embodiment 1 promotor Reporter gene vector

Genome extraction: get LargeYorkshire ear tissue, according to DNA(days roots of TIANamp genomic) test kit specification sheets carries out extracting genome DNA.

Promoterless DsRed carrier is as negative control; The DsRed of take cuts as Sma I (Fermentas) enzyme for skeleton, reclaims 4107bp fragment, obtains without promoter fragment.Complete CMV promotor forward is connected in this carrier, obtains the positive control carrier of this carrier.

The bacterium liquid that comprises negative control and positive control carrier is seeded to respectively containing in kantlex 100 μ g/ml LB substratum according to the ratio of 1:500, and 200rpm acutely rocks 14h.4 ℃ of 6000g collect thalline to 50ml centrifuge tube, and for without intracellular toxin plasmid extraction, concrete operations are according to EndoFree Plasmid Maxi(QIAGEN) specification sheets carries out, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.

According to NCBI (GeneID:397198), design following primer, SP-D (F) gene order is as shown in Seq ID No:2, SP-D (R) gene order is as shown in Seq ID No:3, and it has comprised the scope from transcription initiation site downstream 47bp to upstream 2916bp.

PCR program setting is as follows

Product after amplification is mixed to loading to 0.8%TAE sepharose with 6 * Loading buffer, after 5V/cm electrophoresis 20min, cutting glue reclaims, by the operation of Promega sepharose purification kit purifying specification sheets, obtain purified product, use T4Polynucleotide Kinase(Fermentas simultaneously) carry out phosphatizing treatment, promoterless for DsRed carrier Sma I (Fermentas) enzyme cut, add FastAP simultaneously ^tMthermosensitive Alkaline Phosphatase(Fermentas) carry out dephosphorylation processing, by Rapid DNA Ligation(Fermentas for SP-D promotor) be connected with dephosphorylized carrier, and transform DH5 α, after coated plate, choose the forward and reverse and order-checking that bacterium checking is inserted, the errorless rear acquisition SP-D promotor DsRed plasmid that checks order, promoter sequence is as shown in Seq ID No:1.By comprising the correct bacterium liquid of order-checking, according to the ratio of 1:500, be seeded to containing in kantlex 100 μ g/ml LB liquid nutrient mediums, 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to 50ml centrifuge tube, and for without intracellular toxin plasmid extraction, concrete operations are according to EndoFree Plasmid Maxi(QIAGEN) specification sheets carries out, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid SP-D Promoter DsRed carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of SP-D promotor.

Adopt the SP-D promoter fragment electrophorogram of SP-D (F) and SP-D (R) primer PCR amplification acquisition as shown in Figure 1.

Embodiment 2 cell cultures and transfection

V79 hamster pneumonocyte system cultivates in containing RPMI1640 (Gibco) substratum of 10vt% foetal calf serum (Gibco), and culture condition is 37 ℃, 5%CO ₂, keep cell degree of converging 50%.

3T3-L1 mouse embryo fibroblasts system cultivates in containing DMEM (Gibco) substratum of 10vt% new-born calf serum (Gibco), and culture condition is 37 ℃, 5%CO ₂.

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

Marc145 African green monkey kidney cell line is cultivated in containing DMEM (Gibco) substratum of 10vt% foetal calf serum (Gibco), and culture condition is 37 ℃, 5%CO ₂, keep cell degree of converging 50%.

Twice of 1 * PBS for cell (Gibco) rinsing attached cell of transfection will be treated, 0.05% pancreatin (Gibco) that adds 37 ℃ of preheatings, digestion 5min, add the perfect medium of antibiotic-free to adopt RPMI1640 (Gibco) the substratum re-suspended cell containing 10vt% foetal calf serum (Gibco), after cell counting, every hole is by 1 * 10 ⁴be seeded to 24 orifice plates, after 12h, with 37 ℃ of serum-free antibiotic-free RPMI1640 (Gibco) substratum washed cell once, every hole adds the perfect medium of the antibiotic-free that 500 μ l are fresh, carries out transfection after 12h, and now cell degree of converging is 90%～95%.

