CN103509812A - specific transgenic carrier and construction method thereof - Google Patents
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Abstract
The invention discloses a salivary gland tissue specific transgenic carrier and a construction method thereof. The salivary gland tissue specific transgenic carrier is obtained by emerging an upstream regulation region sequence of a mice parotid gland protein promoter and a visual screened neo-EGFP combined double-label to a piggyBac transposon system and a Lox-Cre enzyme system; the transgenic carrier can make protein express in a particular tissue to achieve a high-efficient large-section emerging efficiency. The carrier also has a green luminescence label which is convenient for screening and a neomycin resistance gene which is convenient for eukaryotic cell screening, and makes the screening of transgenic cells and identification of transgenic animals be simpler, more direct, and more accurate. The transgenic carrier provided by the invention is a salivary gland specific expression high-efficient integration universal carrier, and is capable of being applied to fields of transgenic animals such as transgenic mice and pig, and the like. The invention clones the upstream regulation region of FVB mice for the first time, the cloning method is simple and high efficient, and the difficulty in construction of a large section carrier is effectively overcome.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to the specific high effective integration transgene carrier of a kind of salivary organization and construction process thereof, the invention still further relates to pcr amplified fragment and the cloning process thereof of mouse parotid gland albumen upstream regulatory region.
Background technology
Conventional carriers builds and mainly by genetically engineered restriction enzyme, T4 ligase enzyme and round pcr, object fragment is connected on corresponding carrier framework, then through enzyme, cuts evaluation, and sequence verification, completes vector construction.According to experiment, need to sometimes need to connect a plurality of fragments, often, because of restriction enzyme site restriction, make experimental design more and more difficult, carrier segments is increasing and cause T4 ligase enzyme inefficiency, and vector construction difficulty is large.Therefore, conventional carriers constructing technology is being difficult to successfully aspect larger vector (more than 12Kb) vector construction.
Parotid gland albumen is the highest albumen of gene expression abundance in the parotid gland.Parotid gland protein gene is positioned on No. 2 karyomit(e)s of mouse, and in genome, this gene exists with single copy form, and mainly at parotid gland specifically expressing, studies show that in addition its submaxillary gland at sialisterium and sublingual gland also have low expression.Studies show that this gene is mainly by the upstream regulatory region (11.5Kb) of parotid gland protein gene and downstream sequence (approximately 2.5～3kb) (being parotid gland protein promoter PSP) regulation and control, but its regulatory gene is regulating and expressing in sialisterium only, at other histoorgans of whole body, do not express.The transgenic animal principle that sialisterium is expressed foreign protein be exactly by parotid gland protein promoter can target protein (enzyme or medicine) the saliva bodies of gland such as the parotid gland, my humble abode gland and glandula submandibularis after expression direct secretion in animal saliva, direct oral cavity enters animal digestive tract, brings into play its function.
Mouse parotid gland promotor (PSP) carrier application aspect nearest report or calendar year 2001, by Canadian scholar Serguei P.Golovan, when preparing environmental friendliness animal, applied, this carrier structure is fairly simple, only with phytase gene, replace original parotid gland protein gene, there is no selection markers and fluorescent mark, integration efficiency is also lower, and be integrated into the genome form of how connecting end to end, this integration mode is in genome, more easily be silenced and lose, because there is no selection markers, this carrier is only suitable for procaryotic injection or the injection of ICSI(endochylema) method production transgenic animal, in animal, economic worth is more greatly pig, but pig zygote is opaque, can't see protokaryon.And pig body early embryo collection difficulty is large, and technical barrier is large.Its carrier structure as shown in Figure 1.
Her extra large aromatization of 2006 Nian You China Agricultural University is built 2 pig parotid gland protein promoters (PSP) carrier, except carrier framework, these two carriers all adopt pig PSP part upstream regulatory region sequence (10kb) to connect phytase gene, its difference tailing signal after it: is bGH-pA; Another is the Poly-PA in pig parotid gland albumen PSP downstream.These two carriers are all with a drug screening gene neo.Although these two carriers can carry out drug screening, there is no visable indicia, screening transgenic cell is difficult to confirm, be still conventional carriers, it is similar that its integration mode and Serguei P.Golovan build mouse parotid gland protein carrier.But pig parotid gland protein promoter is because its research is thorough not enough, present analysis, in 10Kb control region, controlling element is incomplete at its upstream, may also exist in other region other to strengthen subarea, causes its downstream albumen enzyme gene expression amount low.2 pig parotid gland protein promoters (PSP) carrier structure as shown in Figure 2.
