CN103421780A - Promoters of pig adipose tissue specific expression gene plin and use thereof - Google Patents

Abstract

The invention belongs to the technical field of animal gene engineering and relates to separation and identification and functional verification of different-length promoter regions of a pig adipose tissue specific expression gene plin. According to the invention, 6 upstream different-length promoters of the pig adipose tissue specific expression gene plin in a pig genome are cloned; nucleotide sequences of the promoters are shown in the formulas of SEQ ID NO: 2 to SEQ ID NO: 7; and a detection result shows that the promoter fragment from -130bp to -910bp and the promoter fragment from -435bp to -896bp have independent promoter activity. The invention also discloses a preparation method of the 6 different-deletion promoter fragments and a corresponding recombinant expression vector, and a use of a dual-luciferase activity detection system in promoter activity analysis.

Description

Adipose Tissue different expression gene plin promotor and application

Technical field

The invention belongs to animal gene engineering technology field, be specifically related to isolation identification and the functional verification of the different lengths promoter region of fatty tissue different expression gene plin in pig.

Background technology

The expression of higher organism gene is subject to the finely regulating of cell internal and external environment, thereby has strict time and spacial ordering.The expression regulation of gene is a complexity and orderly process, by multistage regulation and control level, jointly completed, this mainly comprise transcribe before, transcribe, transcribe after, translate, translate rear five levels.Wherein the regulation and control of transcriptional level are the links of most critical.Promotor is an important controlling element on transcriptional level, is the DNA sequence dna that is positioned at structure gene 5 ' end upstream, can with the expression (military force, 1999) of numerous transcription factor interaction regulatory gene.Therefore the structure, function, binding mode etc. of furtheing investigate promotor are conducive to function and the intergenic interaction that we better understand corresponding gene.

Promotor can be divided into to 3 classes according to the difference of the promotor mode of action and function: constitutive promoter, inducible promoter and tissue-specific promoter.Constitutive promoter refers under the regulation and control of such promotor, and the expression of gene is substantially constant on certain level, and at the different tissues position, expression level does not have notable difference.Inducible promoter refers to that, under the stimulation of some specific physics or chemical signal, this type of promotor can improve the transcriptional level of gene significantly.Tissue-specific promoter refers under the regulation and control of such promotor, and gene is often only expressed at some specific organ or tissue position, and shows and grow the feature of regulating.

Can foreign gene express efficiently and stably in body is the key of genetically engineered research.In eukaryotic cell, utilizing genetic engineering technique to build the expression that multiple eukaryotic expression system carries out foreign gene has been a technology commonly used, but the problem often faced is that the expression amount of foreign gene is lower, and express and often be present in nearly all tissue of body, CMV and the SV40 promotor of utilizing as extensive as present stage.Both all can foreign gene-carrying and the cell of nearly all type in express efficiently.The problem of these two kinds efficient promotor existence is apparent: do not possess the regulatory gene targeted expression in the ability of a certain particular organization.Key that can head it off is the acquisition that possesses the tissue-specific promoter of greater activity.Tissue-specific promoter usually take specific histocyte structure and chemistry, physical signalling and is basis, can carry out the target location to foreign gene, gene is fixed in to a certain tissue or cells, the loss that has reduced human body energy and material reaches the impact on the organism metabolism activity, can play again the directed effect of improving a certain tissue characteristics, this prospect aspect the treatment of human diseases is especially considerable simultaneously.Possess such speciality in view of tissue specificity starts, more and more obtain investigator's favor in genetically engineered.

Tissue-specific promotor can start foreign gene and express at specific position in animal body, keep specificity, this has not only overcome constitutive promoter and has expressed excessively widely because of foreign gene the shortcoming that causes excessive waste, and can impel foreign gene efficient and specific expressed at some specific position, there is obvious Space-time speciality.

Up to the present the research for tissue-specific promoter has also had some achievements, early start is some researchs aspect plant, some tissue-specific promoters have successfully been separated from plant, as: people such as Sunikumar separate and have obtained cotton alpha globulin B gene promoter, after it is connected with the gus reporter gene, import successively respectively cotton, tobacco and and Arabidopis thaliana in, find after deliberation, reporter gene only is expressed in seed, has the tissue specificity of height; (Liu Jingge, 2010); Aspect plant after the separation of tissue-specific promoter plant pest-resistant, disease-resistant, will be widely used aspect cultivating improved seeds.The relative plant of research aspect animal just lags behind a bit, but certain effect is still arranged, most of or for the treatment to some diseases.