By positive control support C MV Promoter DsRed, the promoterless DsRed of negative control carrier, experiment carrier S P-D tri-kinds of plasmids of Promoter DsRed transfection simultaneously V79 hamster pneumonocyte system, 3T3-L1 mouse embryo fibroblasts system, C2C12 mice skeletal clone and Marc145 African green monkey kidney cell line, every hole is diluted to 750ng plasmid in 100 μ l opti-MEM, adds 0.75 μ l Lipofectamine ^tMpLUS (Invitrogen) thoroughly mixes, and adds 3 μ l Lipofectamine after 5min ^tMlTX (Invitrogen) transfection reagent is put upside down and is mixed, and hatches for 25 ℃, after 30min, adds to gently in each hole, mixes.After transfection 12h, change inducing culture to C2C12 clone, Cell differentiation inducing activity merges.After transfection 48h with under fluorescence inverted microscope, observe RFP expression: the transfection efficiency of positive control carrier at 30%(as shown in Fig. 5 and Fig. 7), redfree fluorescent protein expression after the transfection of negative control carrier.After transfection SP-D Promoter DsRed carrier, only have V79 hamster pneumonocyte system to have red fluorescent protein to express (as shown in Figure 3), in rat embryo fibroblast cell system, equal redfree fluorescent protein expression in mice skeletal clone and African green monkey kidney cell line., (as shown in Figure 4,5, 6).

Transfection results only show in hamster pneumonocyte system pig SP-D promotor just have start active, in embryo fibroblast, in Skeletal Muscle Cell, do not start active, also active without starting in nephrocyte, show that pig SP-D promotor has lung tissue specificity and starts activity, can drive goal gene specific expressed in lung tissue.

Embodiment 3SP-D promoter variants function

According to the explanation of manufacturers, utilize Stratagene QuikChange ^tMrite-directed mutagenesis test kit (Stratagene), design mutant primer builds this promoter variants, comprises the disappearance of nucleic acid, replaces and inserts.The promotor of change is verified sudden change by sequencing.Promotor after sudden change is recombinated to goal gene upstream, drive the expression of goal gene.

The application of embodiment 4 series connection SP-D promotors

According to NCBI (Gene ID:397198), design following primer, upstream and downstream primer is introduced respectively Xho I, Kpn I restriction enzyme site and protection base.SP-D2 (F) gene order is as shown in Seq ID No:4, and SP-D2 (R) gene order is as shown in Seq ID No:5.

PCR program setting is as follows

Product after amplification is mixed to loading to 0.8%TAE sepharose with 6 * Loading buffer, after 5V/cm electrophoresis 20min, cut glue and reclaim 2.87kb fragment, by the operation of Promega sepharose purification kit purifying specification sheets, obtain purified product.With Xho I, Kpn I (Fermentas) enzyme, cut PCR purified product and reclaim enzyme and cut product.Enzyme is cut to the Rapid DNA Ligation(Fermentas for SP-D control region that purifying is crossed) carrier after cutting with enzyme is connected, and transform TOP10, after coated plate, choose the forward and reverse of bacterium checking insertion, the bacterium liquid that comprises correct direction of insertion is seeded to containing in kantlex 100 μ g/ml LB liquid nutrient mediums according to the ratio of 1:500, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to 50ml centrifuge tube, and for without intracellular toxin plasmid extraction, concrete operations are according to EndoFree Plasmid Maxi(QIAGEN) specification sheets carries out, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid Double SP-D Promoter DsRed carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of two SP-D promotors.

By positive control support C MV Promoter DsRed, the promoterless DsRed of negative control carrier, and tri-kinds of plasmids of above-mentioned experiment carrier Double SP-D Promoter DsRed transfection simultaneously V79 hamster pneumonocyte system, 3T3-L1 mouse embryo fibroblasts system, C2C12 mice skeletal clone and Marc145 African green monkey kidney cell line, every hole is diluted to 750ng plasmid in 100 μ l opti-MEM, adds 0.75 μ l Lipofectamine ^tMpLUS (Invitrogen) thoroughly mixes, and adds 3 μ l Lipofectamine after 5min ^tMlTX (Invitrogen) transfection reagent is put upside down and is mixed, and hatches for 25 ℃, after 30min, adds to gently in each hole, mixes.After transfection 48h with under fluorescence inverted microscope, observe RFP expression: the transfection efficiency of positive control carrier is 30%, redfree fluorescent protein expression after the transfection of negative control carrier.After transfection Double SP-D Promoter DsRed carrier, only have V79 hamster pneumonocyte system to have red fluorescent protein to express, in rat embryo fibroblast cell system, equal redfree fluorescent protein expression in mice skeletal clone and African green monkey kidney cell line.