Summary of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides the specific transgene carrier of a kind of salivary organization and construction process thereof.
For achieving the above object, the present invention has taked following technical scheme:
A construction process for the specific transgene carrier of salivary organization, comprises the following steps:
(1), take respectively plasmid pcDNA3.1 (+) and pT2AL200R175-CAGGS-EGFP as template, SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:7 and SEQ ID NO:8 are that primer carries out pcr amplification for the first time, obtain neo gene and EGFP gene with some overlaps; Respectively get after 0.5-3 μ L mixes and carry out pcr amplification for the second time as template, purifying obtains neo-EGFP fusion gene; With EcoR I and Xba I double digestion pcDNA3.1 (+) and neo-EGFP fusion gene, purifying reclaims enzyme and cuts product, connects, and builds and obtains pcDNA-Neo-T2A-EGFP carrier;
(2), take plasmid pPB-UbC-EGFP-neo as template, using SEQ ID NO:9 and SEQ ID NO:10 and SEQ ID NO:11 and SEQ ID NO:12 respectively as primer, carry out pcr amplification and obtain Not I-5-PB-Not I, Xho I-3-PB-Xho I, after adopting respectively Not I and Xho I enzyme to cut, be connected to carrier pPSPBGPneo-XynB upper, obtain carrier pPSPBGPneo-PB-XynB;
(3), take the pcDNA-neo-T2A-EGFP carrier that step (1) obtains is template, use LA-PCR system, SEQ ID NO:13 and SEQ ID NO:14 carry out pcr amplification as primer and obtain infusion-CMV-neo-T2A-EGFP fragment, cut glue purification; The carrier pPSPBGPneo-PB-XynB obtaining by Cla I and Bgl II double digestion step (2), cuts glue purification to object fragment; Infusion-CMV-neo-T2A-EGFP fragment is recombinated in the middle of two loxp sites of pPSPBGPNeo-PB-XynB carrier, obtain pPSPBGPneo-T2A-EGFP-PB-XynB carrier;
(4), take the pPSPBGPneo-T2A-EGFP-PB-XynB carrier that step (3) obtains is template, SEQ ID NO:15 and SEQ ID NO:16 are as primer, carry out pcr amplification, obtain the pPB-lox-neoEGFP-loxp fragment in carrier, and add AgeI restriction endonuclease sites, purifying, obtains novel vector skeleton;
(5), take the pcr amplified fragment of mouse parotid gland protein gene upstream control region is template, SEQ ID NO:17 and SEQ ID NO:18 are primer, amplification obtains the downstream 4009bp fragment of mouse parotid gland albumen upstream regulatory region, recombination sequence in 4009bp fragment and novel vector skeleton pPB-lox-neoEGFP-loxp is recombinated, obtain intermediate carrier 1;
(6), the pcr amplified fragment of mouse parotid gland protein gene upstream control region of take is template, take SEQ ID NO:19 and SEQ ID NO:20 is primer, amplification obtains the downstream 4706bp fragment of mouse parotid gland albumen upstream regulatory region; The StuI site of the intermediate carrier 1 of the step of being recombinated (5), is intermediate carrier 2;
(7), the pcr amplified fragment of mouse parotid gland protein gene upstream control region of take is template, take SEQ ID NO:21 and SEQ ID NO:22 is primer, utilizes AgeI site, amplification obtains the downstream 3956bp fragment of mouse parotid gland albumen upstream regulatory region; On the intermediate carrier 2 of the step of being recombinated (6), obtain carrier pPB-MusPSP-neo-EGFP, be the specific transgene carrier of salivary organization.
Therein in an embodiment, the cloning process of the pcr amplified fragment of the mouse parotid gland protein gene upstream control region in described step (5)-(7) is: take FVB mouse gene group DNA as template, SEQ ID NO:1 and SEQ ID NO:2 are primer, and KOD-FX polysaccharase carries out pcr amplification for the first time; The product increasing for the first time of take is template, and SEQ ID NO:3 and SEQ ID NO:4 are primer, carry out pcr amplification for the second time, obtain.