Also there are some problems in these tissue-specific promoters that obtain, as: specificity is not very high; It not is a lot of separating and obtaining the specificity promoter of applying, and has limited the application in genetically engineered; The problems such as the promoter activity of separating is very low.We want to improve tissue-specific promoter's activity can be from the following aspects: (1) builds double-promoter: the basis of SP promotor of mouse that had the investigator to build, added again the CMV strong promoter, claim double-promoter by the promotor rebuild, the method by building double-promoter that found that can improve the expression level (Zhang Qianqian, 2007) of foreign gene in the C2C12 cell.(2) build chimeric promoters: soon to same tissue, have specific two or more different promotors or controlling element to combine.The most frequently used method is the tissue-specific element of constantly screening multiple combination, finally selects optimal combination and uses.The skeletal muscle α-actin gene promoter of mouse, the strongest chimeric promoters SP promotor of activity that TEF-1, MEF-1, MEF-2 oligonucleotide random combine filter out, can be by foreign gene-carrying efficient and stable expression in skeletal muscle.

The process of adipocyte steatolysis is the main core reaction of energy i (in vivo) metabolism, and lipase is by the process of triglyceride hydrolysis.It is neutral lipid that fat drips core, be cholesteryl ester or triglyceride level etc., outside is being coated with by the phospholipid molecule of individual layer, wherein in the phosphatide of individual layer, exist some and drip relevant albumen to fat, the regulate process that the metabolic process that the existence of these albumen is dripped fat and fat thereof drip plays very important effect (Greenberg A, et al., 1991).There is result of study to show, the variation that lipase can occurrence positions in the fat splitting process will be transferred to the surface that fat drips from enchylema, and in whole lipolysis in vitro process, fat drips surperficial existing perilipin and played most important effect (Zhao Xiaoling, Zhu Qing fat, 2006).

Find that the earliest plin finds in the epididymal adipose tissues cell of rat, it is a kind of albumen, is present in fat and drips surface.Because plin participates in the process of steatolysis, and has brought into play great role in whole process, thus more and more to plin research, also become gradually the focus (Jang Y, et al., 2006) in research steatolysis process.The PAT family protein mainly contains ADRP, perilipin, TIP47 and S3-12, and the formation that this family protein drips with fat, maturation and fatty degraded are relevant, regulate the running balance (Miura S, et al., 2002) that fat drips interior fat.Plin can specificly express in adipocyte, and steatolysis is had to two effects of regulating, and not only can regulate and control the efficiency of steatolysis but also can regulate and control the amount (Miyoshi H, et al., 2006) of the triglyceride level of fatty tissue output.Under basic state, plin can suppress steatolysis and lipase be played to the effect of barrier, if under the situation that need to increase at energy requirement, catecholamine (steatolysis adjusting hormone) can impel Serine generation phosphorylation in plin, thereby promoting hormone-sensitive lipase (HSL) to be indexed into fat from cytosol drips surface and promotes fatty decomposition (Liu BH, et al., 2005).According to relevant supposition, show, plin may play crucial unlatching effect in the fat splitting process and in regulation process.Can be subject to the impact of some factors in the expression process of plin, as: (Londos C, et al., 2005) such as tumour necrosis factor, leptin, PPAR γ.The research such as ChiaH is found when using serum amyloid A protein (serum amyloid A protein, while SAA) breaking up porcine adipocyte, plin expresses decline, and the alternative Apolipoprotein A1 of SAA (apolipoprptein A1) lipophorin (the Saha PK main as HDL, et al., 2004).Numerous plin of studies have shown that play the role of a nucleus in the adipocyte lipolysis in vitro, and the hormone regulation of the hydrolytic action of centering lipid is essential, and the deposition for fat in fatty tissue also is absolutely necessary simultaneously.

