CN103695451B - The transgene carrier of salivary gland tissue specific expression foreign protein and transgenic pig and construction process thereof - Google Patents
The transgene carrier of salivary gland tissue specific expression foreign protein and transgenic pig and construction process thereof Download PDFInfo
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Abstract
The invention discloses a kind of transgene carrier and construction process thereof of salivary gland tissue specific expression foreign protein, described expression vector obtains by being inserted in the specific transgene carrier of parotid gland tissues to build by the fusion gene of glucanase gene, xylanase gene and phytase gene structure restructuring; By the genome of this vector introduction pig, and somatic cell nuclear transfer technique is adopted to produce transgenic pig.The present invention is directed to barley material dextranase, zytase in wheat, corn, and the phytase in plant feed there is hydrolytic action, utilize pig sialisterium as reactor, secreted throughout life, reaches the permanent interpolation replacing these zymins in feed, better plays the effect of these enzymes; Expression vector of the present invention has piggyBac transposon, drastically increases transgene efficiency, and the integrated transgene pattern of ordinary plasmids series connection restructuring is become unit single point copy integration, the better environment of simulation biological gene.
Description
Technical field
The technology of the present invention belongs to genetically engineered field, more specifically, the present invention relates to a kind of transgene carrier of salivary gland tissue specific expression foreign protein and transgenic pig and construction process thereof.
Background technology
Beta-glucan belongs to the structural non-starch polysaccharide in plant cell wall, by β-1,3 and β-1, the D type glucose polimer that 4 glycosidic links are formed by connecting, molecular weight, greatly about more than 6500KDa, divides water-soluble and water-insoluble two kinds, its solvability is subject to the impact of glycosidic link content and the polymerization degree in structure, extensively be present in higher plant cell wall, in the Cereal farm crop Formation of Endosperm Cell Walls such as barley, wheat, rye, content is high, and in barley, beta-glucan content is about 4%-10%.Beta-glucanase (β-glucanase) can be hydrolyzed the beta-glucan in the cereal such as barley, wheat and rye, β-1,3 and β-Isosorbide-5-Nitrae glycosidic link in catalytic pyrolysis beta-glucan molecule, and degraded generates small molecules oligosaccharides and glucose.
The content of xylan in plant cell wall is only second to Mierocrystalline cellulose, is the one that in hemicellulose, content is the abundantest, and in all feeds raw material particularly wheat class, cereal byproduct and the assorted dregs of rice, content is more.Xylan is (with β-1 by D-wood sugar main chain, 4 keys are connected) and the polymkeric substance that forms of L-arabinose branch (a-1,2 and a-1,3 is connected), because it is polymerized by pectinose and wood sugar two kinds of monose, therefore also claim araboxylan.Glycan itself is difficult to be absorbed by simple stomach animal digestion, and araboxylan forms a kind of afterbirth agent, the substrates such as plant cell wall internal protein, carbohydrate, fat are hindered to be combined with digestive ferment, make the stickiness of digest improve simultaneously, impeding nutritious substance is fat, the digestion of protein, absorption and utilization especially, reduces the transformation efficiency of feed.Glycan can directly in conjunction with the digestive ferment such as trypsinase, lipase in digestive tube, make it actively reduce, simultaneously and biliary salts, lipid, cholesterol combination, affect the absorption of lipid at small intestine.Araboxylan improves the viscosity of animal intestinal Zhong Shi Porridge, reduces the translational speed of Shi Porridge in digestive tube, and promote a large amount of propagation of bacterium, livestock and poultry diarrhea rate is increased, and the movement comprising nitrogen and phosphorus also increases, and causes the pollution to environment.Zytase is the enzyme of single-minded degradation of xylan, belongs to hydrolase, can be wood oligose by xylanolitic.Zytase is formed primarily of the de-side chain enzyme such as β-Isosorbide-5-Nitrae-D-endo-xylanase, β-circumscribed xylosidase of Isosorbide-5-Nitrae-D-and α-L-arabinose glycosides enzyme, α-D-amino acid aldehydic acid enzyme.
Phytic acid is ubiquitous a kind of antinutritional factor in plant feed, and all plant feeds all contain the phytate of 1%-5%, and the phosphorus content of these salt accounts for the 60%-80% of the total phosphorus content of feed.Due in monogastric animal digestive tube not containing phytase, cause it that phosphorus in plant feed, phytate phosphorus major part cannot or can not be utilized very well to be difficult to by pig and fowl utilize and excrete with ight soil, contaminate environment.Phytic acid has very strong complex ability in addition, can form stable mixture, thus reduce the utilization ratio of these nutritive substances with many mineral elements and protein complexation.Phytase can make phytate phosphorus be degraded into inositol and phosphoric acid, thus reduces the addition of the inorganic phosphorus such as Dietary phosphorus acid hydrogen calcium, and in addition, result of study has also found the potential nutritive value of phytase: the digestibility that can improve protein and energy in feed.Being applied in of phytase certain degree can be alleviated China's phosphor resource scarcity, the waste reducing phosphor resource, reduce phosphorus and discharge the pollution brought.
Dextranase, zytase, phytase are the most frequently used exogenous enzyme preparations of animal feedstuff additive.Digestibility and the utilization ratio that fodder enzyme preparation can improve feed is applied in livestock product cultivation; improve the production performance of livestock and poultry; the excretion of the nitrogen in farm animal excrement, phosphorus can be reduced again; protection water body and soil from pollution, be a class efficient, have no side effect and environment-friendly type " green " fodder additives.Feed enzyme preparation adds the utilization of raising Animal nutrition, reduces costs certain active effect, also there is many problems simultaneously.The character of exploitation fodder enzyme is excellent not absolutely mostly at present, and few enzyme can at pH, thermostability, antipepsin, and each advantage is gathered in the ability aspects such as trypsinase; The interpolation cost restriction enzyme preparation of feed enzyme preparation uses, and in order to not increase aquaculture cost, feed enzyme preparation cost per ton is all no more than 100 yuan (Yang Peilong etc., 2009).In addition, these external source digestive ferment animals self can not secrete, all first utilize fermentable to obtain corresponding enzyme all the time, add in feed artificial, adding additive in feed to easily affects by factors such as feed granulating, expanded, storages, and its vigor and stability aspect thereof often affect by various factors, zymin is caused to be added on DeGrain in actual production, and need constantly to add, effect is undesirable, and cost is high.
Transgenic technology is that the solution of this problem provides technical support.Transgenic animal technology is generally utilize genetic engineering means to build transgene carrier, by the microorganisms such as intestinal bacteria massive duplication in its body, and then obtain a large amount of target DNA fragment, then transgenic animal are obtained by technology such as procaryotic injection, intracytoplasmic sperm injection (intracytoplasmicsperminjection, ICSI) or screening transgenic Cell binding somatic cell clones.Transgene carrier imports zooblast by electroporation, liposome or virus, and then can obtain transgenic cell through drug screening, the technology of carrying out somatic cell clone is also obtain transgenic pig most popular method at present.But all there is various shortcoming and defect in these methods.As low in transgenic animal preparation efficiency; The foreign gene proceeded to is heredity, unstable expression etc. in animal body.Traditional transgene carrier fragment is often in multi-copy in tandem mode (concatamer), passively, random integration is to the one or more site of genome, not only inefficiency, also cause transgene Inheritance and expression instability (Garricketal, 1998; Scrableetal, 1999).The research of transgenosis environmental protection pig also relates to the problem such as polygene coexpression, carrier excessive (> 15kbp), the transgenic cell line of the more difficult acquisition genetic stability of traditional transgene carrier.Though virus vector integration efficiency is high, the safety problem that its supporting capacity finite sum may exist limits it and applies further.