Transfection results only show in hamster pneumonocyte system pig Double SP-D promotor just have start active, in embryo fibroblast, in Skeletal Muscle Cell, do not start active, also active without starting in nephrocyte, show that pig Double SP-D promotor has lung tissue specificity and starts activity, can drive goal gene specific expressed in lung tissue.

The application of embodiment 5SP-D promoter fragment

According to NCBI (Gene ID:733580), design following primer, upstream and downstream primer is introduced respectively Xho I, Kpn I restriction enzyme site and protection base.SP-D3 (F) gene order is as shown in Seq ID No:6, and SP-D3 (R) gene order is as shown in Seq ID No:7.

PCR program setting is as follows

Product after amplification is mixed to loading to 0.8%TAE sepharose with 6 * Loading buffer, after 5V/cm electrophoresis 20min, cut glue and reclaim 2.87kb fragment, by the operation of Promega sepharose purification kit purifying specification sheets, obtain purified product.With Xho I, Kpn I (Fermentas) enzyme, cut PCR purified product and reclaim enzyme and cut product.Enzyme is cut to the Rapid DNA Ligation(Fermentas for SP-D control region that purifying is crossed) carrier after cutting with enzyme is connected, and transform TOP10, after coated plate, choose the forward and reverse of bacterium checking insertion, the bacterium liquid that comprises correct direction of insertion is seeded to containing in kantlex 100 μ g/ml LB liquid nutrient mediums according to the ratio of 1:500, and 200rpm acutely rocks 14h.4 ℃ of 6000 * g collect thalline to 50ml centrifuge tube, and for without intracellular toxin plasmid extraction, concrete operations are according to EndoFree Plasmid Maxi(QIAGEN) specification sheets carries out, and the plasmid A260/280 that purifying obtains is between 1.8-1.9.Obtain recombinant nucleic acid Segment SP-D Promoter DsRed carrier, and utilize the method for transient transfection cell on cell levels, to verify the start-up characteristic of two SP-D promotors.

By positive control support C MV Promoter DsRed, the promoterless DsRed of negative control carrier, and tri-kinds of plasmids of above-mentioned experiment carrier S egment SP-D Promoter DsRed transfection simultaneously V79 hamster pneumonocyte system, 3T3-L1 mouse embryo fibroblasts system, C2C12 mice skeletal clone and Marc145 African green monkey kidney cell line, every hole is diluted to 750ng plasmid in 100 μ l opti-MEM, adds 0.75 μ l Lipofectamine ^tMpLUS (Invitrogen) thoroughly mixes, and adds 3 μ l Lipofectamine after 5min ^tMlTX (Invitrogen) transfection reagent is put upside down and is mixed, and hatches for 25 ℃, after 30min, adds to gently in each hole, mixes.After transfection 48h with under fluorescence inverted microscope, observe RFP expression: the transfection efficiency of positive control carrier is 30%, redfree fluorescent protein expression after the transfection of negative control carrier.After transfection Segment SP-D Promoter DsRed carrier, only have V79 hamster pneumonocyte system to have red fluorescent protein to express, in rat embryo fibroblast cell system, equal redfree fluorescent protein expression in mice skeletal clone and African green monkey kidney cell line.

Transfection results only show in hamster pneumonocyte system pig Segment SP-D promotor just have start active, in embryo fibroblast, in Skeletal Muscle Cell, do not start active, also active without starting in nephrocyte, show that pig Segment SP-D promotor has lung tissue specificity and starts activity, can drive goal gene specific expressed in lung tissue.

Claims (1)

1. a lung tissue specificity SP-D promotor, is characterized in that: the gene order of this promotor is as shown in Seq ID No:1.