CN102604947B - Porcine adipose tissue-specific chimeric promoters - Google Patents
Porcine adipose tissue-specific chimeric promoters Download PDFInfo
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Abstract
The invention belongs to the technical field of animal genetic engineering, and specifically relates to cloning, functional verification and application of two porcine adipose tissue-specific chimeric promoters. According to the invention, the activity of an adi-pro regulating element is verified, and different chimeric promoters containing a pCMVIE promoter and an SV40 enhancer are constructed through a chimeric method, so that the activity of the adi-pro regulating element is greatly improved and new specific expression promoter resources are provided for genetic engineering and molecular breeding. The nucleotide sequences of the promoters are respectively as shown in the sequence tables SEQ ID NO: 4 (sequence identity number: 4) and SEQ ID NO: 5.
Description
Technical field
The invention belongs to animal gene engineering technology field.Be specifically related to the structure of a pork fat organizing specific promotor, functional verification and application, and improve its transcriptional activity by the method for setting up chimeric promoters, be built into two chimeric promoters, but do not destroy its fatty tissue specificity simultaneously again.
Background technology
Promotor (promoter) is an integral part of gene, refers to one section thymus nucleic acid that gene is transcribed (deoxyribonuclein acid, DNA) sequence in genetics.Promotor can (identification such as transcription regulaton factor be also with it in conjunction with formation transcription initiation mixture zone for ribonuclein acid, RNA) polysaccharase, thereby initial gene is transcribed by Yeast Nucleic Acid.Promotor is usually located at upstream region of gene, and the initiation site that its controlling gene is transcribed and the degree of expression are that RNA polymerase is accurately effectively transcribed necessary
[1]The promotor high with RNA polymerase avidity, its initial frequency and efficient are all high, are also referred to as strong promoter.A typical promotor comprises CAAT-box and TATA-box, and they depend on identification and the binding site of the RNA polymerase of DNA respectively, generally is positioned at tens base places, transcription initiation site upstream.Usually have some special dna sequence dnas in the core promoter upstream, i.e. cis-acting elements (ciselement), thus transcription factor is with it in conjunction with activating or the transcribing of suppressor gene
[1]In case the RNA polymerase location also is combined on the promotor and can transcribes by promotor gene, therefore promotor is the critical elements of gene expression regulation, and the interaction of it and trans-acting factors such as RNA polymerase and other albumen cofactors is the essence of promoter regulation genetic transcription.
For foreign gene can be in mammalian cell high level expression, promotor then is a very The key factor.How matching the design promotor in Study on Transgenic Animal is the key link that determines that can foreign gene correctly express in recipient cell, generally for non-specific expressing gene, can only recombinate with it with composing type or broad spectrum type, and for different expression gene, selected promotor must have strict space-time effect specificity, as the histocyte specificity promoter, specificity promoter and inducing specific promotor etc. in period grows.Sometimes specific expressed for enhancing gene also must be designed the single type enhanser or compound enhanser matches.There are many reports to show, adopt increase promotor number can improve expression of exogenous gene efficient.Zhang Qianqian etc.
[2]The scholar combines the promoter sequence of specific high-efficiency expression in the skeletal muscle and cytomegalovirus (CMV) promotor and improves foreign gene widely at the expression efficiency of muscle tissue.Simultaneously enhanser also can strengthen the transcriptional activity of promotor as the far-end control region, and that wherein be studied description at first is SV40.The gene transcription regulation mechanism of animal and human viroid gene transcription regulation mechanism and higher eucaryotic cells roughly the same, the promotor of research virus provides model for understanding the regulation and control of eukaryotic gene group, the genetic transcription Principles of Regulation such as promotor of using virus make up all kinds of engineering carriers, can effectively improve transcribing and expression efficiency of goal gene.
It is an active the highest enhanser that people's CMV early gene enhanser and other virus enhancer such as simplexvirus, Rous sarcoma virus are compared with hepatitis b virus
[3]In many research trials, usually thereby cmv enhancer and promotor are made up the transcriptional activity that reaches enhancing gene
[4,5,6,7,8,9,10]The current research report
[11]From pig BAC storehouse, separate obtaining adiponectin gene 5 ' flanking region sequence, determine 3 transcription initiation sites through 5 '-RACE.In this test, by making up a series of deletion fragments, find that promoter region-1671-+263 is the service area of pig adiponectin genetic transcription activity, wherein-1671--1455 impels the adiponectin gene high expression necessary.Luciferase reporting carrier and EMSA analyze at-important cis-acting elements cAMP-responsive element (CRE) of 1150--1130 district existence, with cAMP-responsive element binding protein (CREB) combination, the result shows that CREB is the important regulating and controlling factor of pig adiponectin genetic transcription activity.But whether this segment can be used as the special high efficient expression starter of fatty tissue is not very clear.In the preparation process of transgenic animal, the promotor that need be able in specific tissue, efficiently express, and available promotor is seldom.Therefore this research is to choose the pig adiponectin gene of pig-1671-+263 zone, and it is chimeric respectively with cmv enhancer and SV40 enhanser, produces needed promotor thereby be transformed into suitable transgenosis.For follow-up transgenic animal provide material to the demand of fatty tissue specific promoter.
Summary of the invention
The objective of the invention is to determine pork fat tissue specificity controlling element adi-pro and verify its activity that by setting up the method for chimeric promoters, the final structure keeps its organizing specific, and can have the chimeric promoters of efficient transcriptional activity afterwards.In the present invention, the applicant has increased and has contained the 640bp fragment of CRE binding site before pig adiponectin gene first exon in the promoter region, with its called after adi-pro, make up the two luciferase reporting carriers of pGL3-Basic, with pRL-TK plasmid transient cotransfection differentiation mouse Preadipocyte In Vitro 3T3-L1 and mice skeletal cell C2C12 after 7 days, verify its activity.Having increased afterwards, (its sequence is seen sequence table SEQ ID NO:2 to pCMV IE promoter fragment, fragment length is 548bp) and SV40 enhanser fragment (its sequence is seen sequence table SEQ ID NO:3, fragment length is 237bp), make up with the adi-pro controlling element respectively, make up the pGL3-CMV-adi-pro promotor and (see sequence table SEQ ID NO:4, length is 1188bp) and pGL3-SVenh-adi-pro (see sequence table SEQ ID NO:5, length is 877bp) chimeric promoters, these two promotor segments are not broken up and break up 7 days 3T3-L1 and C2C12 respectively with pRL-TK plasmid transient cotransfection, finally obtain two fatty tissue specific chimeric promotors (its sequence is shown in SEQ ID NO:4 and 5) that activity is higher.