Along with the fast development of China's pig industry, Swine Production is faced with feed cost constantly soaring pressure and the challenge cultivating environmental protection.Improve pig very urgent to discharges of major pollutant such as nitrogen phosphorus in feed nutrition element absorption and deposition, reduction pig farm refuse.
Environment-friendly type transgenic animal production principle utilizes existing transgenic technology and method by organizing specific expression transgene carrier, the controlling element of external source digestive ferment gene and organizing specific expression thereof is transported and is incorporated in Animal genome, these external source digestive ferment genes are under the regulation and control of its tissue specific promoter, realize continuous expression and secretion external source digestive ferment in particular animals organ-tissue, these secretion digestive ferments in the specific secretory of animal together with animal autodigestion enzyme direct secretion to animal digestive tract, go the antinutritional factor that digestion animal autodigestion enzyme can not digest.Comparing successful story is at present obtained by procaryotic injection to turn escherichia coli phytase gene pig SergueiP.Golovan (2001), agricultural university of China turns phytase pig (2006), but due to feed antinutritional factor complicated component, kind is many, as in non-starch polysaccharide: corn xylan, dextran in wheat class plant, xylan, mannosans in soybean, these environmentally friendly pigs only can meet the decomposition of phytate phosphorus, absorb, non-starch polysaccharide (dextran is digested and assimilated to other influences Animal nutrition, xylan, mannosans etc.) antinutritional factor is invalid, also do not reach the object that joint grain reduces discharging.And these transgenic pigs are all adopt the preparation of traditional random integration technology, transgene fragment often repeats with multiple copied of connecting, and Stability of Transgenic reduces; Random integration, transgenic loci is not easily identified.
Parotid gland albumen is the albumen that in the parotid gland, gene expression abundance is the highest.Parotid gland protein gene is positioned on mouse No. 2 karyomit(e)s, and in genome, this gene exists with single copy form, and main at parotid gland specifically expressing, also has research its submaxillary gland at sialisterium of display and sublingual gland also to have low expression.This gene of research display is is mainly regulated and controled by the upstream regulatory region (11.5Kb) of parotid gland protein gene and downstream sequence (about 2.5 ~ 3kb) (i.e. parotid gland protein promoter PSP), but its regulatory gene is regulating and expressing in sialisterium only, do not express at other histoorgans of whole body.Sialisterium express the transgenic animal principle of 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, plays its function.
Summary of the invention
Based on this, in order to overcome the defect of above-mentioned prior art, the invention provides the transgene carrier of a kind of salivary gland tissue specific expression foreign protein multiple enzymes such as (self secretion) dextranase-zytase-phytases and transgenic pig and construction process thereof, thus improve animal to feed nutrition utilization ratio, reach and subtract phosphorus, nitrogen discharged object, realizes research and production that joint grain reduces discharging environment-friendly type transgenic animal.
In order to realize foregoing invention object, this invention takes following technical scheme:
A construction process for the transgene carrier of salivary gland tissue specific expression foreign protein, comprises the following steps:
(1), utilize the 2A peptide sequence of E2A, P2A and T2A that the phytase gene APPA shown in xylanase gene XynB and SEQIDNO:4 shown in glucanase gene Eg1314, the SEQIDNO:3 shown in glucanase gene Bg17, the SEQIDNO:2 shown in SEQIDNO:1 is built recombinant beta-glucanase gene-xylanase gene-phytase gene fusion gene Bg17-E2A-Eg1314-flag-P2A-XynB-T2A-APPA-HA;
(2), respectively with plasmid pcDNA3.1 (+) and pT2AL200R175-CAGGS-EGFP for template, SEQIDNO:5 and SEQIDNO:6 and SEQIDNO:7 and SEQIDNO:8 is that primer carries out first time pcr amplification, obtains the neo gene with some overlaps and EGFP gene; Respectively get after 0.5-3 μ L mixes and carry out second time pcr amplification as template, purifying obtains neo-EGFP fusion gene; With EcoRI and XbaI double digestion pcDNA3.1 (+) and neo-EGFP fusion gene, purifying reclaims digestion products, connects, and builds and obtains pcDNA-Neo-T2A-EGFP carrier;
(3), with plasmid pPB-UbC-EGFP-neo for template, respectively using SEQIDNO:9 and SEQIDNO:10 and SEQIDNO:11 and SEQIDNO:12 as primer, carry out pcr amplification and obtain NotI-5-PB-NotI, XhoI-3-PB-XhoI, after adopting NotI and XhoI enzyme to cut respectively, be connected on carrier pPSPBGPneo-XynB, obtain carrier pPSPBGPneo-PB-XynB;
(4) the pcDNA-neo-T2A-EGFP carrier, obtained with step (2) is for template, use LA-PCR system, SEQIDNO:13 and SEQIDNO:14 carries out pcr amplification as primer and obtains infusion-CMV-neo-T2A-EGFP fragment, cuts glue purification; With the carrier pPSPBGPneo-PB-XynB that ClaI and BglII double digestion step (2) obtains, glue purification is cut to object fragment; Infusion-CMV-neo-T2A-EGFP fragment is recombinated to pPSPBGPNeo-PB-XynB carrier two loxp sites in the middle of, obtain pPSPBGPneo-T2A-EGFP-PB-XynB carrier;
(5) the pPSPBGPneo-T2A-EGFP-PB-XynB carrier, obtained with step (4) is for template, SEQIDNO:15 and SEQIDNO:16 is 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;
(6), with the pcr amplified fragment of mouse parotid gland protein gene upstream control region for template, SEQIDNO:17 and SEQIDNO:18 is 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, obtains intermediate carrier 1;
(7), with the pcr amplified fragment of mouse parotid gland protein gene upstream control region for template, with SEQIDNO:19 and SEQIDNO:20 for primer, amplification obtains the downstream 4706bp fragment of mouse parotid gland albumen upstream regulatory region; The StuI site of the intermediate carrier 1 of step of being recombinated to (6), is intermediate carrier 2;
(8), with the pcr amplified fragment of mouse parotid gland protein gene upstream control region for template, with SEQIDNO:21 and SEQIDNO:22 for primer, utilize AgeI site, amplification obtains the downstream 3956bp fragment of mouse parotid gland albumen upstream regulatory region; On the intermediate carrier 2 of step of being recombinated to (7), obtain carrier pPB-MusPSP-neo-EGFP;
(9) the ASCI site of the carrier pPB-MusPSP-neo-EGFP that the beta-glucanase gene-xylanase gene, by step (1) obtained-phytase gene fusion gene Bg17-E2A-Eg1314-flag-P2A-XynB-T2A-APPA-HA in-fusion recombinase (Clontech company) reconstitution steps (8) obtains, the transgene carrier of salivary gland tissue specific expression foreign protein
PPB-MusPSP-neo-EGFP-BgEgXyAp, sequence number is SEQIDNO:35.
Wherein in an embodiment, described in step (1), the construction process of fusion gene BgEgXyAp comprises the following steps:
(1), with SEQIDNO:27, SEQIDNO:28 for primer, SEQIDNO:36 is template amplification Bg17-E2A;
(2), with SEQIDNO:29, SEQIDNO:30 for primer, SEQIDNO:36 is template amplification E2A-Eg1314;
(3), utilize Overlap extension PCR, with SEQIDNO:27, SEQIDNO:30 for primer, with mol ratio be 1: 1 mixing Bg17-E2A and E2A-Eg1314 for template, obtain Bg17-E2A-Eg1314-flag;
(1) in-(3), the response procedures of pcr amplification is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 1min; 35 circulations; 72 DEG C of 2min.