In an embodiment, in described step (5)-(7), the response procedures of pcr amplification is for the first time therein: 94 ℃ of 2min; 98 ℃ of 10s, 74 ℃ of 13min, 5cycles; 98 ℃ of 10s, 72 ℃ of 13min, 5cycles; 98 ℃ of 10s, 70 ℃ of 13min, 5cycles; 98 ℃ of 10s, 68 ℃ of 13min, 30cycles; 68 ℃ of 7min; The response procedures of described pcr amplification is for the second time: 94 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 13min, 30cycles; 68 ℃ of 7min.
In an embodiment, described in step (1), the response procedures of PCR is for the first time therein: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 50s, 35 circulations; 72 ℃ of 10min; PCR response procedures for the second time: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 1min40s, 35 circulations; 72 ℃ of 10min.
In an embodiment, described in step (2), the response procedures of PCR is therein: 94 ℃ of 30s; 55 ℃ of 30s, 35 circulations; 72 ℃ of 30s, 72 ℃ of 3min, 30 circulations; Described in step (3), the response procedures of PCR is: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 4min, 35 circulations; 72 ℃ of 10min; Described in step (4)-(7), the response procedures of PCR is: 98 ℃ of 1min; 98 ℃ of 10s; 60 ℃ of 5s; 72 ℃ of 2min; 35 circulations; 72 ℃ of 2min.
The present invention also provides above-mentioned construction process to build the specific transgene carrier of salivary organization obtaining.
Compared with prior art, the present invention has following beneficial effect:
1, the specific transgene carrier of salivary organization of the present invention is to be incorporated in piggyBac high-efficiency transposon system and Lox-Cre enzyme system and to build and obtain by the upstream regulatory region sequence of mouse parotid gland protein promoter (MusPSP) upstream 12.1kb and visual screening neo-EGFP being merged to double-tagging, this transgene carrier can be realized target protein and express and (have mouse in particular organization, the ability of the animal parotid gland specifically expressings such as pig), can under transposase is auxiliary, realize the high effective integration efficiency of large fragment (more than 10kb); And this carrier also has is convenient to the green fluorescence mark EGFP of screening and the neo neomycin resistance gene of eukaryotic cell screening, and render transgenic cell screening and transgenic animal evaluation are more simply, directly, accurately; Consider that the public is to selection markers safety concerns, this mark can be deleted by the disposable Accurate Measurement of loxP-cre enzyme system again simultaneously.Only goal gene need to be inserted to ASCI site in transgene carrier of the present invention, just can produce for animals such as transgenic animal mouse and pigs, there is simple, convenient, safe feature.
2, transgene carrier of the present invention is a kind of sialisterium specifically expressing high effective integration universal support, can be used for the animal aspect transgenosis application such as transgenic mice and pig, both can be for traditional procaryotic injection, endochylema injection, can, for somatic cell clone cell line selection etc., there is good compatibility with modern transgenosis platform again.
3, the present invention is by 4 fragment recombination methods, utilize multipair overlapping primer pair larger sequence fragment amplification, by its people for after interrupting, the homologous recombination joint that utilizes amplified fragments to produce, under recombinase is auxiliary, a section is binned in together, clone first FVB mouse upstream regulatory region, and be cloned in carrier, through order-checking, this control region is different from the control region of bibliographical information.This cloning process is simply efficient, has effectively overcome technically large fragment carrier (more than 12Kb) and has built difficulty.