The present invention is achieved in that
Techniqueflow by Fig. 1, at first utilize PCR method to amplify adi-pro fragment (electrophoresis result as shown in Figure 2), its nucleotide sequence is shown in sequence table SEQ ID NO:1, and sequence length is 640bp, utilize after the order-checking American National biotechnology NCBI of information center (
Http:// blast.ncbi.nlm.nih.gov/Blast.cgi) carry out sequence alignment, determine that the sequence that increases is correct.Next, amplification has the adi-pro fragment of Kpn I and Mlu I restriction enzyme site, and with this sequence clone to the pMD-18T carrier (Figure 22).Use restriction enzyme Kpn I and Mlu I double digestion pMD-18T-adi-pro carrier (the double digestion electrophorogram as shown in Figure 3) and pGL3-Basic carrier (shown in Figure 23) subsequently.Adi-pro fragment and pGL3-Basic carrier that will enzyme cuts out from the pMD-18T-adi-pro carrier carry out ligation.With the two luciferase reporting carrier called after pGL3-adi-pro that build, whether successfully be implemented in this carrier (the double digestion electrophoresis result as shown in Figure 4) with restriction enzyme Kpn I and this carrier electrophoresis detection of Mlu I double digestion adi-pro fragment.Afterwards with pGL3-adi-pro and the differentiation of pRL-TK (shown in Figure 26) transient cotransfection 7 days mouse Preadipocyte In Vitro and mice skeletal sarcoplast (shown in Figure 5), measure fluorescent value by microplate reader, determine that with fluorescence ratio the activity whether the adi-pro controlling element shows will be higher than the activity that shows in the myocyte in the adipocyte of maturation.The two luciferase reporting carriers of the pGL3-adi-pro that will build and pRL-TK transient cotransfection do not break up and 7 days mouse Preadipocyte In Vitro of differentiation simultaneously, the changing conditions of observation adi-pro controlling element activity.Test-results shows that the activity that the adi-pro controlling element shows will be higher than the activity (shown in Figure 6) that shows in the myocyte in the adipocyte of maturation; Adi-pro controlling element activity in undifferentiated adipocyte is lower simultaneously, then shows higher activity (shown in Figure 7) in the adipocyte of maturation.
Second step was the pCMVIE promoter fragment (electrophoresis result as shown in Figure 9) that template amplification has Kpn I and Mlu I restriction enzyme site with carrier for expression of eukaryon pIRES2-EGFP (shown in Figure 25), and with this fragment called after CMV, sequence length is 548bp.With this sequence clone to the pMD-18T carrier, use restriction enzyme Kpn I and Mlu I double digestion pMD-18T-CMV and pGL3-Basic carrier (electrophoresis result as shown in figure 11) subsequently, the CMV fragment that will cut out from the pMD-18T-CMV carrier and linear pGL3-Basic carrier carry out ligation.With the two luciferase reporting carrier called after pGL3-CMV that build.Whether successfully be implemented in this carrier (electrophoresis result as shown in figure 12) with restriction enzyme Kpn I and this carrier electrophoresis detection of Mlu I double digestion CMV fragment; Amplification simultaneously has the adi-pro fragment (electrophoresis result as shown in figure 10) of Mlu I and Xho I restriction enzyme site, carries out ligation with the two luciferase reporting carriers that contain pCMV IE promotor.With the two luciferase reporting carrier called after pGL3-CMV-adi-pro that build.Whether successfully be implemented in this carrier (electrophoresis result as shown in figure 13) with restriction enzyme Mlu I and this carrier electrophoresis detection of Xho I double digestion adi-pro fragment, illustrate and successfully set up chimeric promoters CMV-adi-pro, its nucleotide sequence is shown in sequence table SEQ ID NO:4.
The 3rd step was the SV40 enhanser fragment (electrophoresis result as shown in figure 14) that template amplification has Kpn I and Mlu I restriction enzyme site with carrier for expression of eukaryon pGL3-Enhancer (shown in Figure 24), and with this fragment called after SVenh, fragment length is 237bp.With this sequence clone to the pMD-18T carrier, use restriction enzyme Kpn I and Mlu I double digestion pMD-18T-SVenh carrier (electrophoresis result as shown in figure 15) and two luciferase reporting carrier pGL3-CMV-adi-pro afterwards, the SVenh fragment that will cut out from the pMD-18T-SVenh carrier replaces the CMV fragment in the two luciferase reporting carriers of pGL3-CMV-adi-pro, with the two luciferase reporting carrier called after pGL3-SVenh-adi-pro that build, whether restriction enzyme Kpn I and this carrier electrophoresis detection of Mlu I double digestion SVenh fragment successfully are implemented in this carrier (the double digestion electrophoresis result as shown in figure 16), illustrate and successfully set up chimeric promoters SVenh-adi-pro, its nucleotide sequence is shown in sequence table SEQ ID NO:3.Use restriction enzyme Kpn I and Mlu I double digestion pMD-18T-adi-pro carrier and pGL3-Enhancer carrier for expression of eukaryon at last, adi-pro fragment and the pGL3-Enhancer carrier that cuts out carried out ligation.With the two luciferase reporting carrier called after pGL3-Enhancer-adi-pro that build, whether restriction enzyme Kpn I and this carrier electrophoresis detection of Mlu I double digestion adi-pro fragment successfully are implemented in this carrier (electrophoresis result as shown in Figure 8).
The 4th step, with pGL3-CMV-adi-pro carrier reporting system and pGL3-adi-pro carrier reporting system, as negative control, break up 7 day mouse Preadipocyte In Vitro and differentiation 7 day mice skeletal sarcoplast with pRL-TK plasmid transient cotransfection with pGL3-Basic.Transfection experiment finishes the back collecting cell, measures fluorescent value with microplate reader, observe CMV-adi-pro and whether show high reactivity, and the activity that shows in the adipocyte of maturation will be higher than the activity that shows in the myocyte.Afterwards with pGL3-CMV-adi-pro chimeric promoters carrier reporting system equally with pGL3-Basic as negative control, do not break up and break up 7 days mouse Preadipocyte In Vitro with pRL-TK plasmid transient cotransfection, record fluorescence ratio, the changing conditions of observation CMV-adi-pro chimeric promoters activity.Test-results: the activity of CMV-adi-pro chimeric promoters will be higher than the activity of non-chimeric promoters adi-pro, and the activity that shows in the adipocyte of maturation will be higher than the activity (shown in Figure 17) that shows in the myocyte; And CMV-adi-pro is active very low in the adipocyte of maturation then active higher (shown in Figure 180) in undifferentiated adipocyte.The result shows that chimeric promoters CMV-adi-pro not only makes the increased activity of single controlling element adi-pro also keep the histocyte specificity of adi-pro controlling element simultaneously.
The 5th step, with pGL3-SVenh-adi-pro chimeric promoters carrier reporting system and pGL3-Enhancer-adi-pro carrier reporting system, with pGL3-Enhancer as negative control, with 7 days mouse Preadipocyte In Vitro of pRL-TK plasmid transient cotransfection differentiation and 7 days mice skeletal sarcoplast of differentiation, whether the activity that shows by fluorescence ratio observation chimeric promoters SVenh-adi-pro will be higher than non-chimeric promoters, and whether the activity that the SVenh-adi-pro chimeric promoters shows in the adipocyte of maturation also is higher than the activity that shows in the myocyte.Next with pGL3-SVenh-adi-pro carrier reporting system equally with pGL3-Enhancer as negative control, do not break up and break up 7 days mouse Preadipocyte In Vitro with pRL-TK plasmid transient cotransfection, record fluorescence ratio, the changing conditions of observation SVenh-adi-pro chimeric promoters activity.Test-results: the activity of SVenh-adi-pro chimeric promoters will be higher than the activity of non-chimeric promoters, and the activity that shows in the adipocyte of maturation will be higher than the activity (shown in Figure 19) that shows in the myocyte; The activity that the SVenh-adi-pro chimeric promoters shows in undifferentiated adipocyte is lower, then shows greater activity (shown in Figure 20) in the adipocyte of maturation.Can draw equally: the SVenh-adi-pro chimeric promoters not only makes the increased activity of single controlling element adi-pro also keep the histocyte specificity of adi-pro controlling element simultaneously.