(4), with SEQIDNO:31, SEQIDNO:32 for primer, to insert the pcDNA3.1 carrier of XYNB gene fragment for template, amplification obtains P2A-XynB-T2A;
(5), with SEQIDNO:33, SEQIDNO:34 for primer, to insert appa gene fragment to pcDNA3.1 carrier for template, amplification obtains T2A-APPA-HA;
(6), with SEQIDNO:27, SEQIDNO:34 for primer, mix as template using Bg17-E2A-Eg1314-flag, P2A-XynB-T2A, T2A-APPA-HA that mol ratio is 1: 1: 1, amplification obtains fusion gene BgEgXyAp;
(4) in-(6), the response procedures of pcr amplification is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
Wherein in an embodiment, the cloning process of the pcr amplified fragment of the mouse parotid gland protein gene upstream control region in described step (6)-(8) is: with FVB mouse gene group DNA for template, SEQIDNO:23 and SEQIDNO:24 is primer, and KOD-FX polysaccharase carries out first time pcr amplification; With the product of first time amplification for template, SEQIDNO:25 and SEQIDNO:26 is primer, carries out second time pcr amplification, to obtain final product.
Wherein in an embodiment, in described step (6)-(8), the response procedures of pcr amplification is for the first time: 94 DEG C of 2min; 98 DEG C of 10s, 74 DEG C of 13min, 5cycles; 98 DEG C of 10s, 72 DEG C of 13min, 5cycles; 98 DEG C of 10s, 70 DEG C of 13min, 5cycles; 98 DEG C of 10s, 68 DEG C of 13min, 30cycles; 68 DEG C of 7min; The response procedures of described second time pcr amplification is: 94 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 13min, 30cycles; 68 DEG C of 7min.
Wherein in an embodiment, described in step (2), the response procedures of PCR is for the first time: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 50s, 35 circulations; 72 DEG C of 10min; Second time PCR response procedures: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 1min40s, 35 circulations; 72 DEG C of 10min.
Wherein in an embodiment, described in step (3), the response procedures of PCR is: 94 DEG C of 30s; 55 DEG C of 30s, 35 circulations; 72 DEG C of 30s, 72 DEG C of 3min, 30 circulations; Described in step (4), the response procedures of PCR is: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 4min, 35 circulations; 72 DEG C of 10min; Described in step (5)-(8), the response procedures of PCR is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
Present invention also offers the transgene carrier pPB-MusPSP-neo-EGFP-BgEgXyAp that above-mentioned construction process builds the salivary gland tissue specific expression foreign protein obtained, sequence number is SEQIDNO:35, can the multiple enzyme such as oneself expression dextranase-zytase-phytase.
Present invention also offers a kind of construction process of transgenic pig of salivary gland tissue specific expression foreign protein, comprise the following steps:
(1), by above-mentioned transgene carrier pPB-MusPSP-neo-EGFP-BgEgXyAp and transposase plasmids PCMV-mpb mixing By Transfecting Porcine fetal fibroblast cell line, the mono-clonal transgenic cell line of screening Absorbable organic halogens expression vector;
(2), using the mono-clonal transgenic cell line of acquisition as nuclear donor cell, be expelled in ovocyte, build reconstruct embryo, adopt traditional breeding method to cultivate, the transgenic pig of polygene coexpression can be obtained.
Wherein in an embodiment, the mole ratio of described transgene carrier pPB-MusPSP-neo-EGFP-BgEgXyAp and transposase plasmids PCMV-mpb is 1: 1.
Wherein in an embodiment, the screening step of the mono-clonal transgenic cell line of described Absorbable organic halogens expression vector is: by the plating cells after transfection after 36 hours, go down to posterity according to 1: 6, first in cell screening substratum, G418300ug/ml is added, to the cell screening after transfection after 5 days, use 200ug/ml concentration again instead and continue screening 10 days, then change 80ug/ml into and maintain screening, in 15%FBS peptide bovine serum substratum, add 2.5-5ng/mLBFGF simultaneously, obtain the cell mass being with green fluorescence, grow up to after sheet until cell mass, carefully scrape off and do not have fluorocyte around, go down to posterity with 0.05% trysinization, after reaching 2-3 generation, detect green fluorescence again to express.Selecting green fluorescence expresses bright, the normal cell of cellular form, i.e. mono-clonal transgenic cell line; The formula of described cell screening substratum is: 42.5%GlutaMAX
tM, 42.5% DMEM in high glucose, 15%FBS.
Present invention also offers the transgenic pig that above-mentioned construction process builds the salivary gland tissue specific expression foreign protein obtained, can the multiple enzyme such as oneself expression dextranase-zytase-phytase.
The present invention passes through two for restructuring codon optimized for pig glucanase gene (Bg17/eg1314, source Bisporasp.MEY-1/BacilluslicheniformisEGW039), acidic xylanase gene (the XYNB that pig is codon optimized, Aspergillus niger origin), the phytase gene (APPA, source intestinal bacteria) that pig is codon optimized.Remove himself signal peptide sequence respectively, artificial interpolation pig parotid gland protein signal peptide sequence (PSPsignalpiptide), builds the recombinase had at parotid gland tissues secreting function.Then by 2A sequence small peptides such as T2A, P2A, E2A, by 4 gene fusion to together, fusion gene BgEgXyAp is obtained.Be placed on FVB mouse regulation and control upstream downstream (i.e. the ASCI site of carrier pPB-MusPSP-neo-EGFP), be built into pPB-MusPSP-neo-EGFP-BgEgXyAp transgene carrier.
By pPB-MusPSP-neo-EGFP-BgEgXyAp transgene carrier and transposase being mixed, cotransfection porcine fetus fibroblasts, through drug screening, obtains transgenic cell line.Using the somatocyte of acquisition as nuclear donor cell, be expelled in ovocyte, build reconstruct embryo, through external incubated overnight, moved in the acceptor sow uterine tube of Estrus synchronization.Acceptor sow through gestation and childbirth, and carries out transgenic pig fluoroscopic examination, inverse PCR, southernbloting, transgenic pig saliva Enzyme activity assay.
Compared with prior art, the present invention has following beneficial effect:
1, the present invention sets about from breeding technique, disposable transfer according to the codon optimized glucanase gene (Bg17/Eg1314) of pig, zytase (XynB), phytase (AppA) 4 resistance to acid genes.For wheat class raw material dextranase, corn zytase, and the phytase in plant feed has hydrolytic action, document display Bg17 also has certain degradation capability to Xylo-Mucine (CMC-Na).Utilize pig sialisterium as reactor, secreted throughout life, reach the permanent interpolation replacing these zymins in feed, better play the effect of these enzymes;
2, in transgenic technology, the present invention adopts piggybac system, solve transgene efficiency problem, integrated transgene pattern is recombinated by multiple copied of connecting end to end, become unit single point copy to integrate, the environment of better simulation biological gene, realizes transgenic animal Rapid identification and integration site detects.Transgenic pig of the present invention, can coexpression restructuring dextranase, zytase, phytase 3 kinds of enzymes in its sialisterium, and transgene both can Hydrolysis of Phytic Acid enzyme, can be hydrolyzed again.At present the antinutritional factor such as non-starch polysaccharide in main feedstuff raw material, turning phytase transgenic pig than mentioning in document, advantageously, achieving polygene coexpression technology, improve large fragment gene transfering efficiency problem.