Be subject to the impact of a lot of regulatory factors at fatty atomization, wherein PPAR γ is one of most important regulatory factor.It can be directly combines with the gene specific expressed at fatty tissue and by its activation and then maintain the state of adipocyte, in the myotube cell formation process, the expression amount of plin obviously work improves, and is consistent with PPAR γ expression amount, has also determined that plin is one of target protein of PPAR γ (Yan W, Chen S, Huang J, Shen Y, Qiang B, Gu D, 2004).Two kinds of hypotype PPAR γ of PPAR γ ₁With PPAR γ ₂Can improve the perilipin gene promoter activity, but having the people to think only has PPAR γ ₂To participate in adipogenic important factor.And discovery plin gene promoter is transcribed by a lonely nuclear receptor-estrogen receptor associated receptor (estrogen receptor-related receptor α, ERR α) activate (Tansey JT, et al., 2001), identified the response element of promoter region this receptor by the method for examining report gene, can pass through PPAR γ co-activation (peroxisome proliferator-actived receptor γ coactivator(PGC)-1 α simultaneously) enhanced activity (Tanaka NJ, Nakamura Y, 1998).

From the promoter Analysis obtained plant and in animal at present, find, the problem of ubiquity the following aspects: specificity is not very high; It not is a lot of separating and obtaining the specificity promoter of applying, and has limited the application in genetically engineered; Active low.Particularly the research on animal also relatively is short of.Therefore, higher promotor becomes problem demanding prompt solution than strongly expressed efficiency to obtain specificity.

Up to the present, except the applicant, still do not see the report of the promoter function of research Adipose Tissue skeletal muscle gene plin.So the applicant has carried out the research of different lengths promoter function to this gene, to utilizing better this promotor.

Summary of the invention

The object of the invention is to the different fragments of the fatty tissue different expression gene plin promoter region of separating clone pig, and it is carried out to functional verification, to obtaining the higher startup activity of possessing of this gene and keeping the specific regulation and control section of fatty tissue.Overcome the deficiency of available tissue-specific promoter number in current animal genetic engineering.

The present invention separates and identifies the plin gene and has the specific promotor of fatty tissue from the genome of Large White.The poba gene group DNA of Large White of take has obtained 1691bp as template amplification, 1373bp, 1097bp, 780bp, 316bp, 461bp is the disappearance promoter fragment of totally 6 different lengthss, these segment composition reporter gene firefly luciferase genes (being called for short the LUC gene) are built to pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5, pGL3-basic-C46 carrier for expression of eukaryon, by these carriers difference transfections C2C12, PK and 3T3-L1 cell, relative reactivity by Dual-Luciferase reporting system analysis report gene Photinus pyralis LUC, keep the specific core promoter of fatty tissue district thereby obtain.

The present invention realizes by following technology:

Technological line of the present invention as shown in Figure 1.

Utilize BLASTn database (http://www.ncbi.nlm.nih.gov) search to obtain the upstream regulatory sequence of Adipose Tissue specific gene plin, its nucleotide sequence is as shown in SEQ ID NO:1.Utilize TESS, TFSEARCH and MethPrimer bioinformatics software forecast analysis core promoter zone, cis-trans functional element and CpG island distribution situation.According to the design 5 pairs of deletion-primers (its nucleotide sequence is in Table 1) that predict the outcome, utilize the PCR method to separate from the genome of Large White and obtain the fatty tissue specificity promoter.Build reporter gene LUC fusion vector, the applicant by its respectively called after pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, its fragment length of pGL3-basic-Q5 be respectively 1690bp, 1440bp, 1119bp, 780bp, 320bp, its nucleotide sequence is respectively shown in following SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.Transient transfection C2C12, PK and 3T3-L1 cell, the Dual-Luciferase reporting system detects finds that pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5 are only at the 3T3-L1 cells, and the expression amount in all the other two kinds of cells is with negative control pGL3-basic there was no significant difference.The activity of pGL3-basic-Q4 is greatly improved in 3T3-L1, with the activity of above-mentioned front 5 kinds of carriers, Comparatively speaking extremely significant difference is arranged, and in the PK cell, there is no considerable change.Result shows that the fragment intermediate sequence of pGL3-basic-Q4 possesses the function that independently starts reporter gene expression and also keeping the expression specificity of fatty tissue simultaneously.But the pGL3-basic-Q4 fragment has 780bp somewhat long as the core promoter zone, the associated transcription factor existed according to this section sequence lacks this section again, select the promoter region that 460bp is core to mean with pGL3-basic-C4, sequence SEQ ID NO:7.Transient transfection C2C12, PK and 3T3-L1 cell, the Dual-Luciferase reporting system detects to be found, in the 3T3-L1 cell, a large amount is expressed, higher than pGL3-basic-Q4 expression amount, so determine that pGL3-basic-C4 is that core starts zone.

