CN105779455A - Efficient pig constitutive promoter and application thereof - Google Patents

Efficient pig constitutive promoter and application thereof Download PDF

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CN105779455A
CN105779455A CN201610192603.5A CN201610192603A CN105779455A CN 105779455 A CN105779455 A CN 105779455A CN 201610192603 A CN201610192603 A CN 201610192603A CN 105779455 A CN105779455 A CN 105779455A
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gene
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CN105779455B (en
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段子渊
侯凯丽
王小琪
张瑞莹
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Institute of Genetics and Developmental Biology of CAS
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Abstract

The invention relates to an efficient pig constitutive promoter and application thereof. The pig constitutive promoter provided by the invention is from a region ranging from 1893bp to 184bp of a pig TMSB10 gene, and a sequence of the promoter is selected from nucleotide sequences shown in SEQ ID NO:1-7. The invention also provides a pig hybrid promoter obtained by fusing the pig constitutive promoter with an hCMV enhancer. According to the invention, the efficient constitutive promoter is obtained by cloning from a pig, a pig hybrid promoter containing the hCMV enhancer sequence is successfully constructed, and the activity of the promoter is detected. Results show that the activity of the pig hybrid promoter is about 1.5 times that of an hCMV promoter. The promoter provided by the invention can be applied to research of a transcriptional control mechanism of the pig TMSB 10 gene and also can be used in mammalian cells for carrying out research on related protein expression and transgene, and thus a technical support is provided for a transgene technology.