3, the present invention builds the transgenic pig obtained and also carries EGFP green fluorescent label, is convenient to detect, and its loxp recombination site, also can facilitate the later stage to delete.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention 1 Bg17, Eg1314, XYNB, APPA and fusion gene BgEgXyAp at the enzyme activity determination result figure of mammalian cell;
Fig. 2 is fusion gene in the embodiment of the present invention 1
Bg17-E2A-Eg1314-flag-P2A-XynB-
The recombination to construct schematic diagram of T2A-APPA-HA (BgEgXyAp);
Fig. 3 is the PCR collection of illustrative plates of cloning the MusPSP obtained in embodiment 1, and wherein A is MusPSP upstream regulatory region clone (-11503bp ~+1390bp); B is MusPSP upstream regulatory region clone (-11325bp ~+980bp);
Fig. 4 is that the enzyme of Neo-T2A-EGFP in embodiment 1 cuts qualification electrophorogram, wherein 1,2,3 represents T2A-EGFP (765bp) respectively, neo-T2A (835bp), neo-T2A-EGFP (1600bp);
Fig. 5 is that the enzyme of pcDNA-Neo-T2A-EGFP in embodiment 1 cuts qualification electrophorogram, wherein 1, the 2 object band 1590bp representing pcDNA-Neo-T2A-EGFP, 3 plasmid control representing 1 and 2;
Fig. 6 is that the enzyme of pPSPBGPneo-PB-XynB carrier in embodiment 1 cuts qualification electrophorogram, and wherein M represents Marker2000; 1,2 is pPSPBGPneo-PB-XynB plasmid control, and 3,4 enzymes representing this plasmid XhoI and NotI respectively cut result;
Fig. 7 is that the enzyme of pPSPBGPneo-T2A-EGFP-PB-XynB carrier in embodiment 1 cuts qualification electrophorogram, and wherein M represents DLDNA15000Marker; Swimming band 1 represents the contrast of pPSPBGPneo-T2A-EGFP-PB-XynB vector plasmid, and 2,3 enzymes representing 1 cut result, band 2935bp for the purpose of arrow indication;
Fig. 8 is the structure schematic diagram that embodiment 1 small mouse parotid gland albumen upstream regulatory region (MusPSP) is cloned;
Fig. 9 is MusPSP control region fragment 1 (-3143bp ~+865bp) cloned plasmids AscI in embodiment 1, and NotI enzyme cuts qualification collection of illustrative plates; Wherein be with 1,2,3,4,5 clone enzyme for difference cuts qualification, arrow instruction object band;
Figure 10 is that in embodiment 1, MusPSP control region fragment 2 (-7849bp ~-3143) AgeI, stuI enzyme cuts qualification collection of illustrative plates; Wherein be with 1,2,3 cut qualification for different clone strain enzyme;
Figure 11 is that in embodiment 1, MusPSP control region fragment 3 (-11805pb ~-7849bp) AgeI enzyme cuts qualification collection of illustrative plates; Wherein be with 1,2,3,4 clone enzyme for difference cuts qualification;
Figure 12 is that the ASCI enzyme building the PPB-MusPSP-neoEGFP-BgEgXyAp transgene carrier obtained in embodiment 1 cuts qualification collection of illustrative plates; Wherein M represents DLmarker15000, and the enzyme of the different clone of band 1,2,3,4 cuts qualification;
Figure 13 is the structure collection of illustrative plates building the PPB-MusPSP-neoEGFP-BgEgXyAp transgene expression vector obtained in embodiment 1;
Figure 14 is the fluoroscopic examination figure building the transgenic pig obtained in embodiment 2;
Figure 15 is that the PCR of the transgenic pig of the embodiment of the present invention 2 detects collection of illustrative plates, and wherein No. Lane1-12 is followed successively by M:DL2000Marker; P: plasmid positive control (positive); W: water; N: wild-type pig (nagative) DNA; Lane5-12: transgenic pig DNA sample;
Figure 16 is that the SouthernBloting in the transgenic pig F0 generation of the embodiment of the present invention 2 detects collection of illustrative plates, wherein, and M: the Marker of digoxigenin labeled; 6C, 10C4C are respectively the positive control of 6,10,4 copies; 607 is the contrast of negative pig; 605 and 707 is transgenic positive pig;
Figure 17 is the transgenic pig 605 and 707 integration site sequencing analysis figure of the embodiment of the present invention 2;
Figure 18 is to dextranase in the saliva of transgenic pig in embodiment 3, zytase, the mensuration figure of phytase activity power.
Embodiment
The present invention is described in detail below in conjunction with the drawings and specific embodiments.
If no special instructions, the reagent used in following examples all derives from commercially available, and working method is existing conventional practices.
The structure of embodiment 1 transgene expression vector pPB-MusPSP-neo-EGFP-BgEgXyAp
Comprise the following steps:
1, the acquisition of goal gene
According to bibliographical information, screen a series of acidproof glucanase gene (cel4T (alicyclic acid genus bacillus) lived at protokaryon or lower eukaryotes (Yeast system) high enzyme, beta-1, 3 (4)-glucanase (paecilomyces), Bisporasp.MEY-1 source Bg17A, Bacillus licheniformis source eg1314, the C-APPA in Citrobacterfreundii source, and the AppA of Escherichia coli, the XYNB in aspergillus niger source, these genes are removed corresponding its own signal peptide sequence, add pig parotid gland protein signal peptide mature peptide sequence, and codon optimized according to pig after through row synthetic.Then at mammalian expression system pig kidney PK15 cell expressing, screening can secrete at pig cell the gene to have high enzymatic activity, the present invention filter out be adapted at mammlian system secreting, expressing pig codon optimized after glucanase gene Bg17 (SEQIDNO:1) and Eg1314 (SEQIDNO:2), xylanase gene (XynB, SEQIDNO:3), phytase gene (APPA, SEQIDNO:4).
2, the structure of fusion gene
Utilize E2A P2A the 2A peptide sequence such as T2A by these gene constructed recombinant beta-glucanase gene-xylanase gene-phytase gene fusion genes (Bg17-E2A-Eg1314-flag-P2A-XynB-T2A-APPA-HA), and after its Eg1314 gene, add flag-label, add after appa gene HA-label be convenient to the later stage to expression Protein Detection, the fusion gene of structure is abbreviated as BgEgXyAp.Bg17, Eg1314, XYNB, APPA and fusion gene BgEgXyAp mammalian cell enzyme activity determination result as shown in Figure 1.Fig. 1 display can express the enzyme gene Bg17 with biological function at mammlian system through pig codon optimized and mammalian cell expression screening acquisition 4 kinds, Eg1314, XYNB, APPA, by 4 genes through adding detection label and obtaining BgEgXyAp after merging restructuring, there is the biological function of 4 enzymes.
Refer to Fig. 2, be the recombination to construct schematic diagram of beta-glucanase gene-xylanase gene-phytase gene fusion gene Bg17-E2A-Eg1314-flag-P2A-XynB-T2A-APPA-HA (BgEgXyAp), comprise the following steps:
(1), with N-3G-1F (SEQIDNO:27), Bg17-E2A-R (SEQIDNO:28) is as primer, pUC-Bg17-a3-eg1314-flag (SEQIDNO:36 entrusts Nanjing Jin Sirui synthetic) is template amplification Bg17-E2A (fragment 1);
(2), with E2A-Eg-1F (SEQIDNO:29), E2A-2R (SEQIDNO:30) is as primer, pUC-Bg17-a3-eg1314-flag (SEQIDNO:36 entrusts Nanjing Jin Sirui synthetic) is template amplification E2A-Eg1314 (fragment 2);
(3), Overlap extension PCR is utilized, with N-3G-1F (SEQIDNO:27), E2A-2R (SEQIDNO:30) is primer, to mix Bg17-E2A (fragment 1) and E2A-Eg1314 (fragment 2), for amplification template, (fragment 1: fragment 2=1: 1), obtains Bg17-E2A-Eg1314-flag;
In (1)-(3), the response procedures of pcr amplification is above: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 1min; 35 circulations; 72 DEG C of 2min.