In the 6th step, with carrier reporting system pGL3-CMV-adi-pro and pGL3-SVenh-adi-pro, with 7 days mouse Preadipocyte In Vitro of pRL-TK plasmid transient cotransfection differentiation, by the fluorescence ratio that records, compare the activity of two different chimeric promoters.Test-results: in the adipocyte of maturation, the activity of CMV-adi-pro chimeric promoters is higher than the activity (shown in Figure 21) of SVenh-adi-pro chimeric promoters far away.
The invention has the advantages that:
(1) the detailed checking of the present invention the activity of adi-pro controlling element, made up the different chimeric promoters that contain pCMV IE promotor and SV40 enhanser by chimeric method, the activity of adi-pro controlling element is improved greatly.
(2) the present invention has obtained at mature fat cell active higherly and have two special chimeric promoters of histocyte, and the promotor resource of new specifically expressing is provided for genetically engineered and molecular breeding.
More detailed technical scheme is referring to the content of " embodiment ".
Description of drawings
Sequence table SEQ ID NO:1 chooses the adi-pro nucleotide sequence that contains the CRE binding site before pig adiponectin gene first exon in the promoter region among the present invention, its length is 640bp.
Sequence table SEQ ID NO:2 is the pCMVIE promoter fragment that increases among the present invention, and its length is 548bp.
Sequence table SEQ ID NO:3 is the SV40 enhanser fragment that increases among the present invention, and its length is 237bp.
Sequence table SEQ ID NO:4 is the nucleotide sequence of chimeric promoters CMV-adi-pro of the present invention, and its length is 1188bp.
Sequence table SEQ ID NO:5 is the nucleotide sequence of chimeric promoters SVenh-adi-pro of the present invention, and its length is 877bp.
Fig. 1: be technological line figure of the present invention.
Fig. 2: be to utilize Adi-F, the Adi-R primer adi-pro sequence electrophoresis detection figure that promoter region before pig adiponectin gene first exon contains the CRE binding site that chooses to increasing among the present invention.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Fig. 3: be the adi-pro gene fragment of utilizing Adi-F1, Adi-R1 primer amplification to go out among the present invention, this fragment is connected into the pMD-18T carrier, use the electrophoresis detection figure of restriction enzyme Kpn I and MluI double digestion pMD-18T carrier afterwards.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Fig. 4: be the adi-pro gene fragment that has Kpn I and Mlu I restriction enzyme site among the present invention with carry out ligation with restriction enzyme Kpn I and Mlu I double digestion pGL3-Basic and construct pGL3-adi-pro carrier reporting system after, the electrophoresis detection figure that carries out the evaluation of Kpn I and Mlu I double digestion.Among the figure: swimming lane M:DNA molecular criteria (DL10000ladder).
Fig. 5: be the cell picture that the undifferentiated mouse Preadipocyte In Vitro of cultivating respectively among the present invention, undifferentiated mice skeletal sarcoplast, 7 days mouse Preadipocyte In Vitro of differentiation and the mice skeletal sarcoplast that broke up 7 days are taken under inverted microscope.Among the figure: the cell magnification is 10 * 20.
Fig. 6: be the active comparative analysis of adi-pro controlling element in different cells among the present invention, namely 7 days mouse Preadipocyte In Vitro of differentiation and 7 days mice skeletal sarcoplast of differentiation.Among the figure: the negative contrast of pGL3-Basic, fluorescence ratio be the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Fig. 7: be that the adi-pro controlling element is not namely breaking up and 7 days mouse Preadipocyte In Vitro of differentiation with the activation analysis in a kind of cell different times among the present invention.Among the figure: fluorescence ratio is the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Fig. 8: be to have the adi-pro gene fragment of Kpn I and Mlu I restriction enzyme site and the electrophoresis detection figure that carries out with restriction enzyme Kpn I and Mlu I double digestion pGL3-Enhancer after pGL3-Enhancer-adi-pro carrier reporting system is constructed in ligation among the present invention.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Fig. 9: be the pCMVIE promoter sequence electrophoresis detection figure that utilizes CMV-F, CMV-R primer amplification to go out among the present invention.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 10: be the adi-pro sequence electrophoresis detection figure that utilizes Adi-F2, Adi-R2 primer amplification to go out among the present invention.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 11: be restriction enzyme Kpn I and Mlu I double digestion pGL3-Basic and the electrophoresis detection figure that is connected into the pMD-18T carrier that has Kpn I and Mlu I restriction enzyme site pCMV IE promotor among the present invention.Among the figure: swimming lane M:DNA molecular criteria (DL10000ladder), swimming lane 1 and 2: restriction enzyme Kpn I and Mlu I double digestion pGL3-Basic carrier; Swimming lane 3 and 4: the pMD-18T-CMV carrier of restriction enzyme Kpn I and Mlu I double digestion.
Figure 12: be the CMV fragment that has Kpn I and Mlu I restriction enzyme site among the present invention with carry out ligation with restriction enzyme Kpn I and Mlu I double digestion pGL3-Basic and construct pGL3-CMV-adi-pro carrier reporting system after, the electrophoresis detection figure that carries out the evaluation of Kpn I and Mlu I double digestion.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 13: be the adi-pro fragment that has Mlu I and Xho I restriction enzyme site among the present invention with carry out ligation with restriction enzyme Mlu I and Xho I double digestion pGL3-Basic and construct two luciferase carrier reporting systems after, the electrophoresis detection figure that carries out the evaluation of Mlu I and Xho I double digestion.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 14: be the SV40 enhancer sequence electrophoresis detection figure that utilizes SVenh-F, SVenh-R primer amplification to go out among the present invention.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 15: be the SVenh fragment of utilizing SVenh-F, SVenh-R primer amplification to go out among the present invention, this fragment is connected into the pMD-18T carrier, use the electrophoresis detection figure of restriction enzyme Kpn I and Mlu I double digestion pMD-18T-SVenh carrier afterwards.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 16: be the SVenh fragment that has Kpn I and Mlu I restriction enzyme site among the present invention with carry out ligation with restriction enzyme Kpn I and Mlu I double digestion pGL3-CMV-adi-pro and construct pGL3-SVenh-adi-pro carrier reporting system after, the electrophoresis detection figure that carries out the evaluation of Kpn I and Mlu I double digestion.Among the figure: swimming lane M:DNA molecular criteria (DL2000ladder).
Figure 17: be the active comparative analysis of CMV-adi-pro chimeric promoters in different cells among the present invention, namely 7 days mouse Preadipocyte In Vitro of differentiation and 7 days mice skeletal sarcoplast of differentiation, carry out specific activity with non-chimeric adi-pro controlling element simultaneously.Among the figure: the negative contrast of pGL3-Basic, fluorescence ratio be the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Figure 18: be among the present invention the CMV-adi-pro chimeric promoters in the activation analysis with a kind of cell different times, namely in the mouse Preadipocyte In Vitro of not breaking up and breaking up 7 days.Among the figure: the negative contrast of pGL3-Basic, fluorescence ratio be the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Figure 19: be the active comparative analysis of SVenh-adi-pro chimeric promoters in different cells among the present invention, namely 7 days mouse Preadipocyte In Vitro of differentiation and 7 days mice skeletal sarcoplast of differentiation, carry out specific activity with non-chimeric adi-pro controlling element simultaneously.Among the figure: the negative contrast of pGL3-Enhancer, fluorescence ratio be the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Figure 20: be among the present invention the SVenh-adi-pro chimeric promoters in the activation analysis with a kind of cell different times, namely in the mouse Preadipocyte In Vitro of not breaking up and breaking up 7 days.Among the figure: the negative contrast of pGL3-Enhancer, fluorescence ratio be the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Figure 21: be chimeric promoters CMV-adi-pro and the activation analysis of chimeric promoters SVenh-adi-pro in 7 days mouse Preadipocyte In Vitro of differentiation among the present invention.Among the figure: fluorescence ratio is the STDEV function calculation standard deviation of average usefulness excel spreadsheet lattice as a result.