Concrete steps of the present invention are as follows:

Select to possess the specific expressing gene plin of Adipose Tissue on the basis of forefathers' research, at the state-run biological study website NCBI(http of institute of the U.S.: //www.ncbi.nlm.nih.gov/) the BLASTn database, search obtains the upstream regulatory sequence of this gene, and its nucleotide sequence is as shown in SEQ ID NO:1.Utilize the bioinformatics softwares such as TESS, TFSEARCH and MethPrimer to predict its core promoter zone, cis-trans functional element and CpG island distribution situation to sequence SEQ ID NO:1.According to the consequence devised deletion-primers of neural network forecast, take the Large White genomic dna as template, pcr amplification obtains the promoter region of 6 different lengthss, and sequence is respectively as shown in sequence table SEQ ID NO:2-SEQ ID NO:7.These promotor candidate segment are loaded into to pGL3-basic reporter gene LUC upstream multiple clone site (information such as carrier figure and multiple clone site are shown in Fig. 2) and locate, be assembled into fusion expression vector (Fig. 3).By liposome-mediated infection protocol, by instantaneous C2C12, PK and the 3T3-L1 cell of proceeding to of fusion expression vector, proceed to renilla luciferase reporter gene pRL-TK is the internal reference plasmid simultaneously.The present invention arranges negative control pGL3-basic(Fig. 2), positive control pGL3-control.By the Dual-Luciferase reporting system, reporter gene LUC is carried out to quantitative analysis after transfection 48h, and then investigate the start-up performance of different deletion fragments in the different sources cell of this promotor of checking.Detected result shows that pGL3-basic-Q1, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5, pGL3-basic-C4 are only at the 3T3-L1 cells, and the expression amount in all the other two kinds of cells is with negative control pGL3-basic there was no significant difference.The activity of pGL3-basic-C4 is greatly improved in 3T3-L1, with above-mentioned the first six activity of planting carrier, Comparatively speaking extremely significant difference is arranged, and in the PK cell, there is no considerable change.Result shows that the fragment intermediate sequence of 460bp pGL3-basic-C4 possesses the function that independently starts reporter gene expression and also keeping the expression specificity of fatty tissue simultaneously.

The invention has the advantages that:

(1) the detailed checking of the present invention each section ability that promotor gene is expressed in the different sources cell of plin promotor, for the expression regulation of plin gene has brought deeper cognition.

(2) the present invention has identified the core section of the promotor plin of Adipose Tissue specifically expressing, for genetically engineered and molecular breeding provide the promotor resource of new specifically expressing.

More detailed technical scheme is referring to the content of " embodiment ".

The accompanying drawing explanation

Sequence table SEQ ID NO:1, disclose the upstream regulatory nucleotide sequence of the present invention clone's Adipose Tissue specific expression gene plin, and length is 2000bp.

Sequence table SEQ ID NO:2 is the nucleotide sequence that lacks promotor pGL3-basic-Q1 obtained from described SEQ ID NO:1 nucleotide sequence clone, and length is 1691bp.

Sequence table SEQ ID NO:3 is the nucleotide sequence that lacks promotor pGL3-basic-Q2 obtained from described SEQ ID NO:1 nucleotide sequence clone, and length is 1373bp.

Sequence table SEQ ID NO:4 is the nucleotide sequence that lacks promotor pGL3-basic-Q3 obtained from described SEQ ID NO:1 nucleotide sequence clone, and length is 1097bp.

Sequence table SEQ ID NO:5 is the nucleotide sequence that lacks promotor pGL3-basic-Q4 obtained from described SEQ ID NO:1 nucleotide sequence clone, and length is 780bp.