(4), with N-XYB3-3F (SEQIDNO:31), N-XYB3-4R (SEQIDNO:32) is primer, with pCD-XYNB (basic skills: EcoRI and XhoI site XYNB gene fragment being inserted into pcDNA3.1 (invtrogen) carrier) for template, amplification obtains P2A-XynB-T2A;
(5), with N-APPA-5F (SEQIDNO:33), N-APPA-6R (SEQIDNO:34), with pCD-APPA (basic skills: EcoRI and XhoI site appa gene fragment being inserted into pcDNA3.1 (invtrogen) carrier) for template, amplification obtains T2A-APPA-HA;
(6), with N-3G-1F (SEQIDNO:27), N-APPA-6R (SEQIDNO:34), with pcr amplification product: get 1 ~ 5ul after Bg17-E2A-Eg1314-flag (step 3), P2A-XynB-T2A (step 4), T2A-APPA-HA (step 5) 1: 1: 1 mixing in molar ratio and increase.Obtain beta-glucanase gene-xylanase gene-phytase gene fusion gene Bg17-E2A-Eg1314-flag-P2A-XynB-T2A-APPA-HA (BgEgXyAp).
In (4)-(6), the response procedures of pcr amplification is above: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
3, the structure of expression vector pPB-MusPSP-neo-EGFP
(1), the clone of mouse parotid gland albumen upstream regulatory region (PSP) (comprises parotid gland albumen to turn
record initiation site and First Intron)
Get FVB Strains of Mouse tail tissue sample, its genomic dna of extracting, then with FVB mouse gene group DNA for template, adopt KOD-FX polysaccharase (TOYOBO, Japan) to carry out pcr amplification.First primer mpsp3497-F1 (SEQIDNO:23) is adopted, mpsp16390-R1 (SEQIDNO:24) carries out first time pcr amplification, obtain mouse parotid gland albumen upstream regulatory region (-11503bp ~+1390bp), result as shown in Figure 3A; Then nested primers mPSP-3675-F2 (SEQIDNO:25) and mPSP-15980-R2 (SEQIDNO:26) is adopted, with the product of first time amplification for template, carry out second time pcr amplification, amplification mouse parotid gland protein domain (-11325bp ~+980bp), result as shown in Figure 3 B, obtain clone FVB strain parotid gland albumen upstream regulatory region MusPSP, wherein primer sequence is as shown in table 1, and the analytical results of the alignment of MusPSP and bibliographical information is in table 2.Through strengthening subarea (U73190.1 with the mouse PSP parotid gland albumen upstream regulatory region submitted in NCBI, X68699.1) sequence alignment analysis, two strengthen subarea acquaintance property and reach 99%, core promoter district (transcription initiation site upstream 300bp) is completely the same with mouse database (UCSC) comparison result, analytical results shows this research and obtains MusPSP control region and Golovan, S.P etc. (2001) to prepare parotid gland albumen upstream regulatory region that transgenic pig and mouse adopt be not same source.
The cloning 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 DEG C of 2min for the first time; 98 DEG C of 10s, 74 DEG C of 13min, 5cycles; 98 DEG C of 10s, 72 DEG C of 13min, 5cycles; 98 DEG C of 10s, 70 DEG C of 13min, 5cycles; 98 DEG C of 10sec, 68 DEG C of 13min, 30cycles; 68 DEG C of 7min.The response procedures of described second time pcr amplification is 94 DEG C of 2min; 98 DEG C of 10sec, 68 DEG C of 13min, 30cycles; 68 DEG C of 7min.
The comparing result of the sequence of MusPSP sequence and bibliographical information cloned by table 2
(2), build and merge pcDNA-Neo-T2A-EGFP double-tagging carrier, can be used for the later stage
the visual green fluorescent protein (EGFP) deleted and neomycin resistance gene (neo) fusion gene
A, amplification neo
With plasmid pcDNA3.1 (+) (invitrogen company) for template, design primer EcoRI-neo-1 (SEQIDNO:5), neo-T2A-2 (SEQIDNO:6) increases neo gene.PCR response procedures: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 50s; 35 circulations; 72 DEG C of 10min.PCR primer 1% agarose gel electrophoresis detects.Then with EasyPurePCRPurificationKit (Beijing Quanshijin Biotechnology Co., Ltd), PCR primer is purified (be called for short one and cross column purification), remove primer and other impurity;
B, amplification EGFP
With plasmid pT2AL200R175-CAGGS-EGFP (Japan, TheNationalInstituteofGenetics center, professor KoichiKawakami presents) template, design primer T2A-EGFP-3 (SEQIDNO:7), EGFP-4 (SEQIDNO:8) increases EGFP.Wherein, about 15bp and neo-T2A-2 (SEQIDNO:6) overlap is had in upstream primer T2A-EGFP-3 (SEQIDNO:7); the terminator codon removing neo adds GCC tri-bases, downstream primer restricted property restriction endonuclease XbaI enzyme cutting site and protection base.PCR response procedures is identical with step a with product purification condition.
C, neo-EGFP fusion gene
The PCR primer of purified recovery neo and EGFP carries out concentration determination, after adjustment concentration, respectively gets after 1 μ L mixes and carries out pcr amplification as template.PCR response procedures: 98 DEG C, 2min; 98 DEG C, 10s; 68 DEG C, 1min40s; 35 circulations; 72 DEG C, 10min.Obtain neo-EGFP fusion gene.Enzyme cuts qualification, and as shown in Figure 4, restriction enzyme digestion and electrophoresis figure object stripe size is consistent with expection for qualification result.
D, pcDNA-Neo-T2A-EGFP vector construction
The neo-EGFP fusion gene obtained above by over-lap PCR is crossed purification column, removes primer and PCR waste reaction solution.With
ecoRI (Fermentas company) and
xbaI (Fermentas company) double digestion pcDNA3.1 (+) plasmid and PCR primer neo-EGFP fusion gene.Digestion products is crossed column purification and is reclaimed.Connect test kit (Takara company) constant temperature in 16 DEG C of connection instrument with LigationKitVer.2.0 to spend the night connection.Finally be built into pcDNA-Neo-T2A-EGFP carrier, and carry out enzyme and cut qualification, qualification result is as shown in Figure 5, completely the same with expection through electrophoresis detection restriction enzyme mapping stripe size.
In step (2), the primer sequence of synthesis is as table 3.
Table 3 builds the primer merging neo-EGFP double-tagging carrier and adopt
(3), piggyBac transposon system element clone and vector construction
With pPB-UbC-EGFP-neo (WellcomeTrustSanger gives at center) for template, use primer NotI-5-PB-F (SEQIDNO:9) and NotI-5-PB-R (SEQIDNO:10) respectively, XhoI-3-PB-F (SEQIDNO:11) and XhoI-3-PB-R (SEQIDNO:12), with TaKaRaLATaq enzyme pcr amplification NotI-5-PB--NotI, XhoI-3-PB-XhoI, after using corresponding digestion with restriction enzyme respectively, be connected in carrier pPSPBGPneo-XynB, it is completed
piggyBactransposon vector is transformed, after transformation, and called after pPSPBGPneo-PB-XynB, its enzyme cuts qualification result as shown in Figure 6, cut pPSPBGPneo-PB-XynB enzyme with XhoI and NotI enzyme respectively and cut result display, obtain 285bp and 349bp object band, electrophoresis two stripe size conforms to expection.
The reaction system of PCR: by following component preparation PCR reaction solution.
PCR response procedures is: 94 DEG C of 30S; 55 DEG C of 30S, 35 circulations; 72 DEG C of 30S; 72 DEG C of 3min, 30 circulations.
With pcDNA-neo-T2A-EGFP carrier for template, with primer infu-CMV-neo-EGFP-R (SEQIDNO:13), infu-CMV-neo-EGFP-F (SEQIDNO:14) increases, and obtains infusion-CMV-neo-T2A-EGFP fragment, cuts glue purification and reclaims for subsequent use.