Accompanying drawing explanation
Fig. 1 be Serguei P.Golovan build turn phytase carrier;
Two pig parotid gland protein transgene carriers that the Yi Haifang of Tu2Wei China Agricultural University (2006) builds;
Fig. 3 is the structure schematic diagram of the pPB-MusPSP-neoEGFP carrier of the embodiment of the present invention 1;
Fig. 4 is the PCR collection of illustrative plates of the MusPSP that in embodiment 1, clone obtains, and wherein A is MusPSP upstream regulatory region clone (11503bp～+ 1390bp); B is MusPSP upstream regulatory region clone (11325bp～+ 980bp);
Fig. 5 is that the enzyme of Neo-T2A-EGFP in embodiment 1 is cut evaluation electrophorogram, wherein 1,2,3 represents respectively T2A-EGFP(765bp), neo-T2A(835bp), neo-T2A-EGFP(1600bp);
Fig. 6 is that the enzyme of pcDNA-Neo-T2A-EGFP in embodiment 1 is cut evaluation electrophorogram, 1,2 object band 1590bp that represent pcDNA-Neo-T2A-EGFP wherein, and 3 represent 1 and 2 plasmid contrast;
Fig. 7 is that the enzyme of pPSPBGPneo-PB-XynB carrier in embodiment 1 is cut evaluation electrophorogram, and wherein M represents Marker2000; 1,2 are the contrast of pPSPBGPneo-PB-XynB plasmid, and 3,4 represent that respectively the enzyme of this plasmid Xho I and Not I cuts result;
Fig. 8 is that the enzyme of pPSPBGPneo-T2A-EGFP-PB-XynB carrier in embodiment 1 is cut evaluation electrophorogram, and wherein M represents DL DNA15000Marker; Swimming is with 1 to represent pPSPBGPneo-T2A-EGFP-PB-XynB vector plasmid contrast, and 2,3 represent that 1 enzyme cuts result, and arrow indication is object band 2935bp;
Fig. 9 is embodiment 1 small mouse parotid gland albumen upstream regulatory region (MusPSP) clone's structure schematic diagram;
Figure 10 is MusPSP control region fragment 1(-3143bp～+ 865bp in embodiment 1) cloned plasmids AscI, NotI enzyme is cut evaluation collection of illustrative plates; Wherein be with 1,2,3,4,5 for different clone's enzymes, to cut evaluation, arrow is indicated object band;
Figure 11 is MusPSP control region fragment 2(-7849bp～-3143 in embodiment 1) AgeI, stuI enzyme cut evaluation collection of illustrative plates; Wherein be with 1,2,3 for different clone strain enzymes, to cut evaluation;
Figure 12 is MusPSP control region fragment 3(-11805pb～-7849bp in embodiment 1) AgeI enzyme cuts evaluation collection of illustrative plates; Wherein be with 1,2,3,4 to be that different clone's enzymes are cut evaluation;
Figure 13 utilizes the comparative analysis of pPB-MusPSP-neoEGFP carrier transfection PEF cell screening result in embodiment 2; Upper figure photo is under 40 times of mirrors, the observed result of (right figure) under (left figure) and ordinary light under 488nm wavelength exciting light respectively, and observed result corresponding to transfection respectively organized in C1, C2, Z3, Z4 representative; Wherein, C1, C2 are respectively the transfection of single plasmid transfection contrast 1(cyclic plasmid) and the transfection of single plasmid transfection contrast 2(linearization plasmid); Z3, Z4 are each group of two plasmid co-transfections;
Figure 14 is pPB-MusPSP-neo-EGFP carrier transfection efficiency analysis chart in embodiment 2.
Embodiment
Below in conjunction with specific embodiment, describe the present invention in detail.
The primer using in following examples is all to entrust Shanghai Sheng Gong Bioisystech Co., Ltd synthetic.
The construction process of the embodiment 1 specific transgene carrier of salivary organization
Refer to Fig. 3, the constructing plan figure for the specific transgene carrier of salivary organization of the present invention, comprises the following steps:
(1), the clone of mouse parotid gland albumen upstream regulatory region (PSP) (comprises that parotid gland albumen turns
record initiation site and First Intron)
Get FVB Strains of Mouse tail tissue sample, its genomic dna of extracting, then take FVB mouse gene group DNA as template, adopts KOD-FX polysaccharase (TOYOBO, Japan) to carry out pcr amplification.First adopt primer mpsp3497-F1 (SEQ ID NO:1), mpsp16390-R1 (SEQ ID NO:2) carries out pcr amplification for the first time, obtains mouse parotid gland albumen upstream regulatory region (11503bp～+ 1390bp), and result as shown in Figure 4 A; Then adopt nested primers mPSP-3675-F2 (SEQ ID NO:3) and mPSP-15980-R2 (SEQ ID NO:4), the product increasing for the first time of take is template, carry out pcr amplification for the second time, amplification mouse parotid gland albumen region (11325bp～+ 980bp), result as shown in Figure 4 B, obtain cloning FVB strain parotid gland albumen upstream regulatory region MusPSP, wherein primer sequence is as shown in table 1, and the analytical results of the sequence contrast of MusPSP and bibliographical information is in Table 2.Through with NCBI in the mouse PSP parotid gland albumen upstream regulatory region submitted to strengthen subarea (U73190.1, X68699.1) sequence alignment analysis, two strengthen subarea acquaintance property and reach 99%, core promoter district (transcription initiation site upstream 300bp) is in full accord with mouse database (UCSC) comparison result, analytical results shows that this research obtains MusPSP control region and Golovan, and it is not same source that S.P etc. (2001) prepare parotid gland albumen upstream regulatory region that transgenic pig and mouse adopt.