Figure 22: be the T carrier structure synoptic diagram that is used for the clone among the present invention.
Figure 23: be for the two luciferase reporting carrier structure synoptic diagram of the pGL3-Basic that analyzes pig adi-pro controlling element and chimeric promoters CMV-adi-pro, SVenh-adi-pro activity among the present invention.
Figure 24: be the two luciferase reporting carrier structure synoptic diagram of pGL3-Enhancer that are used for amplification SVenh sequence among the present invention and analyze chimeric promoters SVenh-adi-pro activity.
Figure 25: be the pIRES2-EGFP carrier for expression of eukaryon structural representation that is used for amplification CMV promoter sequence among the present invention.
Figure 26: be to be used for analyzing pig adi-pro controlling element and the active used pRL-TK confidential reference items carrier structure synoptic diagram of chimeric promoters CMV-adi-pro, SVenh-adi-pro among the present invention.
Figure 27: be to be used for calculating the used STDEV function derivation formula figure of fluorescence ratio standard deviation among the present invention.
Figure 28: be the former sequence of first promotor of obtaining of the present invention, length is 1188bp.
Figure 29: be the former sequence of second promotor obtaining of the present invention, length is 877bp.
Embodiment
Embodiment 1: promoter region contains obtaining of CRE binding site sequence adi-pro before pork fat organizing specific expression gene adiponectin first exon.
1. main agents:
Phenol (Chemical Reagent Co., Ltd., Sinopharm Group), chloroform (Chemical Reagent Co., Ltd., Sinopharm Group), primary isoamyl alcohol (Chemical Reagent Co., Ltd., Sinopharm Group), plasmid extraction kit (available from OMEGA company), pMD-18T carrier (precious biotechnology Dalian company limited), LA Taq polysaccharase (precious biotechnology Dalian company limited), dNTP (available from Fermentas company), DL-2000Marker (available from Fermentas company), UNIQ-10 pillar DNA glue reclaims test kit (available from Beijing hundred Tyke Bioisystech Co., Ltd)
2. the preparation of main solution:
(1) blood sample is gathered the preparation of related solution
1M Tris.cl storing solution: take by weighing 121.4g Tris alkali and be dissolved in the 800mL distilled water, add concentrated hydrochloric acid and transfer to pH=8.0, be settled to 1000mL.Autoclaving, room temperature preservation.
0.5MEDTA (pH=8.0): take by weighing 186.1gNa2EDTA2H
2O is dissolved in the 800mL distilled water, adds NaOH (about 20g) and transfers to pH=8.0, is settled to 1000mL.Autoclaving, room temperature preservation.
(2) preparation of extracting genome DNA related solution
Proteinase K: the 200mg Proteinase K is dissolved in the 10mL distilled water, with the 2mL packing, and-20 ℃ of freezing preservations.
The saturation balance phenol solution: commercially available phenol is through 180 ℃ of vapor enrichments, adds oxine to final concentration 0.1% after being chilled to room temperature, adds isopyknic 0.5mol/L TrisCl (pH8.0), fully shakes mixing.Standing demix is removed the upper strata damping fluid, adds isopyknic 0.1mol/L TrisHCl (pH8.0), shakes 20min, inhales and removes the upper strata water, repeats to reach more than 7.9 until phenol phase pH value, places brown bottle, room temperature preservation.Chloroform/primary isoamyl alcohol (volume ratio 24: 1): measure the 480mL chloroform, add the 20mL primary isoamyl alcohol, mixing.
10 * SET damping fluid: 15m mol/L TrisCl (pH8.0), 15m mol/L EDTA, 5mmol/L sodium-chlor.
10%SDS: take by weighing the 50gSDS heating and be dissolved in the 400mL distilled water, be settled to 500mL.Autoclaving, room temperature preservation.
TE damping fluid: 100mmol/L TrisCl (pH=8.0), 1mmol/L EDTA (pH=8.0).(3) preparation of agarose gel electrophoresis and detection related solution
50 * TAE storing solution: take by weighing 242gTris alkali and be dissolved in the 750mL distilled water, add 57.1mLHAC, 100mL 0.5M EDTA (pH=8.0) adds water and is settled to 1000mL.
5 * TBE storing solution: take by weighing 54gTris alkali and 27.5g boric acid and be dissolved in the 750mL distilled water, add 20mL 0.5M EDTA (pH=8.0), add water and be settled to 1000mL.
6 * sample-loading buffer: 0.25% bromjophenol blue.0.25% dimethylbenzene green grass or young crops, the 15%Ficoll ficoll.
Ethidium bromide (EB, 10mg/mL): add 0.1gEB in the 100mL distilled water, stir and make dissolving fully, change brown reagent bottle over to, the tinfoil parcel.
(4) preparation of related solution
LB liquid nutrient medium: get microbial culture Tryptones 10g, microbial culture yeast extract 5g, NaCl 5g is dissolved in the 950mL distilled water, shaking container dissolves fully until solute, regulate pH value to 7.0 with 5mol/L NaOH (about 0.2mL), add water and be settled to 1000mL, autoclaving, room temperature preservation.
The LB solid medium: obtaining liq substratum as stated above, every 100mL adds the 1.5g agar powder, and the 30min that sterilizes under 121 ℃ of high pressure steam at sterile state lower berth flat board, seals standby after the cooling.
0.1mol/L CaCl
2Solution: get the anhydrous CaCl of 1.1g
2Be dissolved in the 90mL distilled water, add distilled water and be settled to 100mL, the 30min that sterilizes under 121 ℃ of high pressure steam preserves under room temperature.
3. tissue sample: collect the blood sample of " Large White " (be foreign pig blood relationship) and " plum mountain pig " (for the Chinese Pigs blood relationship), utilize conventional phenol/chloroform extraction method extraction pig genomic dna, detailed step is as follows:
4. the extraction of pig genomic dna:
(1) every pig precaval vein is taken a blood sample about 20mL, adds one and is equipped with in advance in the 50mL centrifuge tube of 1mL antithrombotics (0.5mol/LEDTA).
(2) use high speed freezing centrifuge 3000rpm, 4 ℃ of centrifugal 10min abandon upper serum.
(3) add the distilled water of 1.5 times of volumes, jog 10min, broken red blood cell.
(4) 5000rpm, 4 ℃ of centrifugal 10min discard upper strata red corpuscle slurry.
(5) add 20mL 0.9%NACl solution washing, 5000rpm, 4 ℃ of centrifugal 10min, supernatant discarded obtains the white corpuscle precipitation.
(6) add 1 * SET damping fluid suspension white corpuscle, add Proteinase K (10mg/L) to concentration 100 μ g/mL, add 10%SDS again and spend the night to 0.5%, 55 ℃ of digestion of final concentration.