Description

Efficiently pig constitutive promoter and application
Technical field
The present invention relates to gene engineering technology field, specifically, relate to a kind of efficiently pig composition Type promoter and application.
Background technology
Carnis Sus domestica provides animal proteinum and other nutrient substance of high-quality, therefore, excellent pig for the mankind That plants cultivates for promoting the development of agricultural to have important function.Simultaneously as pig is in anatomy, The aspect such as physiology and hereditism and the mankind have high similarity, thus are considered as that the mankind are different Plant preferable supply source and the model animal of human diseases research of organ transplantation.Transgenic technology is permissible Realize the genome of pig is carried out above-mentioned autotelic lose by the technological means such as genetic engineering Pass and modify.
Promoter is one section of DNA sequence, is the important component part of gene, can lead to Cross and carry out the time of transcribing of controlling gene with trans acting factor interaction and transcribe degree.Eucaryon Biological promoter according to the time played a role and Region-specificity can be divided into inducible promoter, Tissue specific promoter and constitutive promoter.Constitutive promoter can at different tissues and Different cells start the stable expression of genes of interest.In view of promoter starts activity for purpose This important regulating and controlling effect of gene expression dose, has efficiently and starts purpose continually and steadily The promoter of the gene expression researcher often first-selection when carrying out transgeneic procedure.
At present, conventional in transgenic technology promoter include hCMV, EEF1 α, PGK, CAG etc., although these promoteres can start the expression of exogenous gene in vitro very well, but The promoter of Hosts, in the genome being incorporated into pig after may cause host Cellular defence mechanisms and inactivate.Therefore, the clone of efficient pig constitutive promoter has important Meaning.
It addition, existing relevant report proves, hCMV enhancer can effectively strengthen eukaryotic promoter Startup activity, in mice experiment made on the living, hybrid promoter still is able to play good function.
At present, not yet have from pig, clone the relevant report of efficient constitutive promoter and application.
Summary of the invention
It is an object of the invention to provide the efficient constitutive promoter of Novel pig and pig hybrid promoter.
It is a further object of the present invention to provide the application of above-mentioned promoter.
The present invention is based on the idea that utilize Ensemble data base and ncbi database to search pig 5 ' upstream regulatory sequences of extrasin beta-10 gene (TMSB10), utilize online eukaryotic promoter Forecasting software NNPP, TSSW, Promoter 2.0, Promoter Scan, FirstEF are to this base Core promoter region cis-trans functional element and the CpG island distribution situation of cause are predicted.If Counting a series of primer, from the genome of length pig, PCR amplification obtains corresponding purpose fragment, and Purpose fragment is inserted in report carrier pGL3-basic and build recombinant vector, transient transfection 293T Cell, utilizes β-gal as the activity of object of reference detection different promoters fragment.Last in activity Insert hCMV enhancer sequence with before length all suitably fragment, use identical method pair The activity of hybrid promoter is analyzed, and demonstrates the starting efficiency of different promoters.
In order to realize the object of the invention, the pig constitutive promoter that the present invention provides, described startup Son is from-1893bp～the 184bp district of pig TMSB10 gene, and its sequence is selected from such as SEQ ID Nucleotide sequence shown in NO:1-7.
The present invention also provides for pig hybrid promoter, its be by described pig constitutive promoter with HCMV enhancer merges the promoter obtained.Preferably by the pig group as shown in SEQ ID NO:5 Constitutive promoter merges, with the hCMV enhancer as shown in SEQ ID NO:8, the promoter obtained.
It is highly preferred that described pig hybrid promoter is PhCMVE-TMSB10, its sequence is:
I) nucleotide sequence shown in SEQ ID NO:9；Or
Ii) nucleotide sequence shown in SEQ ID NO:9 is substituted, lacks and/or increases by one Or multiple nucleotide and there is the nucleotide sequence of identical function；Or
Iii) under strict conditions with sequence hybridization shown in SEQ ID NO:9 and there is identical function Nucleotide sequence, described stringent condition is at 0.1 × SSPE containing 0.1%SDS or containing 0.1% In 0.1 × SSC solution of SDS, hybridize at 65 DEG C, and wash film with this solution；Or
Iv) with i), ii) or nucleotide sequence iii) there is more than 90% homology and there is phase The nucleotide sequence of congenerous.
The present invention also provides for the expression cassette containing described promoter.
The present invention also provides for the carrier containing described promoter, preferably carrier for expression of eukaryon, more excellent Select pGL3-basic.
The present invention also provides for the engineering bacteria containing described promoter.
The present invention also provides for the transgenic cell line containing described promoter, and preferred mammal is thin Born of the same parents, more preferably 293T cell.
The present invention also provides for the application in regulation and control downstream gene expression of the described promoter, Qi Zhongsuo State downstream gene and include reporter gene LUC, GFP and gene TMSB10 etc..
The present invention further provides the application in preparation transgenic animal of the described promoter, wherein Described animal includes pig etc..
The present invention first from pig clone obtain efficient constitutive promoter, and successfully construct and contain There is the pig hybrid promoter hCMVE-TMSB10 of hCMV enhancer sequence, and to promoter Activity detected.Result shows, the activity of hCMVE-TMSB10 is about hCMV 1.5 times of promoter activity.What the promoter of the present invention can be used for pig TMSB10 gene transcribes tune Control Mechanism Study, and carry out correlative protein expression in the mammalian cell and transgenic grinds Study carefully, provide technical support for transgenic technology.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of PCR primer in the embodiment of the present invention 1.
Fig. 2 is the recombiant plasmid enzyme action qualification figure in the embodiment of the present invention 1.
Fig. 3 is the active testing result of the promoter purpose fragment of different length in the embodiment of the present invention 3.
Fig. 4 is the fine jade cloning hCMV enhancer (hCMVE) sequence obtained in the embodiment of the present invention 4 Sepharose electrophoretogram.
Fig. 5 is the enzyme action qualification figure of recombiant plasmid pGL3-hCMVE-TMSB10 in the embodiment of the present invention 4.
Fig. 6 is the active testing result of pig hybrid promoter hCMVE-TMSB10 in the embodiment of the present invention 4.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not Specializing, embodiment is all according to conventional laboratory conditions, such as Sambrook equimolecular cloning experimentation Handbook (Sambrook J&Russell DW, Molecular cloning:a laboratory manual, 2001) condition, or according to manufacturer's description advised.
The clone of embodiment 1 pig constitutive promoter and the structure of recombinant vector