Sequence table SEQ ID NO:6 is the nucleotide sequence that lacks promotor pGL3-basic-Q5 obtained from described SEQ ID NO:1 nucleotide sequence clone, and length is 316bp.

Sequence table SEQ ID NO:7 is the nucleotide sequence that lacks promotor pGL3-basic – C4 obtained from described SEQ ID NO:1 nucleotide sequence clone, and length is 461bp.

Fig. 1: Technology Roadmap of the present invention.

Fig. 2: mean carrier pGL3-basic structural representation, comprise reporter gene luc+ below multiple clone site.

Fig. 3: what build take the fusion vector sketch that pGL3-basic is skeleton.The promotor series deletion fragment of fatty tissue specific expression gene plin drives LUC genetic expression, and the deleted promoter in figure means.Amp is ammonia benzyl resistance screening gene; Luc+ is the reporter gene Photinus pyralis LUC; MCS means multiple clone site; The direction of arrow means the direction of gene or promoter expression.

Fig. 4: the Kpn I of fusion vector pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5, pGL3-basic-C4 and Nhe I double digestion are identified figure.M means DL marker2000,1-6 means respectively the double digestion result of fusion vector pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5, pGL3-basic-C4, the band that the above is larger is carrier ribbon, lower end is disappearance promotor band, and band length is respectively 1691bp, 1373bp, 1097bp, 780bp, 316bp, 461bp.

Fig. 5: by the expression of series disappearance promoters driven LUC gene in the cell of different sources of Adipose Tissue specific expression gene plin, wherein Fig. 5 A is pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5 double digestion figure; Fig. 5 B is pGL3-basic-C4 double digestion figure.

Embodiment

Embodiment 1: the promotor candidate segment of the fatty tissue different expression gene plin of the extraction of Large White genomic dna and pig and the acquisition of corresponding deletion fragment

1, the extraction of Large White genomic dna:

By Large White that adopt at scene (from Hua Zhong Agriculture University's elaboration pig farm, kind for the routine report, lower with) to add the 0.5M ethylenediamine tetraacetic acid (EDTA) (be EDTA to fresh blood, purchased from river, Wuhan City profit fine chemistry industry limited liability company) as antithrombotics (press blood flow volume volume ratio 1/10), and shake up anti-hemostasis-coagulation.

White corpuscle separates:

(1) 4 ℃, 6000rpm, centrifugal 10min, serum deprivation.

(2) add 2 times of volume distilled waters, shake up gently precipitation 10min, broken red blood cell.

(3) 4 ℃, 6000rpm, centrifugal 10min, remove upper strata red corpuscle slurry.

(4) utilize the 0.9%NaCl washing precipitation, shake up gently 10min.

(5) 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant, obtains the white corpuscle precipitation.

Total DNA extraction:

(1) in white corpuscle precipitation or muscle tissue grind slurries, add equal-volume 1 * SET(1ml), Proteinase K (10mg/mL) is to final concentration 100 μ g/mL, and 10% sodium lauryl sulphate (SDS) is to final concentration 0.5%, shake up, digestion is incubated overnight in 55 ℃ of shaking baths.

(2) add the saturated phenol of isopyknic Tris in Digestive system, shake up gently 15min, in 4 ℃, the centrifugal 10min of 11000rpm, carefully draw supernatant liquor and be transferred in another centrifuge tube, note putting on corresponding mark.

(3) add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25:24:1), slowly put upside down centrifuge tube 10min, in the cryogenic freezing whizzer, 4 ℃, the centrifugal 10min of 11000rpm, carefully draw supernatant, is transferred in another clean centrifuge tube.

(4) add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24:1), slowly put upside down centrifuge tube 10min, 4 ℃, the centrifugal 10min of 11000rpm in the cryogenic freezing whizzer.

(5) supernatant liquor is sucked mark good centrifuge tube in, add the precooling dehydrated alcohol of 2 times of volumes, carefully mix the cotton-shaped DNA precipitation of rear visible white.

(6) with the rifle head, the DNA precipitation is chosen, be placed in the EP pipe that corresponding number is housed, under room temperature, allow the ethanol volatilization clean, add appropriate ultrapure water dissolving DNA.