(7) add isopyknic saturated phenol, cover tightly the pipe lid, the about 20min of mixing that turns upside down gently, 5000rpm, 4 ℃ of centrifugal 10min.
(8) draw supernatant liquor with the heavy caliber suction nozzle of cutting sharp mouth, move into new 50mL centrifuge tube, discard lower floor's phenol.
(9) repeat above-mentioned steps 7,8 twice.
(10) add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) mixed solution, reverse centrifuge tube gently, mixing 15min, 1000rpm, 4 ℃ of centrifugal 15min collect supernatant according to step 8 again.
(11) add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1), the same extracting more once.
(12) dehydrated alcohol of 2.5 times of volume precoolings of adding shakes centrifuge tube gently, namely has DNA to be cotton-shaped and separates out.
(13) with the cotton-shaped DNA of pasteur pipet sucking-off in the centrifuge tube of a new clean 1.5mL, with 70% washing with alcohol once, the centrifugal 5min of 3000rpm discards upper strata ethanol.
(14) with centrifuge tube in the vacuum-drying pump 37 ℃ vacuumize dry DNA, add 300 μ L TE dissolving DNAs, in 4 ℃ of preservations.
5. the concentration of pig genomic dna is identified and quality examination:
Utilize the DNA/RNA concentration determination instrument of Pharmacia company to measure DNA concentration, according to size and the extension rate of concentration, be converted into DNA concentration to be measured, and the DNA in the pipe is done corresponding dilution, with 50ng/ μ L packing ,-20 ℃ of preservations.According to the A260/A280 ratio of record, estimation DNA quality, general A260/A280 is between 1.6-1.8, and protein content can satisfy the requirement of this test when being zero.
6.adi-pro the amplification of fragment:
Utilize the NCBI website (
Http:// www.ncbi.nlm.nih.gov/nuccore/FJ461661.1) get access to the sequence information of promotor before pig adiponectin gene first exon, GenBank accession number: FJ461661.1.Choose the promoter sequence that wherein contains the CRE binding site, called after adi-pro.Genomic dna with Large White and Mei Shan pig is template, utilizes that primer Adi-F and Adi-R carry out pcr amplification in the table 1.The PCR reaction totally is 25 μ L:0.5 μ L dNTP, 0.5 μ L special primer, and 0.25 μ L LA Taq enzyme, 2.5 μ L Buffer, 300-500ng genomic dna template adds deionized water to 25 μ L.
PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 45s, 59.5 ℃ of annealing 40s, 72 ℃ of 45s amount to 35 circulations; Last 72 ℃ are extended 10min.
The recovery of PCR product and purifying: the PCR product is after 1.5% agarose gel electrophoresis detects, the gel that will contain the purpose fragment under ultraviolet lamp from the sepharose downcuts, put into 1.5mL Ependorff pipe, reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd) purified pcr product with UNIQ-10 pillar DNA glue then.
Ligation: the PCR product of purifying is connected with the pMD18-T carrier, and the ligation cumulative volume is 10 μ l, comprising 1.5 μ l2 * ligationbuffer, and 0.3 μ l carrier, 5 μ l purified pcr products, 4 ℃ of connections of spending the night.
Transform: get 50～100 μ l competent cells under the sterile state in 1.5ml Ependorff pipe, the connection product of 5～10 μ l is added mixing gently, place 42 ℃ of heat shock 90s behind the 30min on ice, do not shake the Ependorff pipe therebetween, take out back ice bath 2～3min rapidly, the LB nutrient solution that adds 400 μ l antibiotic-frees, 37 ℃, the 220rpm/min temperature is bathed recovery 45-60min.The centrifugal 6min of 6000rpm/min removes an amount of supernatant then, with remaining thalline and substratum mixing gently, contain on the flat board of 100 μ g/mL penbritins (Amp), smoothen, just putting to cultivate to be inverted behind the 1h for 37 ℃ and cultivating, observe after 14-16 hour and have or not bacterium colony to grow.
The evaluation of positive colony and order-checking: the picking mono-clonal inserts 1.5mL to contain 500 μ L penbritin (Amp) concentration is the LB nutrient solution of 100 μ g/mL from flat board, and cultivates about about 4-6h in 37 ℃ of shaking table 220rpm/min.Be template with bacterium liquid, select for use Auele Specific Primer Adi-F and Adi-R (shown in the table 1) to carry out pcr amplification.Electrophoresis detection, the bacterium liquid of picking positive colony is sent to order-checking, and sequencing is finished by Beijing AudioCodes biotechnology limited liability company.Sequencing result is shown as correct adi-pro fragment sequence (seeing that shown in the SEQ ID NO:1, sequence length is 640bp).
Embodiment 2: the structure of controlling element adi-pro transfection carrier
1. main agents
DL10000Marker, the T4DNA ligase enzyme is available from precious biotechnology Dalian company limited; Restriction enzyme KpnI, Mlu I is available from NEB company; Plasmid a small amount of rapid extraction test kit is available from the white biotech firm in Yuanping City; The little extraction reagent kit of Endo-free Plasmid Mini KitII plasmid is available from OMEGA company; The pGL3-Basic carrier for expression of eukaryon is available from Promega company
2. two luciferase reporting carrier pGL3-Basic double digestions
With Kpn I, Mlu I double digestion pGL3-Basic carrier, double digestion system 20 μ l:2 μ l Buffer 2,0.5 μ l Kpn I, 0.5 μ l Mlu I, 1 μ lpGL3-Basic carrier, 0.2 μ l BSA mends sterilization ultrapure water to 20 μ l.Enzyme is cut 4-6h.Enzyme is cut product and is reclaimed test kit (available from Beijing hundred Tyke Bioisystech Co., Ltd) recovery purifying with UNIQ-10 pillar DNA glue.
3. the TA cloning vector double digestion that contains the adi-pro fragment
Utilize that primer Adi-F1 and Adi-R1 amplify the adi-pro fragment that has Kpn I and Mlu I restriction enzyme site in the table 1, be connected with the pMD-18T carrier.With Kpn I, Mlu I double digestion pMD-18T-adi-pro carrier, double digestion system 20 μ l:2 μ l Buffer 2,0.5 μ l KpnI, 0.5 μ lMlu I, 6 μ l contain the TA cloning vector of purpose fragment, and 0.2 μ l BSA mends sterilization ultrapure water to 20 μ l.Enzyme is cut 4-6h.Enzyme is cut product and is reclaimed test kit (available from Beijing hundred Tyke Bioisystech Co., Ltd) recovery purifying with UNIQ-10 pillar DNA glue.
3. the structure of recombinant vectors
Adi-pro fragment after double digestion reclaimed is connected on the pGL3-Basic carrier behind the double digestion, and constructed carrier is called after pGL3-adi-pro respectively.Linked system 10 μ l:1 μ l 10 * Buffer, 0.5 μ l 5U/ μ l T4 ligase, the pGL3-Basic carrier of 2 μ l double digestions, 6 μ l adi-pro fragments are mended sterilization ultrapure water to 10 μ l.16 ℃ of connections of spending the night.Connect product then and be transformed into bacillus coli DH 5 alpha.Utilize PCR to detect positive colony, positive colony send Beijing AudioCodes biotechnology limited liability company to check order.Sequence is carried out enlarged culturing to positive colony after order-checking is confirmed correctly, and the extracting plasmid.The plasmid extraction kit that plasmid extracts with OMEGA company extracts, plasmid purity and concentration purity utilize NanoDrop2000 Spectrophotometer (U.S. NanoDrop company) to detect, and the OD260/280 value of the sample of surveying just can be used for follow-up experiment at 1.8-2.0.In addition, the plasmid that extracts carries out the double digestion evaluation again, and the double digestion qualification result as shown in Figure 4.The double digestion qualification result shows that constructed recombinant vectors is correct.