Clone's primer of table 1 mouse parotid gland albumen upstream regulatory region (PSP)
The reaction system of PCR is as follows:
Response procedures: the response procedures of pcr amplification is 94 ℃ of 2min for the first time; 98 ℃ of 10s, 74 ℃ of 13min, 5cycles; 98 ℃ of 10s, 72 ℃ of 13min, 5cycles; 98 ℃ of 10s, 70 ℃ of 13min, 5cycles; 98 ℃ of 10sec, 68 ℃ of 13min, 30cycles; 68 ℃ of 7min.The response procedures of described pcr amplification is for the second time 94 ℃ of 2min; 98 ℃ of 10sec, 68 ℃ of 13min, 30cycles; 68 ℃ of 7min.
The comparing result of the sequence of table 2 clone's MusPSP sequence and bibliographical information
(2), build to merge pcDNA-Neo-T2A-EGFP double-tagging carrier, can be used for the later stage
the visual green fluorescent protein (EGFP) of deleting and neomycin resistance gene (neo) fusion gene
A, amplification neo
Take plasmid pcDNA3.1(+) (invitrogen company) be template, design primer EcoR I-neo-1 (SEQ ID NO:5), neo-T2A-2 (SEQ ID NO:6) amplification neo gene.PCR response procedures: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 50s; 35 circulations; 72 ℃ of 10min.PCR product detects with 1% agarose gel electrophoresis.Then use EasyPure PCR Purification Kit (Beijing Quanshijin Biotechnology Co., Ltd) to PCR product purification (being called for short-crossing column purification), remove primer and other impurity;
B, amplification EGFP
With plasmid pT2AL200R175-CAGGS-EGFP (Japan, The National Institute of Genetics center, Koichi professor Kawakami present) template, design primer T2A-EGFP-3 (SEQ ID NO:7), EGFP-4 (the SEQ ID NO:8) EGFP that increases.Wherein, in upstream primer T2A-EGFP-3 (SEQ ID NO:7), there are 15bp left and right and neo-T2A-2 (SEQ ID NO:6) overlap; the terminator codon of removing neo adds tri-bases of GCC, the restricted property of downstream primer restriction endonuclease Xba I restriction enzyme site and protection base.PCR response procedures is identical with step a with product purification condition.
C, neo-EGFP fusion gene
The PCR product of purified recovery neo and EGFP carries out concentration determination, adjusts after concentration, respectively gets after 1 μ L mixes and carries out pcr amplification as template.PCR response procedures: 98 ℃, 2min; 98 ℃, 10s; 68 ℃, 1min40s; 35 circulations; 72 ℃, 10 min.Obtain neo-EGFP fusion gene.Enzyme is cut evaluation, and as shown in Figure 5, restriction enzyme digestion and electrophoresis figure object stripe size is consistent with expection for qualification result.
D, pcDNA-Neo-T2A-EGFP vector construction
The above-mentioned neo-EGFP fusion gene obtaining by overlapping PCR is crossed to purification column, remove primer and PCR waste reaction solution.With
ecoR I (Fermentas company) and
xba I (Fermentas company) double digestion pcDNA3.1(+) plasmid and PCR product neo-EGFP fusion gene.Enzyme is cut product and is crossed column purification recovery.With Ligation Kit Ver.2.0, connect the connection of spending the night of test kit (Takara company) constant temperature in 16 ℃ of connection instrument.Finally be built into pcDNA-Neo-T2A-EGFP carrier, and carry out enzyme and cut evaluation, qualification result as
Shown in Fig. 6, in full accord with expection through electrophoresis detection restriction enzyme mapping stripe size.
In step (2), synthetic primer sequence is as table 3.
Table 3 builds and merges the primer that neo-EGFP double-tagging carrier adopts
(3), piggyBac transposon system element clone and vector construction
The pPB-UbC-EGFP-neo (Wellcome Trust Sanger gives at center) of take is template, use respectively primer Not I-5-PB-F (SEQ ID NO:9) and Not I-5-PB-R (SEQ ID NO:10), Xho I-3-PB-F (SEQ ID NO:11) and Xho I-3-PB-R (SEQ ID NO:12), by TaKaRa LA Taq enzyme pcr amplification Not I-5-PB--Not I, Xho I-3-PB-Xho I, use respectively after corresponding digestion with restriction enzyme, be connected in carrier pPSPBGPneo-XynB, it is completed
piggyBactransposon vector transformation, after transformation, called after pPSPBGPneo-PB-XynB, its enzyme is cut qualification result as shown in Figure 7, with Xho I and Not I enzyme, cut pPSPBGPneo-PB-XynB enzyme respectively and cut result demonstration, obtain 285bp and 349bp object band, electrophoresis two stripe size conform to expection.