(7) measure its concentration and purity on DNA concentration determination instrument, and, at 80 volts of about 2h of electrophoresis of 1% sepharose, detect the DNA extraction quality under ultraviolet lamp.

2, the acquisition of the fatty tissue different expression gene plin deletion fragment of pig

The DNA sequence dna of the plin gene of the pig of utilization report (the GenBank accession number: 654411) be probe sequence, at NCBI(http: //www.ncbi.nlm.nih.gov/) select the sequence of First Exon ATG upstream 2000bp as the promotor candidate segment.Utilize the bioinformatics softwares such as TESS, TFSEARCH and MethPrimer to predict core promoter zone, cis-trans functional element and the CpG island distribution situation of this section candidate sequence.According to the consequence devised deletion-primers of neural network forecast, (primer sequence is as shown in table 1; The PCR reaction parameter arranges as shown in table 2).Wherein P1F/R, P2F/R, P3F/R, P4FR, P5F/R, P6F/R represent forward direction primer (forward primer) and the backward primer (reverse primer) of 65 ' end deleted carriers of structure successively, the forward direction primer adds restriction enzyme site Nhe I (GCTAGC, with underscore, mean), carry protectiveness base CTA(shadow representation) simultaneously; The restriction enzyme site that backward primer adds is Kpn I (GGTACC means with underscore), carries protectiveness base CGG(simultaneously and means with shadow zone); By carrier difference called after TA-Q1, TA-Q2, TA-Q3, TA-Q4, TA-Q5, TA-Q6.

Table 1: the design of promoter deletion carrier primer

Table 2PCR Amplification

Embodiment 2: the promotor candidate segment of the fatty tissue different expression gene plin of pig and the transfection carrier of corresponding deletion fragment build.

The Adipose Tissue different expression gene plin deletion fragment that embodiment 1 is obtained is connected with carrier pGL3-basic, enzyme is cut TA cloned plasmids TA-Q1, TA-Q2, TA-Q3, TA-Q4, TA-Q5, the TA-Q6 of carrier (carrier pGL3-basic, purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd) and deletion fragment.See Fig. 2 by the information such as multiple clone site of Kpn I, Nhe I double digestion pGL3-basic and TA-Q1, TA-Q2, TA-Q3, TA-Q4, TA-Q5, TA-Q6(carrier pGL3-basic).It is as shown in table 3 that enzyme is cut system:

Table 3 enzyme is cut system

37 ° of C enzymes are cut the integrity of cutting with 1.5% agarose gel electrophoresis detection enzyme after 2h and are reclaimed purpose fragment: pGL3-basic and reclaim larger fragment, and TA-Q1, TA-Q2, TA-Q3, TA-Q4, TA-Q5, TA-Q6 reclaim the deletion fragment of corresponding size.The glue of producing with Shanghai Lay maple biological gene technology limited liability company reclaims test kit and reclaims (by the specification sheets operation of this test kit), is placed in-20 ℃ of Refrigerator stores.

(2) deletion fragment enzyme cut back to close is connected to (see figure 3) on the pGL3-basic carrier

Linked system is as shown in table 4:

Table 4 linked system

After 16 ° of C water-baths connect 12h, it is proceeded in bacillus coli DH 5 alpha, containing screening positive monoclonal on the LB flat board of penbritin (Amp), recon is carried out to PCR and enzyme is cut evaluation, positive recombinant is chosen and sent Beijing AudioCodes prosperous bio tech ltd order-checking.For the correct positive colony of order-checking in original bacteria liquid: the ratio of culture volume ratio=1:100 is carried out enlarged culturing, 37 ° of C shaking tables shake plasmid a small amount of extraction agent box (D6950-01) the extracting plasmid that can be used for cell transfecting of producing with OMEGA company after 16h, called after pGL3-basic-Q1, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5, pGL3-basic-C4 respectively.Identify the plasmid of extracting by Kpn I, Nhe I double digestion, enzyme is cut system as mentioned above, and now plasmid only need add 1 μ L enzyme and cuts and the results are shown in Figure 4.With

Nucleic acid-determination of protein concentration instrument (U.S. Beckman company product) is measured the concentration of plasmid, is placed in-20 ℃ of refrigerators standby.