The activation analysis of embodiment 3:adi-pro controlling element
1. main agents:
Foetal calf serum (FBS is available from U.S. GIBCO company); Horse serum (HBS is available from U.S. GIBCO company); DMEM high glucose medium (available from U.S. GIBCO company); Liposome Lipofectamine2000 (available from invitrogen company); Dual-Luciferase Reporter AssaySystem (available from Promega company)
2. main consumptive material: disposable filter, the disposable aspiration needle syringe in cell cultures 24 orifice plates (U.S. Corning company), Tissue Culture Flask, disposable plastic transfer pipet, 0.22 μ m aperture, seal film (Parafilm company), rubber gloves, enzyme plate (U.S. Corning company)
3. the preparation of main agents:
(1) 1 * PBS preparation: take by weighing NaCl 8g, KCl 0.2g, Na
2HPO
412H
2O 1.54g, KH
2PO
40.19g be dissolved in the 1000mL distilled water, regulate pH to 7.0, autoclaving.
(2) oil red O dye liquor: take by weighing 0.5g oil red O (available from Amersco company) and add the 100mL Virahol and leave standstill filter paper filtering behind the 8-10min, according to oil red O dye liquor and 3: 2 dilution proportion oil red O dye liquor of deionized water, filter paper filtering 2 times, room temperature is placed 10min, places brown bottle to keep in Dark Place for 4 ℃.
(3) preparation of cells frozen storing liquid: add 4mL sterilization foetal calf serum in the sterile chamber, the serum free medium after 14mL filters, 2mLDMSO (available from Sigma company), the mixing lucifuge is sub-packed in the 1.5mL centrifuge tube ,-20 ℃ of preservations.
The preparation of (4) 0.25% pancreatin solution: transfer to pH to 8.0, filtration sterilization, packing 200 μ L in the 1.5mL centrifuge tube ,-20 ℃ of preservations.
The high sugared DMEM culture medium preparation of (5) 10% foetal calf serums: 50mL specification syringe is drawn the DMEM high glucose medium, adds the 10mL foetal calf serum in every 100mLDMEM high glucose medium, and 0.22 μ M strainer carries out filtration sterilization, seals 4 ℃ of preservations.
The high sugared DMEM culture medium preparation of (6) 2% horse serums: 50mL specification syringe is drawn the DMEM high glucose medium, adds the 2mL horse serum in every 100mL DMEM high glucose medium, and 0.22 μ M strainer carries out filtration sterilization, seals 4 ℃ of preservations.
(7) preparation of Regular Insulin (Insulin) storage liquid: take by weighing 25mg Regular Insulin (available from Sigma company), add the dilute hydrochloric acid (pH2-3) of 12.5mL, the preparation final concentration is the Regular Insulin storage liquid of 1 μ g/mL.
(8) preparation of IBMX (1-methyl-3-isopropylcarbinol xanthine) storage liquid: take by weighing 100mg IBMX (available from Sigma company), add 900 μ LDMSO, the preparation final concentration is the IBMX storage liquid of 0.5mol/L.
(9) preparation of dexamethasone (Dexamethasone) storage liquid: take by weighing 25mg dexamethasone (available from Sigma company), add the 6.37mL dehydrated alcohol, the preparation final concentration is the dexamethasone storage liquid of 1 μ mol/L.
4. cell cultures
Can be to the cell cultivation of going down to posterity when the cell degree of converging of mouse Preadipocyte In Vitro 3T3-L1 and mice skeletal sarcoplast C2C12 reaches 70%-80%.At first draw old substratum in the Tissue Culture Flask, with 1 * PBS flushing 2-3 time, add 0.25% trypsin Trypsin, available from U.S. GIBCO company) 200-300 μ L, trypsinase is fully contacted with attached cell, place 37 ℃ of incubators to digest about 30s, discard trypsinase, add new foetal calf serum high glucose medium, fully blow and beat mixing with suction pipe, make it to become single cell suspension, be inoculated into afterwards in the Tissue Culture Flask, put into CO2gas incubator and continue to cultivate.Can change the nutrient solution suction behind the 24h and abandon not adherent cell.Cultivate afterwards and can change liquid once (undifferentiated cell is seen accompanying drawing 5 undifferentiated mouse Preadipocyte In Vitro and undifferentiated mice skeletal sarcoplast) in every 1-2 days.
5. cell induces differentiation culture
(1) the 3T3-L1 cell induces differentiation culture
From cell culture incubator, take out the form of observation of cell and upgrowth situation under inverted microscope with being inoculated in the cell that 24 orifice plates growths 24h degree of converging reaches 70%-80%.Each orifice plate cell count reaches 8 * 10
4-1 * 10
5With the sucking-off of foetal calf serum high glucose medium, afterwards inducing culture is added according to every hole 600 μ L.Wherein inducing culture is: add 0.5 μ L Regular Insulin in every 1mL foetal calf serum high glucose medium, and 1 μ L IBMX, 1 μ L dexamethasone is rare.In division culture medium, induce differentiation after 2 days cell volume begin to become big change circle, pimple appears in the part, be changed to foetal calf serum high glucose medium with cell culture medium this moment, continues at 5%CO
2Cultivate in the incubator.Induce differentiation after 7 days at mouse Preadipocyte In Vitro 3T3-L1, carry out the transfection test.Once changed liquid in about every 1-2 days during this time.
(2) oil red O stain
At first inhale and abandon original nutrient solution, with PBS damping fluid (being purchased) washing 2-3 time, add fixedly 10min of 10% neutral formalin.Add oil red O dye liquor afterwards and dye, suction discards staining fluid behind the dyeing 10min, places microscopically to observe, and in time takes pictures, if dyeing time is long, fat drips can spontaneous breaking (dyeing back fat drips and sees 7 days mouse Preadipocyte In Vitro of accompanying drawing 5 differentiation).
(3) the C2C12 cell induces differentiation culture
Take out from cell culture incubator being inoculated in the cell that 24 orifice plates growths 24h degree of converging reaches 70%-80%, place observation of cell form and growing state under the inverted microscope.Each orifice plate cell count reaches 8 * 10
4-1 * 10
52% old horse serum high glucose medium is abandoned in suction, changes 2% fresh horse serum high glucose medium into, places 5%CO
2Continue in the incubator to cultivate.Induce differentiation after 6-7 days the mice skeletal sarcoplast, carry out the transfection test.Once changed liquid in about every 1-2 days during this time.
5. transient transfection test:
Select for use Lipofectamine 2000 to carry out the transient transfection test.Wherein select for use pRL-TK plasmid (Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega company) as reference, with the pGL3-adi-pro plasmid co-transfection.
(1) 2h before transfection on-test, (compound method: 50mL specification syringe is drawn the DMEM high glucose medium with the high sugared DMEM substratum of 10% foetal calf serum, add the 10mL foetal calf serum in every 100mLDMEM high glucose medium, 0.22 μ M strainer carries out filtration sterilization, seals 4 ℃ of preservations) cultivate.