1, Ensemble data base and ncbi database is utilized to search Porcine thymosin β-10 gene 5 ' upstream regulatory sequences (hereinafter referred to as full length sequence), utilize the prediction of online eukaryotic promoter soft Part NNPP, TSSW, Promoter 2.0, Promoter Scan, FirstEF are to this gene Core promoter region cis-trans functional element and CpG island distribution situation are predicted.Respectively with Predict the outcome, in full length sequence (TMSB10) and commercial vectors pEGFP-C2 HCMVE/P (i.e. hCMV promoter) sequence is a series of primer of stencil design (table 1), point From the genome of length pig, PCR amplification does not obtains pig TMSB10 gene The purpose fragment of-1893bp～184bp district different length (TMSB10-1, TMSB10-2, TMSB10-3, TMSB10-4, TMSB10-p-1 and TMSB10-p-2), from carrier In pEGFP-C2, amplification obtains hCMVE/P sequence, and purpose fragment is inserted report load respectively Body pGL3-basic builds recombinant vector.
The amplimer (underscore part is restriction enzyme site) of table 1TMSB10 promoter respective segments
PCR reaction system is as follows:
PCR response procedures is as follows:
It is pure that amplified production reclaims test kit through 2% agarose gel electrophoresis detection and Omega company Change, deposit in-20 DEG C standby.PCR electrophoresis result is shown in Fig. 1.
2, the structure of the transfection carrier containing Porcine thymosin β-10 gene different length promoter fragment Build
(1) carrier and the double digestion of PCR primer
With restricted enzyme (purchased from Thermo Fisher company) enzyme action carrier pGL3-basic (purchased from Promega company) and promoter PCR reclaim product.Enzyme action system is as follows:
(2) connect
By the double digestion product of recovery according to purpose fragment and pGL3-basic be 3-10:1 mole Ratio, connects 2h with T4DNA ligase in 16 DEG C.
(3) Top competent cell is converted
1. take appropriate Top10 competent cell, mix with coupled reaction liquid, be placed on ice 10min。
2. 42 DEG C of water-bath 90s.
Ice bath 5min the most immediately.
4. appropriate nonreactive LB liquid medium, 37 DEG C of 170rpm shaken cultivation are added 30min。
5. draw appropriate bacterium solution on the solid LB culture plate containing ampicillin, and use It is coated with bacterium rod to smear uniformly.
6. culture plate incubated overnight in 37 DEG C of incubators it is inverted.
(4) plasmid identification
1. the sizeable bacterium colony of picking from culture plate, adds appropriate containing ammonia benzyl penicillium sp In the LB liquid medium of element, 37 DEG C of shaken cultivation a few hours.According to going, endotoxin plasmid is little The operating instruction of extraction reagent kit (purchased from Omega company) extracts plasmid.
2. double digestion is identified: ibid, enzyme action qualification result is shown in Fig. 2 to enzyme action system.
3. check order qualification: double digestion is identified correct plasmid send to raw work biological engineering (on Sea) limited company checks order.
(5) plasmid extraction
Carry according to the operating instruction removing the little extraction reagent kit of endotoxin plasmid (purchased from Omega company) Take plasmid.
The cell transfecting of embodiment 2 recombiant plasmid
With 293T cell for transfection object, negative control is pGL3-basic, and positive control is PGL3-hCMVE/P, normalization reference is β-gal.
Cell transfecting:
(1) the previous day in transfection 293T cell is carried out trypsinization process, spread after counting Plate (24 orifice plate) so that it is reach 80%-90% in the density transfecting the same day.
(2) change DMEM or Opti-MEM culture medium before carrying out transfection experiment into, cultivate 3-4h.
(3) transfection mixture is prepared: DNA (μ g): PEI (μ l)=1:2-3, plasmid 500ng/ hole, Each plasmid sets 3 multiple holes.
1. dilute with 50 μ l Opti-MEM serum-free mediums (other serum-free mediums also can) Release DNA, immediately mix homogeneously.
The most before use by PEI mix homogeneously, dilute appropriate PEI with 50 μ l Opti-MEM.Mixed Even, room temperature places 5min.
(4) mixing homogeneously careful for DNA with PEI after dilution, room temperature places 20min.
(5) adding in culture hole by said mixture, the light culture plate that shakes is so that transfection cocktail Uniformly diffuse in culture medium.
(6) 37 DEG C it are placed in, 5%CO2Incubator cultivates 18-48h.It is changed to after cultivating 4-6 hour Culture medium containing serum.
Embodiment 3 promoter activity is tested
1, solution preparation
1. ONPG reactant liquor (1 ×): with Z buffer dissolving 0.2g ONPG powder to 50ml, Subpackage also keeps in Dark Place in-20 DEG C.
2. Luciferase Assay Sulfate (1 ×): by Bright-GloTM Luciferase Assay Bright-Glo in System test kitTMLuciferase Assay Buffer and Bright-GloTM Luciferase Assay Substrate mixing is yellow clear solution to solution.
3. Z buffer (1 ×): weigh 10.75g Na2HPO4·12H2O, 3.1g NaH2PO4·2H2O, 0.375g KCl, 0.123g MgSO4·7H2O, adds ultra-pure water and dissolves To 500ml, in 4 DEG C of preservations.
2, cell is collected
(1) sucking culture medium, every hole adds 500 μ l brine once.
(2) discarding cleaning mixture, every hole adds 100 μ l cell pyrolysis liquids, is placed on ice, jog Cracking 20min.
(3) it is transferred to protein mixture in clean EP pipe (place on ice, prevent albumen Degraded, affects experimental result), 4 DEG C of 12000rpm are centrifuged 10min.
3, β-gal value is measured
(1) take 20 μ l protein liquids in new clean EP pipe, add 100 μ l ONPG working solutions With 450 μ l Z buffer, turn upside down and mix for several times.
(2) in 37 DEG C of reactions to faint yellow, 200 μ l NaCO are added3(1M) terminate instead Should, overturn and mix for several times.
(3) often pipe draws 100 μ l reaction mixtures in 96 clean orifice plates, if 2 multiple holes.
(4) open Epoch microplate reader, open computer, click on Gene 5 icon on desktop, Enter reading software home page, click on the new in read now, absorbing wavelength is set to 410nm, Again 96 orifice plates are placed in Epoch microplate reader and carry out reading.
4, Luciferase fluorescent value measures
Separately take 20 μ l protein liquids in new clean EP pipe, add 20 μ l Luciferase Assay Sulfate (1 ×), quickly mixes, and is immediately placed in chemiluminescence detector and detects its RLUs Value, records data.
5, data process and analyze: result is shown in Fig. 3 (vertical coordinate is RLUs meansigma methods).
The acquisition of embodiment 4 pig hybrid promoter
1, the acquisition of hCMV enhancer (hCMVE) sequence
With the hCMV sequence in carrier pEGFP-C2 as template, design PCR primer (table 2), PCR reaction system and response procedures are with described in embodiment 1.Pcr amplification product is through 2% agarose Detected through gel electrophoresis and Omega company reclaim kits, deposit in-20 DEG C standby.PCR Product electrophoresis result is shown in Fig. 4.
Table 2hCMVE primer
2, the structure of recombinant vector pGL3-hCMVE-TMSB10
Above-mentioned amplified production is connected to carrier by the mode using enzyme action to connect On pGL3-TMSB10-4, build and obtain containing pig hybrid promoter PhCMVE-TMSB10 (PhCMVE-TMSB10 is by 447bp human cytomegalic inclusion disease virus enhancer sequence and 325bp pig TMSB10 gene promoter sequence composition hybrid promoter) recombinant vector pGL3-hCMVE-TMSB10.Then carrying out enzyme action qualification, result is shown in Fig. 5.
3, cell transfecting and active testing
Cell transfecting and active testing are tested respectively with described in embodiment 2 and 3.Data process and divide Analysis result is shown in Fig. 6.
Fig. 6 result shows, pig hybrid promoter hCMVE-TMSB10 (SEQ ID NO:9) Activity be about hCMV promoter (SEQ ID NO:10) activity 1.5 times.
Although, the most with a general description of the specific embodiments the present invention has been made in detail Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention Upper these modifications or improvements, belong to the scope of protection of present invention.