By ClaI and BglII restriction enzyme double digestion pPSPBGPNeo-PB-XynB fragment, digestion products does agarose gel electrophoresis, cuts glue purification to object fragment.Preferably apply In-
infusion-CMV-neo-T2A-EGFP fragment is recombinated in the centre, two loxp sites of pPSPBGPNeo-PB-XynB carrier by AdvantagePCRCloningKi test kit (Clontech company), obtain pPSPBGPneo-T2A-EGFP-PB-XynB carrier, its enzyme cuts qualification result as shown in Figure 7, the electrophoresis result of plasmid AscI and the qualification of BglII double digestion, electrophoretic band size conforms to expection, successfully obtains the pPSPBGPneo-T2A-EGFP-PB-XynB transposon vector with neo-EGFP double-tagging and xylanase gene (XynB).
PCR reaction system is as follows:
PCR response procedures is: 98 DEG C of 2min; 98 DEG C of 10s; 68 DEG C of 4min; 35 circulations; 72 DEG C of 10min.
In this step, the primer of piggyBac transposon main element clone and positive and negative qualification 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 qualification
Table 5pPSPBGPneo-T2A-EGFP-PB-XynB vector construction primer
(4), parotid gland albumen upstream regulatory region and loxP-CMV-neoEGFP-loxp and PiggyBac
system globe area
First utilize primer P11160-ASCIF (SEQIDNO:15), P407-AgeIR (SEQIDNO:16), with pPSPBGPneo-T2A-EGFP-PB-XynB carrier for template, use
pPB-lox-neoEGFP-loxp fragment in MaxDNAPolymerase enzyme (Takara company) amplification vector, and artificial interpolation AgeI restriction endonuclease sites, for subsequent use after purifying, be novel vector skeleton.PCR response procedures is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C 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, and carried out vector construction, construction process refers to Fig. 8.
The first step: first utilize primer inf-AgeI-8700F (SEQIDNO:17), inf-AscI-12540R (SEQIDNO:18), the pcr amplified fragment of the mouse parotid gland albumen upstream regulatory region obtained with step (1), for template, is used
maxDNAPolymerase enzyme (Takara company) increases MusPSP downstream 4009bp fragment (fragment 1), add and the recombination sequence in novel vector skeleton pPB-lox-neoEGFP-loxp, it is first recombinated on carrier, its enzyme cuts 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 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
Second step: then utilize the restriction enzyme site StuI in MusPSP fragment 1, design primer inf-AgeI-4090F (SEQIDNO:19), StuI-8763R (SEQIDNO:20), the pcr amplified fragment of the mouse parotid gland albumen upstream regulatory region obtained with step (1), for template, is used
the fragment 2 of MaxDNAPolymerase enzyme (Takara company) 4706bp that increases.The StuI site of the intermediate carrier fragment 1 that the first step of being recombinated to obtains, its enzyme cuts 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 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
3rd step: design primer inf-AgeI-440F (SEQIDNO:21), inf-AgeI-4000R (SEQIDNO:22), the pcr amplified fragment of the mouse parotid gland albumen upstream regulatory region obtained with step (1), for template, is used
the fragment 3 of MaxDNAPolymerase enzyme (Takara company) 3956bp that increases, utilize artificial AgeI site of adding, second step of the fragment of last 3956bp being recombinated to obtains on intermediate carrier, complete whole vector construction pPB-MusPSP-neo-EGFP, be the specific transgene carrier of parotid gland tissues 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 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
In this step, the primer of employing is as shown in table 6.
The primer adopted in table 6pPB-MusPSP-neo-EGFP vector construction
4, the structure of transgene expression vector PPB-MusPSP-neo-EGFP-BgEgXyAp
By the fusion gene BgEgXyAp ASCI site of in-fusion recombinase (Clontech company) restructuring to PPB-MusPSP-neo-EGFP, success builds the polygene transfer vector PPB-MusPSP-neoEGFP-BgEgXyAp of 4 gene co-expressing parotid gland specifically expressings, and sequence number is SEQIDNO:35.The ASCI enzyme of PPB-MusPSP-neoEGFP-BgEgXyAp transgene carrier cuts qualification collection of illustrative plates as shown in figure 12, and as can be seen from Figure 12, the size that enzyme cuts the fusion gene obtained is about 4171bp, consistent with expection.The structure collection of illustrative plates of transgene expression vector as shown in figure 13.
The transgene carrier that embodiment 2 utilizes embodiment 1 to build and obtains builds transgenic pig
1, the screening of transgenic cell
Utilize BXT electroporation, by the pPB-MusPSP-neo-EGFP-BgEgXyAp carrier that builds and transposase plasmids PCMV-mPB, (WellcomeTrustSangerInstitute presents, applicant has removed wherein neo gene) according to mol ratio 1: 1 ratio By Transfecting Porcine fetal fibroblast (boar), transfection conditions: 310V, burst length 1ms, pulse number 3 times.Plating cells after electrotransfection, after 36 hours, goes down to posterity according to 1: 6.Cell screening substratum compound method 42.5%GlutaMAX
tM(life company)+42.5% DMEM in high glucose (life company)+15%FBS, first in cell screening substratum, G418300ug/ml is added, to the cell screening after electrotransfection after 5 days, use 200ug/ml concentration instead and continue screening 10 days, then change 80ug/ml into and maintain screening, in 15%FBS peptide bovine serum substratum, add 2.5-5ng/mLBFGF (basicfibroblastgrowthfactor) simultaneously, obtain the cell mass being with green fluorescence, after growing up to sheet Deng these cell masses, carefully scrape off with rifle head and do not have fluorocyte around, the most handy 0.05% trysinization is gone down to posterity, after reaching 2-3 generation, express at detection green fluorescence.Selecting green fluorescence expresses bright, and the normal cell of cellular form is for subsequent use as nuclear transfer donor cell.Utilize the method can obtain transgenic cell line fast, and cell edge is level and smooth, cellularstructure is normal and quality is good.And general method obtains screening cell, cell is comparatively large, has cavity, present ageing state in cell.
The collection of 2, porcine oocytes-granulosa cell complex body (COCs) and In-vitro maturation
Collect pig ovary from pig slaughterhouse (Milky Way meat processing combine of Guangdong Province), put into 28 ~ 37 DEG C of physiological saline containing 1% dual anti-(the dual anti-product for lifetechnology: Penicillin-Streptomycin-Glutamine) and send laboratory back in 4h.By the ovocyte that the 10mL syringe extraction diameter with No. 18 syringe needles is in 2 ~ 6mm ovarian follicle after cleaning with the physiological saline of 37 DEG C.Pick out that tenuigenin is even, ovarian cumulus is fine and close under the microscope and cumulus cell-the oocyte complex (Cumulusoocytecomplexes, COCs) of parcel more than 3 layers, after washing with M199 maturation culture solution, proceed to and be placed in CO in advance
2hatch in four orifice plates being added with 500 μ LM199 nutrient solutions of more than 4h in incubator, at 39 DEG C, 5%CO
2, saturated humidity incubator in cultivate 42 ~ 44h.
3, after maturation culture, on COCs, granulosa cell is removed and the selecting of mature egg
After oocyte maturation, COCs is transferred to and after blowing and beating with pipettor in the centrifuge tube containing Unidasa, liquid rotating is moved on in 30mm culture dish, with mouth suction pipe, the ovocyte sloughing ovarian cumulus is sorted out.Under stereomicroscope, the ovocyte of discharging first polar body is selected after washing.
4, the preparation of donor cell
With centrifuge washing after trysinization, operate liquid (without calcium H-NCSU-23 micrurgy liquid) with HN cell precipitation is resuspended and be used as nuclear donor after piping and druming evenly.