The reaction system of PCR: by following component preparation PCR reaction solution.
PCR response procedures is: 94 ℃ of 30S; 55 ℃ of 30S, 35 circulations; 72 ℃ of 30S; 72 ℃ of 3min, 30 circulations.
Take pcDNA-neo-T2A-EGFP carrier as template, with primer infu-CMV-neo-EGFP-R (SEQ ID NO:13), infu-CMV-neo-EGFP-F (SEQ ID NO:14) amplification, obtains infusion-CMV-neo-T2A-EGFP fragment, cuts glue purification and reclaims standby.
By Cla I and Bgl II restriction enzyme double digestion pPSPBGPNeo-PB-XynB fragment, enzyme is cut product and is done agarose gel electrophoresis, and object fragment is cut to glue purification.Preferably application
advantage PCR Cloning Ki test kit (Clontech company) is recombinated infusion-CMV-neo-T2A-EGFP fragment in the centre, two loxp sites of pPSPBGPNeo-PB-XynB carrier, obtain pPSPBGPneo-T2A-EGFP-PB-XynB carrier, its enzyme is cut qualification result as shown in Figure 8, the electrophoresis result that plasmid is identified with Asc I and Bgl II double digestion, electrophoretic band size conforms to expection, successfully obtains the pPSPBGPneo-T2A-EGFP-PB-XynB transposon carrier with neo-EGFP double-tagging and xylanase gene (XynB).
PCR reaction system is as follows:
PCR response procedures is: 98 ℃ of 2min; 98 ℃ of 10s; 68 ℃ of 4min; 35 circulations; 72 ℃ of 10min.
In this step, the primer of piggyBac transposon main element clone and positive and negative evaluation is as shown in table 4, and pPSPBGPneo-T2A-EGFP-PB-XynB vector construction primer is as shown in table 5.
The primer of table 4piggyBac transposon main element clone and positive and negative evaluation
Table 5pPSPBGPneo-T2A-EGFP-PB-XynB vector construction primer
(4), parotid gland albumen upstream regulatory region and loxP-CMV-neoEGFP-loxp and PiggyBac
system merges
First utilize primer P11160-ASCIF (SEQ ID NO:15), P407-AgeIR (SEQ ID NO:16), take pPSPBGPneo-T2A-EGFP-PB-XynB carrier as template, uses
pPB-lox-neoEGFP-loxp fragment in Max DNA Polymerase enzyme (Takara company) amplification vector, and manually add AgeI restriction endonuclease sites, standby after purifying, be novel vector skeleton.PCR response procedures is: 98 ℃ of 1min; 98 ℃ of 10s; 60 ℃ of 5s; 72 ℃ of 2min; 35 circulations; 72 ℃ of 2min.Because mouse parotid gland protein promoter is too large, be difficult to together with other component construction, the present invention attempts a kind of new method, completes vector construction, and construction process refers to Fig. 9.
The first step: first utilize primer inf-AgeI-8700F (SEQ ID NO:17), inf-AscI-12540R (SEQ ID NO:18), the pcr amplified fragment of the mouse parotid gland albumen upstream regulatory region that the step (1) of take obtains is template, uses Prime
max DNAPolymerase enzyme (Takara company) amplification MusPSP downstream 4009bp fragment (fragment 1), add with novel vector skeleton pPB-lox-neoEGFP-loxp in recombination sequence, it is first recombinated on carrier, its enzyme is cut qualification result as shown in Figure 10, electrophoretic band size conforms to expection (4009bp), through sequence verification, be successfully completed fragment 1 and be connected with novel vector skeleton pPB-lox-neoEGFP-loxp.PCR response procedures: 98 ℃ of 1min; 98 ℃ of 10s; 60 ℃ of 5s; 72 ℃ of 2min; 35 circulations; 72 ℃ of 2min.