Claims (10)

1. pig constitutive promoter, it is characterised in that described promoter is from Porcine thymosin β-10 -1893bp～the 184bp district of gene, its sequence is selected from the nucleotide as shown in SEQ ID NO:1-7 Sequence.
2. pig hybrid promoter, it is characterised in that it is by pig composing type described in claim 1 Promoter merges, with hCMV enhancer, the promoter obtained.
Pig hybrid promoter the most according to claim 2, it is characterised in that it is by such as Pig constitutive promoter shown in SEQ ID NO:5 and the hCMV as shown in SEQ ID NO:8 Enhancer merges the promoter obtained.
Pig hybrid promoter the most according to claim 3, it is characterised in that its sequence is:
I) nucleotide sequence shown in SEQ ID NO:9；Or
Ii) nucleotide sequence shown in SEQ ID NO:9 is substituted, lacks and/or increases by one Or multiple nucleotide and there is the nucleotide sequence of identical function；Or
Iii) under strict conditions with sequence hybridization shown in SEQ ID NO:9 and there is identical function Nucleotide sequence, described stringent condition is at 0.1 × SSPE containing 0.1%SDS or containing 0.1% In 0.1 × SSC solution of SDS, hybridize at 65 DEG C, and wash film with this solution；Or
Iv) with i), ii) or nucleotide sequence iii) there is more than 90% homology and there is phase The nucleotide sequence of congenerous.
5. contain the expression cassette of promoter described in any one of claim 1-4.
6. contain the carrier of promoter, preferably pGL3-basic described in any one of claim 1-4.
7. contain the engineering bacteria of promoter described in any one of claim 1-4.
8. contain the transgenic cell line of promoter described in any one of claim 1-4, preferably feed Breast zooblast, more preferably 293T cell.
9. the answering in regulation and control downstream gene expression of promoter described in any one of claim 1-4 With, wherein said downstream gene includes reporter gene LUC, GFP and gene TMSB10.
10. the application in preparation transgenic animal of the promoter described in any one of claim 1-4, Wherein said animal includes pig.
CN201610192603.5A 2016-03-30 2016-03-30 Efficient pig constitutive promoter and application Expired - Fee Related CN105779455B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102604947A (en) * 2011-01-24 2012-07-25 华中农业大学 Porcine adipose tissue-specific chimeric promoters
US20120270322A1 (en) * 2011-04-21 2012-10-25 Snu R&Db Foundation Shuttle vectors for mycobacteria-escherichia coli and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102604947A (en) * 2011-01-24 2012-07-25 华中农业大学 Porcine adipose tissue-specific chimeric promoters
US20120270322A1 (en) * 2011-04-21 2012-10-25 Snu R&Db Foundation Shuttle vectors for mycobacteria-escherichia coli and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
UENISHI,H. 等: "GenBank 登录号：NP_001090951.1", 《NCBI》 *
马强 等: "4个猪种IGF-1基因核心启动子区的克隆及生物信息学分析", 《农业大学学报》 *

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