Transfection is carried out according to 1 μ g plasmid transfection amount in the every hole of (2) 24 orifice plates.The plasmid of at first each being treated transfection dilutes with the low blood serum medium of OPTI-MEMI of 50 μ L, at room temperature leaves standstill 5min.
(3) for every porocyte of wanting transfection, low blood serum medium (available from the invitrogen company) dilution of OPTI-MEMI Lipofectamine 2000 with 50 μ L, treat that transfection plasmid and liposome Lipofectamine 2000 add according to 1: 3 (quality and volume ratio), at room temperature leave standstill 5min, after treating that Lipofectamine 2000 dilutions are finished, must mix with the plasmid diluent within the 20min.During mixing 20min, inhale and abandon original substratum in the Tissue Culture Plate, clean each orifice plate with the low blood serum medium of OPTI-MEMI, to dilute residual original substratum, prevent from influencing transfection efficiency.
(4) pRL-TK plasmid and pGL3-adi-pro plasmid co-transfection.PRL-TK plasmid and pGL3-adi-pro plasmid carry out the cotransfection test according to 1: 20 ratio.
(5) pRL-TK plasmid and pGL3-adi-pro and Lipofectamine 2000 mixtures are joined in 24 orifice plates, and rock culture plate gently and make its mixing.
(6) 24 orifice plates are placed CO
2Continue in the cell culture incubator to cultivate, cultivate the substratum that more renews behind the 6h, with cell activity after the assurance transfection.
6. uciferase activity is measured
Select for use the two luciferase reporting systems of Dual-Luciferase Reporter Assay System of Promega company to test.
(1) at (between the 24-36h) within the 48h after the transfection cell is carried out cracking.Before the lysing cell, inhale and to abandon original fluid, clean 2-3 time with PBS, the cell pyrolysis liquid of drawing 100 μ L joins in each cell hole, and wherein cell pyrolysis liquid is according to Passive Lysis Buffer: the deionized water of sterilizing is that 1: 4 ratio is prepared.After lysate adds, the cell orifice plate is rocked 20min all around or blow and beat, make the abundant cracking of cell.After cracking was finished, cell pyrolysis liquid can place-20 ℃ of mensuration of preserving or can directly carry out two uciferase activities.
(2) the Luciferase Assay Buffer II with 10mL joins among the Luciferase Assay Substrate, fully mixing.This mixed solution is BufferI.
(3) with the Stop﹠amp of 200 μ L; Glo Substrate 50X joins the Stop﹠amp of 10mL; Among the Glo Buffer, abundant mixing.This mixed solution is BufferII.
(4) measure with multi-functional microplate reader.Every hole adds 10 μ L cell pyrolysis liquids in the black enzyme plate, places multi-functional microplate reader.The mensuration process at first adds 50 μ L Buffer I in two steps, treats to add 50 μ L Buffer II again after this step end of run.Notice that whole mensuration process answers lucifuge operation.
Embodiment 4: set up chimeric promoters CMV-adi-pro and SVenh-adi-pro
1. main agents: with embodiment 2 agents useful for same; Restriction enzyme Xho I (available from NEB company); The pIRES2-EGFP carrier for expression of eukaryon; PGL3-Enhancer carrier for expression of eukaryon (available from Beijing Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega company).
2. set up chimeric promoters CMV-adi-pro:
Amplification CMV fragment: with pIRES2-EGFP (available from Beijing Pu Luomaige (Beijing) Bioisystech Co., Ltd, being U.S. Promega company) carrier for expression of eukaryon is as template, utilize the pCMV IE promoter fragment of primer CMV-F and CMV-R amplification 548bp (seeing sequence table SEQ ID NO:2) in the table 1, with this fragment called after CMV.The PCR reaction totally is 25 μ L:0.5 μ L dNTP, 0.5 μ L special primer, and 0.25 μ L LA Taq enzyme, 2.5 μ L Buffer,, 1 μ L pIRES2-EGFP (100 times of plasmid dilutions) adds deionized water to 25 μ L.PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 45s, 65.1 ℃ of annealing 45s, 7 ℃ of 45s amount to 35 circulations; Last 72 ℃ are extended 10min.The PCR product reclaims purifying, is connected with the pMD-18T carrier, transforms, and the picking positive colony checks order.
The double digestion reaction of pGL3-Basic carrier for expression of eukaryon and pMD-18T-CMV carrier:
(1) Kpn I and Mlu I double digestion pMD-18T-CMV carrier: 8 μ L pMD-18T-CMV carriers, 2 μ L buffer, 4,0.7 μ L Kpn I restriction enzymes, 0.6 μ L Mlu I restriction enzyme, 0.2 μ L BSA, 8.5 μ L deionized waters.
(2) Kpn I and Mlu I double digestion pGL3-Basic carrier for expression of eukaryon: 1 μ L pGL3-Basic carrier, 2 μ L buffer, 4,0.7 μ L Kpn I restriction enzymes, 0.6 μ L Mlu I restriction enzyme, 0.2 μ L BSA, 15.5 μ L deionized waters.
Place 37 ℃ of constant incubators and carry out endonuclease reaction 4-6h.
Ligation: carries out ligation after the purpose fragment reclaims, ligation system cumulative volume is 10 μ L, 6 μ L CMV wherein, 2 μ LpGL3-Basic carriers, 1 μ L T4 buffer, 0.5 μ L T4 ligase enzyme, 0.5 μ L deionized water.16 ℃ of connections of spending the night.
Connect product and transform, the picking positive colony extracts plasmid (the little extraction reagent kit of Endo-free Plasmid Mini KitII plasmid is available from OMEGA company).
Amplification has the adi-pro fragment of Mlu I and Xho I restriction enzyme site: utilize primer Adi-F2 and Adi-R2 amplified fragments in the table 1.The double digestion reaction of pGL3-CMV carrier and pMD-18T-adi-pro carrier:
(1) Xho I and Mlu I double digestion pMD-18T-adi-pro carrier: 8 μ L pMD-18T-adi-pro carriers, 2 μ L buffer, 3,0.5 μ L Xho I restriction enzymes, 0.5 μ L Mlu I restriction enzyme, 0.2 μ L BSA, 8.8 μ L deionized waters.
(2) Xho I and Mlu I double digestion pGL3-CMV carrier: 1 μ L pGL3-CMV carrier, 2 μ L buffer, 3,0.5 μ L Xho I restriction enzymes, 0.5 μ L Mlu I restriction enzyme, 0.2 μ L BSA, 15.8 μ L deionized waters.
Place 37 ℃ of constant incubators and carry out endonuclease reaction 4-6h.
The purpose fragment reclaims, and connects, and transforms, and the picking positive colony extracts plasmid pGL3-CMV-adi-pro.The restriction enzymes double zyme cutting plasmid detects as Figure 12 and shown in Figure 13 after extracting plasmid, and enzyme cuts out CMV fragment and adi-pro fragment.
3. set up chimeric promoters SVenh-adi-pro:
Amplification SVenh fragment: be template with the pGL3-Enhancer carrier for expression of eukaryon, utilize the increase SV40 enhanser fragment of 237bp (seeing sequence table SE ID NO:3) of primer SVenh-F and SVenh-R in the table 1, this gene fragment called after SVenh.The PCR reaction totally is 25 μ L:2 μ L dNTP, 1 μ L special primer, and 0.25 μ L LA Taq enzyme, 2.5 μ L Buffer,, 0.2 μ L pGL3-Enhancer adds deionized water to 25 μ L.PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 45s, 67 ℃ of annealing 35s, 72 ℃ of 30s amount to 35 circulations; Last 72 ℃ are extended 10min.Reclaim the SVenh fragment that amplification obtains, 4 ℃ are spent the night and carry out ligation with the pMD-18T carrier.Transform, the picking positive colony, plasmid is extracted in the correct back of order-checking.