Second step: then utilize the restriction enzyme site StuI in MusPSP fragment 1, design primer inf-AgeI-4090F (SEQ ID NO:19), StuI-8763R (SEQ ID NO:20), the pcr amplified fragment of the mouse parotid gland albumen upstream regulatory region that the step (1) of take obtains is template, uses
the fragment 2 of Max DNA Polymerase enzyme (Takara company) amplification 4706bp.The StuI site of the intermediate carrier fragment 1 that the first step of being recombinated obtains, its enzyme is cut qualification result as shown in Figure 11, and restriction enzyme digestion and electrophoresis stripe size conforms to expection 4706bp, through sequence verification, is successfully completed the clone of fragment 2.PCR response procedures: 98 ℃ of 1min; 98 ℃ of 10s; 60 ℃ of 5s; 72 ℃ of 2min; 35 circulations; 72 ℃ of 2min.
The 3rd step: design primer inf-AgeI-440F (SEQ ID NO:21), inf-AgeI-4000R (SEQ ID NO:22), the pcr amplified fragment of the mouse parotid gland albumen upstream regulatory region that the step (1) of take obtains is template, uses
the fragment 3 of Max DNA Polymerase enzyme (Takara company) amplification 3956bp, utilize artificial AgeI site of adding, the fragment of last 3956bp is recombinated on second step acquisition intermediate carrier, complete whole vector construction pPB-MusPSP-neo-EGFP, be the specific transgene carrier of salivary organization of the present embodiment, its enzyme cut qualification result as shown in Figure 12 restriction enzyme digestion and electrophoresis stripe size conform to expection, through sequence verification, be successfully completed the clone of fragment 3.Reserved ASCI site can be inserted for any gene fragment.PCR response procedures: 98 ℃ of 1min; 98 ℃ of 10s; 60 ℃ of 5s; 72 ℃ of 2min; 35 circulations; 72 ℃ of 2min.
In this step, the primer of employing is as shown in table 6.
The primer adopting in table 6 pPB-MusPSP-neo-EGFP vector construction
The carrier that embodiment 2 utilizes embodiment 1 to build screens transgenic cell
Adopt ordinary method, the pPB-MusPSP-neo-EGFP carrier By Transfecting Porcine fetal fibroblast building with embodiment 1, by G418 screening observations after 14 days.As shown in figure 13, result shows that this carrier, under auxiliary enzymes is collaborative, improves the efficiency of transgenic cell screening greatly to result.
Its screening efficiency is carried out to flow cytometer showed result as shown in figure 14, as can be seen from Figure 14, along with mPB/PSP mol ratio raises, Z1～Z6 group obtains green fluorescence cell count and is the rear downward trend that first rises, mPB/psp mol ratio obtains green fluorescence cell positive rate when reaching 1/4 the highest, having reached 22.16%, 1116 positive cell clone, is 65.65 times of common transfection linearization plasmid (contrasting 1).The transgenic positive number of cell clones that mPB/PSP mol ratio obtains 1/1 time is maximum, reaches 1303 cells, and positive rate is 19.86%, 76.65 times of common transfection linearization plasmid (contrasting 2).Screening efficiency is greatly improved.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
- The construction process of the specific transgene carrier of 1.Yi Zhong salivary organization, is characterized in that, comprises the following steps:(1), take respectively plasmid pcDNA3.1 (+) and pT2AL200R175-CAGGS-EGFP as template, SEQ ID NO:5 and SEQ ID NO:6 and SEQ ID NO:7 and SEQ ID NO:8 are that primer carries out pcr amplification for the first time, obtain neo gene and EGFP gene with some overlaps; Respectively get after 0.5-3 μ L mixes and carry out pcr amplification for the second time as template, purifying obtains neo-EGFP fusion gene; With EcoR I and Xba I double digestion pcDNA3.1 (+) and neo-EGFP fusion gene, purifying reclaims enzyme and cuts product, connects, and builds and obtains pcDNA-Neo-T2A-EGFP carrier;(2), take plasmid pPB-UbC-EGFP-neo as template, using SEQ ID NO:9 and SEQ ID NO:10 and SEQ ID NO:11 and SEQ ID NO:12 respectively as primer, carry out pcr amplification and obtain Not I-5-PB-Not I, Xho I-3-PB-Xho I, after adopting respectively Not I and Xho I enzyme to cut, be connected to carrier pPSPBGPneo-XynB upper, obtain carrier pPSPBGPneo-PB-XynB;(3), take the pcDNA-neo-T2A-EGFP carrier that step (1) obtains is template, use