HN operates the formula following (all reagent is analytical pure rank, purchased from Kang Long bio tech ltd, Guangzhou) of liquid:
5, ovocyte stoning with note core
Select and discharge first polar body and the good ovocyte of form, with the locking pin that external diameter is 100 ~ 120 μm, internal diameter is that the kernel removing needle of 15 ~ 20 μm adopts the stoning of blind suction method: the operation drop adding about 50 μ L in 65mm sterile culture dish, and cover with paraffin oil, then the ovocyte of about 30 and appropriate somatocyte are moved into wherein.Fix ovocyte with locking pin, stir ovocyte with kernel removing needle and make polar body in the position at about 5 o'clock.Lunge along 3 o ' clock positions with kernel removing needle, remove polar body and neighbouring kytoplasm, afterwards pin is withdrawn from and polar body and kytoplasm are spued, select all gaps of an individual cells injection ovum and namely complete embryo's restructuring procedure.Reconstruct embryo puts into embryo medium renewal cultivation 1h.
6, ovocyte and somatic fusion and activation
Reconstructed eggs is transferred to after balancing 2min in fusion liquid in batches and wash 3 times with fusion/activation solution, the integration slot being paved with and merging liquid is put into by often criticizing 5 ~ 8, cell membrane contact face and the electrode runs parallel that reconstructed eggs makes donor cell-acceptor ovum is stirred with solid glass pin, apply 120v/mm, the electric pulse induced fusion of 100 μ s, 2DC activates reconstructed embryo simultaneously, proceeds in the embryo medium of mineral oil covering after then washing 3 times with embryo medium immediately, be placed in 39 DEG C, 5%CO
2in the incubator of saturated humidity, after 4h under stereoscopic microscope decision fusion situation.After the reconstructed embryo embryo medium merged is washed 5 times, proceed to the embryo medium that pre-equilibration is good, be placed in 39 DEG C, saturated humidity, hypoxemia (5%O
2+ 5%CO
2+ 90%N
2) condition under cultivate.
7, operation transplantation clone embryo produces transgenic pig
Acceptor sow is the high-quality sow of Hua Nongwenshi herding limited-liability company of Guangdong Province.The present embodiment adopts common oviduct transplantation method, fetal development be in be 2 cells or 4 cell stage time transplant.Perform the operation the same day to sow fasting, and operation consent Baoding sow also carries out general anesthesia to it.Operative site to be selected second from the bottom, to nipple middle part, first to use clean water operative site and surrounding, dry and first to sterilize on a large scale with the tincture of iodine afterwards, then take off iodine with 75% alcohol.Cover operation cloth and expose operative site simultaneously, cut skin and subdermal muscle along ventrimeson, be then separated subcutaneous lipids and peritonaeum, hand probes into abdominal cavity, slowly pulls out uterus and uterine tube, checks ovulation situation.The suction embryonic tube that embryo is housed is inserted from uterine tube umbrella mouth, carefully embryo is blown into.Then recovery uterus and uterine tube are to intraperitoneal.Routine operation is sewed up, injection of antibiotics anti-inflammatory in postoperative continuous 4 days.Self secretion dextranase-genetically modified transgenic pig of zytase-phytase can be obtained.
8, transgenic pig birth and detection
Transgenosis piggy positive identification, detects with blue light modulation direct irradiation, and transgenic positive pig can fluoresced green, as shown in figure 14, then carry out PCR, Southernbloting to it to be further analyzed, utilize inverse PCR technique, detect it and be incorporated into site in genome.Transgenic pig PCR detects collection of illustrative plates as shown in figure 15, and detect through PCR, the clone pig of the present embodiment obtains 2 transgenic primary (F0), namely 950605 (are called for short 605, dead in one week after birth), 950707 (being called for short 707), and this trans genie individual does not carry AmP
rresistant gene and transposase (PCMV-mPB) gene order.Detect through PCR, confirmation fluoroscopic examination result, only have 707 and 605 pigs for positive, and plasmid backbone part Amp
rit is all negative for detecting with helper plasmid PBase.Transgenic pig F0 detects collection of illustrative plates as shown in figure 16 for SouthernBloting, and Southernbloting results verification transgenosis list copy is integrated; Inverse PCR detects the integration site of transgenic pig 605 and 707, as shown in figure 17, finds this site to be positioned at pig genome No. 7 karyomit(e) Legumain upstream region of gene.
Embodiment 3 embodiment 2 builds the transgene expression effect detection of the transgenic pig obtained
After two monthly ages, the transgene expression effect building the transgenic pig obtained to embodiment 2 detects, mainly have detected dextranase (β-glucannase) in the saliva of transgenic pig (wherein No. 707 pigs), zytase (Xylanase), phytase (phytase) enzyme activity.Detection method is: bite by piggy with sponge or rub bar, gather piggy saliva 50ul, at 39.5 DEG C, measure in acetic acid-sodium acetate buffer solution (HAC-NaAC), often kind of enzyme repeats according to 3 biology, 3 technology replications, get 3 and contrast (wild-type) with after the identical age in days non-transgenic pig saliva mixing of house.Detect beta-glucanase of wherein recombinating, zytase, phytase activity.Mean ± S.D. is adopted to take statistics analysis.Enzyme activity determination result as shown in figure 18, as can be seen from Figure 18, dextranase (β-glucannase) in transgenosis No. 707 pig salivas, zytase (Xylanase), phytase (phytase) enzyme activity, all far away higher than the content in the saliva of non-transgenic pig, shows that the present invention builds the transgene expression effect of the transgenic pig obtained fine.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not 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 (7)
1. a construction process for the transgene carrier of salivary gland tissue specific expression foreign protein, is characterized in that, comprises the following steps:
(1) utilize the 2A peptide sequence of E2A, P2A and T2A that the phytase gene APPA shown in xylanase gene XynB and SEQIDNO:4 shown in glucanase gene Eg1314, the SEQIDNO:3 shown in glucanase gene Bg17, the SEQIDNO:2 shown in SEQIDNO:1 is built recombinant beta-glucanase gene-xylanase gene-phytase gene fusion gene BgEgXyAp;
The construction process of described fusion gene BgEgXyAp comprises the following steps:
(a), with SEQIDNO:27, SEQIDNO:28 for primer, SEQIDNO:36 is template amplification Bg17-E2A;
(b), with SEQIDNO:29, SEQIDNO:30 for primer, SEQIDNO:36 is template amplification E2A-Eg1314;
(c), utilize Overlap extension PCR, with SEQIDNO:27, SEQIDNO:30 for primer, with mol ratio be 1: 1 mixing Bg17-E2A and E2A-Eg1314 for template, obtain Bg17-E2A-Eg1314-flag;
A in ()-(c), the response procedures of pcr amplification is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 1min; 35 circulations; 72 DEG C of 2min;
(d), with SEQIDNO:31, SEQIDNO:32 for primer, to insert the pcDNA3.1 carrier of XynB gene fragment for template, amplification obtain P2A-XynB-T2A;
(e), with SEQIDNO:33, SEQIDNO:34 for primer, to insert appa gene fragment to pcDNA3.1 carrier for template, amplification obtain T2A-APPA-HA;
(f), with SEQIDNO:27, SEQIDNO:34 for primer, using mol ratio be Bg17-E2A-Eg1314-flag, P2A-XynB-T2A, T2A-APPA-HA mixing of 1: 1: 1 as template, amplification obtains fusion gene BgEgXyAp;
D in ()-(f), the response procedures of pcr amplification is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min;