Embodiment 3: liposome-mediated cell transfecting

The present invention does transfection with 24 porocyte culture dish and uses.In order to eliminate the error of experiment, each recombinant vectors all carries out three-wheel independently to be tested, and three multiple holes are done in each test.According to Lipofectamine ^TM2000(invitrogen company) specification sheets, every hole is in the quality of carrier: the ratio transfection of the volume of liposome=1 μ g:3 μ L.The cell that the acceptor of transfection is different sources, mainly comprise that the Mouse Muscle source cell is C2C12, porcine kidney cell PK and 3T3-L1 cell.Recombinant vectors is transfected into after cell 48h and after the collecting cell lysate, utilizes the Dual-Luciferase detection system to be analyzed the activity of disappearance promotor.Being formulated as follows of cell cultures of the present invention, the key step of inducing differentiation, transfection and various solution is described:

(1) preparation of main solution

1) cell growth medium (the foetal calf serum substratum FBS of 10% concentration): foetal calf serum (purchased from GIBCO BRL company) is positioned over to 4 ℃ of refrigerators and melts, after it melts fully, draw the foetal calf serum filtered of 100mL with the syringe of 50mL, add 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates, DMEM/F12 substratum (purchased from invitrogen company), be settled to 1L, it is standby that packing is placed in 4 ℃ of refrigerators.

2) cell dissociation buffer: pancreatin 2.5g, Na ₂HPO ₄1.15g, KCl 0.2g, NaCl 8.0g, KH ₂PO ₄0.2g, be dissolved in the 900mL distilled water, until completely dissolved, be settled to 1L, 0.22 μ m membrane filtration degerming, be placed in after packing in-20 ℃ of refrigerators standby.

3) Na of balanced salt solution PBS:2.89g ₂HPO ₄, the KCl of 0.2g, the K of 0.2g ₂HPO ₄, 8g NaCl adds the 800mL distilled water, after fully dissolving, is settled to 1L, after autoclaving, room temperature is placed stand-by.

4) cells frozen storing liquid: draw respectively the DMEM(of the foetal calf serum substratum (FBS) of dimethyl sulfoxide (DMSO) (DMSO), 6.00mL of 1.50mL and 7.50mL purchased from invitrogen company), after fully mixing, be placed in-20 ℃ of refrigerators with the amount packing of every pipe 1.50mL standby.

(2) cell cultures

1) change in time nutrient solution according to the upgrowth situation of cell: should change nutrient solution when the cell culture fluid flavescence, the nutrient solution that sucking-off is old, add fresh nutrient solution, is generally that 24h changes once.

2) after the degree of converging of Growth of Cells to 80%, gone down to posterity, the substratum that the suction pipe sucking-off is old, rinse twice with 1 * phosphoric acid buffer (PBS) (purchased from invitrogen company).

3) in advance 0.25% trypsinase is placed in to 37 ℃ of incubator temperature and bathes several minutes to reduce the injury to cell.50cm ²Tissue Culture Flask adds 1mL Digestive system (being purchased from invitrogen company) to get final product, and the cell bottle of front and back rotation fast, so that trypsinase can cover all cells, then is placed in the about 1min of incubator, accelerates tryptic digestion.

4) treat that cell attachment is loosening, in culturing bottle, add the FBS cell growth medium (purchased from the biological company limited of Hangzhou folium ilicis chinensis) of 10% fresh concentration to stop tryptic effect fast.

5) repeatedly blow and beat a bottle parietal cell with suction pipe, make it bottle and fully from wall, break away from and form cell suspension.

6) cell suspension of each sucking-off 1/3 moves in new cell bottle, supplements new cell growth medium, makes every bottle of final volume reach 4mL.

(3) liposome-mediated cell transfecting step

1) transfection the day before yesterday, get cell that one bottle of upgrowth situation is good with after tryptic digestion, add 2mL fresh not containing any antibiotic 10% FBS Growth of Cells nutrient solution, after with suction pipe, cell being dispelled form uniform cell suspension fully, with cell counting count board, cell is counted.Calculating 24 porocyte culture plates needs altogether cell concentration (every porocyte number is about 0.5-2 * 10 ⁵), the suspension that then sucking-off needs altogether is extra fresh not containing any antibiotic 10% FBS Growth of Cells nutrient solution (cumulative volume 12mL) to adding in it, after piping and druming evenly, every hole packing 500 μ L cell suspensions, rock gently culture plate and shake up cell and be placed on 37 ℃, 5%CO ₂Cultivate 24h in cell culture incubator.