LA-PCR system, SEQ ID NO:13 and SEQ ID NO:14 carry out pcr amplification as primer and obtain infusion-CMV-neo-T2A-EGFP fragment, cut glue purification; The carrier pPSPBGPneo-PB-XynB obtaining by Cla I and Bgl II double digestion step (2), cuts glue purification to object fragment; Infusion-CMV-neo-T2A-EGFP fragment is recombinated in the middle of two loxp sites of pPSPBGPNeo-PB-XynB carrier, obtain pPSPBGPneo-T2A-EGFP-PB-XynB carrier;(4), take the pPSPBGPneo-T2A-EGFP-PB-XynB carrier that step (3) obtains is template, SEQ ID NO:15 and SEQ ID NO:16 are as primer, carry out pcr amplification, obtain the pPB-lox-neoEGFP-loxp fragment in carrier, and add AgeI restriction endonuclease sites, purifying, obtains novel vector skeleton;(5), take the pcr amplified fragment of mouse parotid gland protein gene upstream control region is template, SEQ ID NO:17 and SEQ ID NO:18 are primer, amplification obtains the downstream 4009bp fragment of mouse parotid gland albumen upstream regulatory region, recombination sequence in 4009bp fragment and novel vector skeleton pPB-lox-neoEGFP-loxp is recombinated, obtain intermediate carrier 1;(6), the pcr amplified fragment of mouse parotid gland protein gene upstream control region of take is template, take SEQ ID NO:19 and SEQ ID NO:20 is primer, amplification obtains the downstream 4706bp fragment of mouse parotid gland albumen upstream regulatory region; The StuI site of the intermediate carrier 1 of the step of being recombinated (5), is intermediate carrier 2;(7), the pcr amplified fragment of mouse parotid gland protein gene upstream control region of take is template, take SEQ ID NO:21 and SEQ ID NO:22 is primer, utilizes AgeI site, amplification obtains the downstream 3956bp fragment of mouse parotid gland albumen upstream regulatory region; On the intermediate carrier 2 of the step of being recombinated (6), obtain carrier pPB-MusPSP-neo-EGFP, be the specific transgene carrier of salivary organization.
- 2. the construction process of the specific transgene carrier of salivary organization according to claim 1, it is characterized in that, the cloning process of the pcr amplified fragment of the mouse parotid gland protein gene upstream control region in described step (5)-(7) is: take FVB mouse gene group DNA as template, SEQ ID NO:1 and SEQ ID NO:2 are primer, and KOD-FX polysaccharase carries out pcr amplification for the first time; The product increasing for the first time of take is template, and SEQ ID NO:3 and SEQ ID NO:4 are primer, carry out pcr amplification for the second time, obtain.
- 3. the construction process of the specific transgene carrier of salivary organization according to claim 2, is characterized in that, the response procedures of described pcr amplification is for the first time: 94 ℃ of 2min; 98 ℃ of 10s, 74 ℃ of 13min, 5cycles; 98 ℃ of 10s, 72 ℃ of 13min, 5cycles; 98 ℃ of 10s, 70 ℃ of 13min, 5cycles; 98 ℃ of 10s, 68 ℃ of 13min, 30cycles; 68 ℃ of 7min; The response procedures of described pcr amplification is for the second time: 94 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 13min, 30cycles; 68 ℃ of 7min.
- 4. the construction process of the specific transgene carrier of salivary organization according to claim 1, is characterized in that, described in step (1), the response procedures of PCR is for the first time: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 50s, 35 circulations; 72 ℃ of 10min; PCR response procedures for the second time: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 1min40s, 35 circulations; 72 ℃ of 10min.
- 5. the construction process of the specific transgene carrier of salivary organization according to claim 1, is characterized in that, described in step (2), the response procedures of PCR is: 94 ℃ of 30s; 55 ℃ of 30s, 35 circulations; 72 ℃ of 30s, 72 ℃ of 3min, 30 circulations; Described in step (3), the response procedures of PCR is: 98 ℃ of 2min; 98 ℃ of 10s, 68 ℃ of 4min, 35 circulations; 72 ℃ of 10min; Described in step (4)-(7), the response procedures of PCR is: 98 ℃ of 1min; 98 ℃ of 10s; 60 ℃ of 5s; 72 ℃ of 2min; 35 circulations; 72 ℃ of 2min.
- 6. the construction process described in claim 1-5 any one builds the specific transgene carrier of salivary organization obtaining.
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