(2), respectively with plasmid pcDNA3.1 (+) and pT2AL200R175-CAGGS-EGFP for template, SEQIDNO:5 and SEQIDNO:6 and SEQIDNO:7 and SEQIDNO:8 is that primer carries out first time pcr amplification, obtains the neo gene with some overlaps and EGFP gene; Respectively get 0.5-3 μ L mix after as template, with SEQIDNO:5 and SEQIDNO:8 for primer carries out second time pcr amplification, purifying obtains neo-EGFP fusion gene; With EcoRI and XbaI double digestion pcDNA3.1 (+) and neo-EGFP fusion gene, purifying reclaims digestion products, connects, and builds and obtains pcDNA-Neo-T2A-EGFP carrier;
(3), with plasmid pPB-UbC-EGFP-neo for template, respectively using SEQIDNO:9 and SEQIDNO:10 and SEQIDNO:11 and SEQIDNO:12 as primer, carry out pcr amplification and obtain NotI-5-PB-NotI, XhoI-3-PB-XhoI, after adopting NotI and XhoI enzyme to cut respectively, be connected on carrier pPSPBGPneo-XynB, obtain carrier pPSPBGPneo-PB-XynB;
(4) the pcDNA-neo-T2A-EGFP carrier, obtained with step (2) is for template, use LA-PCR system, SEQIDNO:13 and SEQIDNO:14 carries out pcr amplification as primer and obtains infusion-CMV-neo-T2A-EGFP fragment, cuts glue purification; With the carrier pPSPBGPneo-PB-XynB that ClaI and BglII double digestion step (3) obtains, glue purification is cut to object fragment; Infusion-CMV-neo-T2A-EGFP fragment is recombinated to pPSPBGPNeo-PB-XynB carrier two loxp sites in the middle of, obtain pPSPBGPneo-T2A-EGFP-PB-XynB carrier;
(5) the pPSPBGPneo-T2A-EGFP-PB-XynB carrier, obtained with step (4) is for template, SEQIDNO:15 and SEQIDNO:16 is 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;
(6), with the pcr amplified fragment of mouse parotid gland protein gene upstream control region for template, SEQIDNO:17 and SEQIDNO:18 is 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, obtains intermediate carrier 1;
(7), with the pcr amplified fragment of mouse parotid gland protein gene upstream control region for template, with SEQIDNO:19 and SEQIDNO:20 for primer, amplification obtains the downstream 4706bp fragment of mouse parotid gland albumen upstream regulatory region; The StuI site of the intermediate carrier 1 of step of being recombinated to (6), is intermediate carrier 2;
(8), with the pcr amplified fragment of mouse parotid gland protein gene upstream control region for template, with SEQIDNO:21 and SEQIDNO:22 for primer, utilize AgeI site, amplification obtains the downstream 3956bp fragment of mouse parotid gland albumen upstream regulatory region; On the intermediate carrier 2 of step of being recombinated to (7), obtain carrier pPB-MusPSP-neo-EGFP;
The PCR amplification method of the mouse parotid gland protein gene upstream control region in described step (6)-(8) is: with FVB mouse gene group DNA for template, SEQIDNO:23 and SEQIDNO:24 is primer, and KOD-FX polysaccharase carries out first time pcr amplification; With the product of first time amplification for template, SEQIDNO:25 and SEQIDNO:26 is primer, carries out second time pcr amplification, to obtain final product; Described first time, the response procedures of pcr amplification was: 94 DEG C of 2min; 98 DEG C of 10s, 74 DEG C of 13min, 5cycles; 98 DEG C of 10s, 72 DEG C of 13min, 5cycles; 98 DEG C of 10s, 70 DEG C of 13min, 5cycles; 98 DEG C of 10s, 68 DEG C of 13min, 30cycles; 68 DEG C of 7min; The response procedures of described second time pcr amplification is: 94 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 13min, 30cycles; 68 DEG C of 7min;
(9) the beta-glucanase gene-xylanase gene, by step (1) obtained-phytase gene fusion gene BgEgXyAp in-fusion recombinase is recombinated the ASCI site of carrier pPB-MusPSP-neo-EGFP that step (8) obtains, obtains the expression vector pPB-MusPSP-neo-EGFP-BgEgXyAp that sequence number is four gene co-expressings of SEQIDNO:35.
2. the construction process of the transgene carrier of salivary gland tissue specific expression foreign protein according to claim 1, is characterized in that, described in step (2), the response procedures of PCR is for the first time: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 50s, 35 circulations; 72 DEG C of 10min; Second time PCR response procedures: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 1min40s, 35 circulations; 72 DEG C of 10min.
3. the construction process of the transgene carrier of salivary gland tissue specific expression foreign protein according to claim 1, is characterized in that, described in step (3), the response procedures of PCR is: 94 DEG C of 30s; 55 DEG C of 30s, 35 circulations; 72 DEG C of 30s, 72 DEG C of 3min, 30 circulations; Described in step (4), the response procedures of PCR is: 98 DEG C of 2min; 98 DEG C of 10s, 68 DEG C of 4min, 35 circulations; 72 DEG C of 10min; Described in step (5)-(8), the response procedures of PCR is: 98 DEG C of 1min; 98 DEG C of 10s; 60 DEG C of 5s; 72 DEG C of 2min; 35 circulations; 72 DEG C of 2min.
4. the construction process described in any one of claim 1-3 builds the transgene carrier pPB-MusPSP-neo-EGFP-BgEgXyAp of the salivary gland tissue specific expression foreign protein obtained, and sequence number is SEQIDNO:35.
5. a construction process for the transgenic pig of salivary gland tissue specific expression foreign protein, is characterized in that, comprises the following steps:
(1), by transgene carrier pPB-MusPSP-neo-EGFP-BgEgXyAp according to claim 4 and transposase plasmids PCMV-mpb mixing By Transfecting Porcine fetal fibroblast cell line, the mono-clonal transgenic cell line of screening Absorbable organic halogens expression vector;
(2), using the mono-clonal transgenic cell line of acquisition as nuclear donor cell, be expelled in ovocyte, build reconstruct embryo, adopt traditional breeding method to cultivate, the transgenic pig of salivary gland tissue specific expression foreign protein can be obtained.
6. the construction process of the transgenic pig of salivary gland tissue specific expression foreign protein according to claim 5, it is characterized in that, the mole ratio of described transgene carrier pPB-MusPSP-neo-EGFP-BgEgXyAp and transposase plasmids PCMV-mpb is 1: 1.
7. the construction process of the transgenic pig of salivary gland tissue specific expression foreign protein according to claim 5, is characterized in that, the screening step of the mono-clonal transgenic cell line of described Absorbable organic halogens expression vector is:
By the plating cells after transfection after 36 hours, go down to posterity according to 1: 6, first in cell screening substratum, G418300ug/ml is added, to the cell screening after transfection after 5 days, use 200ug/ml concentration again instead and continue screening 10 days, then change 80ug/ml into and maintain screening, in 15%FBS peptide bovine serum substratum, add 2.5-5ng/mLBFGF simultaneously, obtain the cell mass being with green fluorescence, grow up to after sheet until cell mass, carefully scrape off and do not have fluorocyte around, go down to posterity with 0.05% trysinization, after reaching 2-3 generation, detect green fluorescence again to express, selecting green fluorescence expresses bright, the normal cell of cellular form, i.e. mono-clonal transgenic cell line,
The formula of described cell screening substratum is: 42.5%GlutaMAX
tM, 42.5% DMEM in high glucose, 15%FBS.
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| CN105671079A (en) * | 2016-02-26 | 2016-06-15 | 华南农业大学 | Mouse with human nerve growth factor transgenes as well as preparation method and application of mouse |
| CN109628487A (en) * | 2018-12-21 | 2019-04-16 | 华南农业大学 | A method of growth factor of human nerve is prepared using transgene pig salivary gland |
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