The structure of pGL3-SVenh-adi-pro carrier: carry out the double digestion reaction with Kpn I and these two kinds of restriction enzymes of Mlu I equally.CMV gene order in the pGL3-CMV-adi-pro carrier is cut away, the substitute is the SVenh fragment of downcutting from the pMD-18T-SVenh carrier.After 16 ℃ of connections of spending the night, transform tests such as picking positive colony, the correct back of the order-checking little extraction reagent kit extraction of Endo-free Plasmid Mini KitII plasmid plasmid.
The primer sequence information of the extension increasing sequence that table 1 the present invention is designed
Table 1 explanation: the underscore under the primed DNA sequence is restriction enzyme site; Thickened portion is the protectiveness base.
Embodiment 5: the activation analysis of chimeric promoters CMV-adi-pro and SVenh-adi-pro
1. main agents and consumptive material: with embodiment 3.
2. chimeric promoters CMV-adi-pro activation analysis:
With vector expression system pGL3-adi-pro, pGL3-CMV-adi-pro and 3T3-L1 mouse Preadipocyte In Vitro and the C2C12 mice skeletal sarcoplast of confidential reference items pRL-TK plasmid transient cotransfection differentiation after 7 days, pGL3-Basic is as negative control, after the transfection off-test, the cracking collecting cell, measure fluorescent value, measurement result shows that pGL3-CMV-adi-pro shows high reactivity in the adipocyte of maturation, and activity will be higher than non-chimeric vector expression system pGL3-adi-pro; Afterwards in order to measure the tissue specificity whether pGL3-CMV-adi-pro still has the adi-pro controlling element, itself and confidential reference items pRL-TK plasmid transient cotransfection are not broken up and 7 days mouse Preadipocyte In Vitro of differentiation, and the result shows that chimeric promoters CMV-adi-pro does not show high reactivity and then show high reactivity in the adipocyte of maturation in undifferentiated adipocyte.
3. chimeric promoters SVenh-adi-pro activation analysis:
Expression vector system pGL3-Enhancer-adi-pro, pGL3-SVenh-adi-pro and confidential reference items pRL-TK plasmid transient cotransfection do not break up and break up 3T3-L1 mouse Preadipocyte In Vitro and the C2C12 mice skeletal sarcoplast after 7 days, and pGL3-Enhancer is as negative control.After the cracking collecting cell, microplate reader is measured fluorescent value, determines its activity by fluorescence ratio.Measurement result shows that pGL3-SVenh-adi-pro shows high reactivity in the adipocyte of maturation, and activity will be higher than non-chimeric vector expression system pGL3-Enhancer-adi-pro; Same in order to measure the tissue specificity whether pGL3-SVenh-adi-pro still has the adi-pro controlling element afterwards, itself and confidential reference items pRL-TK plasmid transient cotransfection are not broken up and 7 days mouse Preadipocyte In Vitro of differentiation, and its result is: chimeric promoters SVenh-adi-pro does not show high reactivity and then show high reactivity in the adipocyte of maturation in undifferentiated adipocyte.
4. the specific activity of chimeric promoters CMV-adi-pro and SVenh-adi-pro is:
Determine two kinds of different chimeric promoters by above two steps transient transfection experiment and only in the adipocyte of maturation, show high reactivity, next, with expression vector system pGL3-CMV-adi-pro and pGL3-SVenh-adi-pro and 7 days mouse Preadipocyte In Vitro of confidential reference items pRL-TK plasmid transient cotransfection differentiation, transfection 36h cracking collecting cell is measured fluorescent value with microplate reader.Determine the result by fluorescence ratio: the activity of chimeric promoters CMV-adi-pro will be higher than the activity of chimeric promoters SVenh-adi-pro.
All liposome transfection experiments last night, will be diluted to 1-4 * 10 with growth medium with cell density behind the cell dissociation before carrying out
5Individual/ml, and get 500 μ l cell suspension inoculations in 24 well culture plates, each sample carries out 3 repetitions.The fluorescent value result who records, 3 with each sample are repeated to represent with the ratio of expression vector and pRL-TK plasmid respectively, this ratio is revised with the ratio of negative control and pRL-TK plasmid, calculate the mean value of three repetitions afterwards, with the STDEV function calculation standard deviation in the Excel form.The calculating derivation formula of function S TDEV is seen shown in the accompanying drawing 27
[12]
The applicant is at first contained the sequence of CRE binding site before clone pig fatty tissue specific expression gene adiponectin first exon in the promoter region, called after adi-pro, and its nucleotide sequence is shown in sequence table SEQ ID NO:1.Utilize American National biotechnology information center ncbi database (http://www.ncbi.nlm.nih.gov/) to carry out the comparison of sequence.Amplification has the adi-pro fragment of restriction enzyme site, be building up to two luciferase reporting carrier pGL3-Basic, by transient transfection differentiation 7 days mouse Preadipocyte In Vitro and mice skeletal sarcoplast, detect this controlling element drives reporter gene in different cells activity.Amplification has pCMV IE promoter fragment and the SV40 enhanser fragment of different restriction enzyme sites, set up into different chimeric promoters with determining active adi-pro controlling element, finally determine the higher chimeric promoters of show activity by the different cells of transient transfection different times.Test-results shows, the chimeric promoters activity that pCMV IE promotor and adi-pro controlling element are set up is higher than the chimeric promoters that non-chimeric promoters adi-pro and SV40 enhanser and adi-pro form, and only shows high reactivity in the adipocyte of maturation.
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Claims (3)
1. the ripe fatty tissue specific chimeric of pig promotor, its nucleotide sequence is shown in sequence table SEQ ID NO:4.
2. the ripe fatty tissue specific chimeric of pig promotor, its nucleotide sequence is shown in sequence table SEQ ID NO:5.
3. claim 1 or 2 described promotors are in the application of regulatory gene in the ripe fatty tissue of pig is expressed.
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| CN112680418A (en) * | 2020-10-30 | 2021-04-20 | 吉林大学 | Cell model for screening GPR109A receptor agonist and screening method |
| CN115322997A (en) * | 2022-05-21 | 2022-11-11 | 河南中医药大学 | Preparation method and application of pBiflu fluorescent label expression vector |
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| CN1213004A (en) * | 1997-09-29 | 1999-04-07 | 成都拜奥生物技术研究所 | Recombination leukocyte mesonium-2 adenovirus carrier and producing method |
| CN1213699A (en) * | 1997-10-08 | 1999-04-14 | 中国预防医学科学院病毒学研究院 | Prodn. system of novel recombination adenovirus adjoint virus |
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| CN1213004A (en) * | 1997-09-29 | 1999-04-07 | 成都拜奥生物技术研究所 | Recombination leukocyte mesonium-2 adenovirus carrier and producing method |
| CN1213699A (en) * | 1997-10-08 | 1999-04-14 | 中国预防医学科学院病毒学研究院 | Prodn. system of novel recombination adenovirus adjoint virus |
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| Ling et al.Cloning and characterization of the 50-flanking region of the pig adiponectin gene.《Biochemical and Biophysical Research Communications》.2009,(第381期),236–240. |
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