2) treat the cell of transfection for every hole: according to the concentration for the treatment of the transfection plasmid and every hole, treat that the amount 1 μ g of transfection plasmid gets appropriate plasmid and joins 50 μ LOPTI-MEM nutrient solutions (purchased from invitrogen company, specification sheets operation by this reagent), in, piping and druming mixes the standing 5min of rear room temperature gently; With plasmid amount: Lipofectamine ^TMIn the situation that 2000 volume is 1:2.5, get the Lipofectamine of 2.5 μ L ^TM2000 join in 50 μ LOPTI-MEM nutrient solutions, mix the standing 5min of rear room temperature.

3) by step 2) in two portions of mixed solutions in 30min, mix, the standing 20min of room temperature.

4) abandon original fluid, clean cell twice with 1 * PBS or OPTI-MEM nutrient solution, and scavenging solution is exhausted.

5) every porocyte adds 3) in to supply OPTI-MEM nutrient solution to cumulative volume after mixed solution 100 μ L be 500 μ L, with the sway,postural culture plate of right-angled intersection all around to mix.

6) 37 ℃, 5%CO ₂Cell culture incubator is replaced with the FBS Growth of Cells nutrient solution that does not contain any antibiotic 10% after cultivating 6h

Embodiment 4: this experiment of detection of the promotor candidate segment of Adipose Tissue different expression gene plin and two luciferase activity of corresponding deletion fragment is executed example negative control plasmid pGL3-basic is set, positive control plasmid pGL3-control, with renilla luciferase reporter gene PRL-TK(carrier purchased from Pu Luomaige (Beijing) Bioisystech Co., Ltd, be U.S. Promega company) do internal reference plasmid (in order to proofread and correct transfection efficiency), with Lipofectamine ^TM2000 do transfection reagent, in the Tissue Culture Dish in 24 holes in the quality of every hole carrier: the ratio of the volume of liposome=1 μ g:3 μ L by the deleted carrier of structure respectively transient transfection Mouse Muscle source cell be C2C12, porcine kidney cell PK, mouse pre-adipose cell lines 3T3-L1(is called for short: 3T3-L1, purchased from Disease Prevention Control Center, Hubei Prov), dye and do contrast with idle running, collecting cell lysate after transfectional cell 48h, utilizing luciferase reporter gene to detect lifetime measurement system is analyzed activity and the tissue specificity of the different deletion fragments of promotor of Adipose Tissue different expression gene plin, determine high reactivity and possess the section of fatty tissue expression specificity.Result shows (as shown in Figure 5): in three kinds of different cells, the 6 deleted carrier pGL3-basic-Q1 that build, pGL3-basic-Q2, pGL3-basic-Q3, pGL3-basic-Q4, pGL3-basic-Q5, pGL3-basic-C4 compare and there is no obvious difference with the very low same negative control pGL3-basic of activity in 3T3-L1 at the PK cell, in the C2C12 cell, pGL3-basic-Q4, pGL3-basic-C4 activity are very high, and wherein the activity of pGL3-basic-C4 is the highest.Result of the present invention finally obtains the promoter fragment of 460bp, and (the recombinant expression vector pGL3-basic-C4 built, sequence information is shown in SEQ ID NO:7, possesses independently start-up performance and has the fatty tissue specificity concurrently.

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Claims (4)

1. the promotor of a regulation and control Adipose Tissue specific expression gene plin, its nucleotide sequence is as shown in sequence table SEQ ID NO:5.

2. the promotor of a regulation and control Adipose Tissue specific expression gene plin, its nucleotide sequence is as shown in sequence table SEQ ID NO:7.

3. the application of the described promotor of claim 1 or 2 in swine improvement.

4. the application of claim 3, comprising the application in regulation and control Adipose Tissue specifically expressing.

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