CN103122359A - Xylanase recombinant expression vector and cultivation method of transgenic pig capable of secreting xylanase - Google Patents
Xylanase recombinant expression vector and cultivation method of transgenic pig capable of secreting xylanase Download PDFInfo
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- CN103122359A CN103122359A CN2011103748684A CN201110374868A CN103122359A CN 103122359 A CN103122359 A CN 103122359A CN 2011103748684 A CN2011103748684 A CN 2011103748684A CN 201110374868 A CN201110374868 A CN 201110374868A CN 103122359 A CN103122359 A CN 103122359A
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Abstract
The invention discloses a xylanase recombinant expression vector and a cultivation method of a transgenic pig capable of secreting xylanase. The recombinant expression vector which is regulated and controlled to express by a regulatory element is constructed by steps of splicing external xylanase genes and the regulatory element and inserting the spliced one to a eukaryotic expression vector. According to the invention, external xylanase genes are guided to pig genome successfully through a genetic engineering technology and expressed under control of the regulatory element, so that the pig can secrete xylanase in the digestive tract to eliminate the antinutritional effect of xylan in feed. Compared with a method that xylanase is added to pig feed to eliminate antinutritional effect of xylan, the invention has the advantages that the degrading efficiency of xylanase in pig feed remarkably improved, the digestion and absorption efficiencies to the nutritional matters in the feed are improved, the growing performance of pig is improved, the discharge of pollutants in excrement is reduced, the feed cost of the pig is remarkably lowered, and the pig raising benefit is improved.
Description
Technical field
The present invention relates to gene engineering technology field, relate in particular to gene engineering technique, be specifically related to a kind of zytase recombinant expression vector, but and utilize this recombinant expression vector to cultivate the method for transgenic pig of self secretion zytase.
Background technology
In pig feed, topmost composition is corn and dregs of beans etc., and these cereal seeds are to belong to vegetality feedstuff.Plant contains the non-starch polysaccharides such as a large amount of Mierocrystalline celluloses, hemicellulose and pectin glycan, because they are main components of plant cell wall.Xylan is the abundantest a kind of hemicellulose of content in plant cell wall, and it accounts for 35% of vegetable cell gross dry weight, is a kind of non-starch polysaccharide main in vegetality feedstuff.
The research discovery, pig self can not produce zytase.Owing to parasitizing gastral microbe species and quantity far fewer than multistomachal animals such as ox or sheep, pig also can't rely on the degrade xylan of plant cell wall in feed of the zytase of the microorganism secretion capacity in digestive tube.Therefore, in pig feed, the intracellular nutritive substance of cereal seed can't fully be discharged in digestive tube for the pig utilization that disappears.On the other hand, because xylan is Soluble Non Starch Polysaccharides, the xylan that is difficult to digest in swine alimentary canal can be combined with a large amount of water, the stickiness of chyme is increased, thereby reduce swine alimentary canal to digestion and the assimilated efficiency of nutritive ingredient in chyme.As seen, because pig self can not secrete zytase, xylan in vegetality feedstuff just becomes a kind of anti-nutrient substance concerning pig, it has not only reduced digestion, absorption and the utilising efficiency of pig to the feed nutrition material, various pollutents in swine excrement have also been increased, discharging as nitrogen, sulphur etc. causes more havoc to environment.
In order to eliminate xylan to the anti-oxidant action of pig, the main method that adopts at present is to add the zytase of producing by microbial fermentation in pig feed.Although adding zytase in feed, the part Study proof can improve pig to digestion and the assimilated efficiency of feed nutrition material, and the growth performance of raising pig, reduce pollutant emission in ight soil, increased feed cost but add zytase in feed, reduced the profit of raising pigs.In addition, because feed need to (for example high temperature) be completed under special conditions in common some operation of preparation process, this easily causes the zytase generating portion enzyme of interpolation to live and loses, and causes the effect of xylan in its hydrolysate feed unstable or undesirable.
Therefore, be necessary that very exploitation is more effective, cheaper technology is eliminated the anti-oxidant action of xylan in pig feed, improves pig to digestion and the assimilated efficiency of feed, thus the discharging of pollutent in the growth performance of raising pig and minimizing ight soil.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of by after the splicing of xylanase gene and controlling element, be inserted into after carrier for expression of eukaryon formed, zytase recombinant expression vector that can modulated element regulating and expressing zytase.
But another object of the present invention is to provide the method for utilizing above-mentioned zytase recombinant expression vector to cultivate self secretion zytase Porcine reconstructed embryos.
Another object of the present invention is to provide utilizes the reconstructed embryo of being cultivated by above-mentioned zytase recombinant expression vector to carry out the method that transgenic pig is cultivated.
Above-mentioned purpose of the present invention is achieved by following scheme:
A kind of zytase recombinant expression vector, this recombinant expression vector are with after the xylanase gene of external source and controlling element splicing, are inserted in carrier for expression of eukaryon, build the zytase recombinant expression vector that obtains by the controlling element regulating and expressing.
In above-mentioned zytase recombinant expression vector, the effect of controlling element is that the regulation and control Exogenous-xylanase Additive is at the swine alimentary canal organizing specific expression, this controlling element can be the controlling element of oral cavity tissue specifically expressing or the controlling element of parotid gland tissues specifically expressing, also can be the controlling element of stomach-tissue specifically expressing or the controlling element of intestinal tissue specifically expressing.
In above-mentioned zytase recombinant expression vector, the xylanase gene of external source refers in any this area disclosed xylanase gene, as derives from the xylanase gene of microorganism, yeast or insect etc.
In above-mentioned zytase recombinant expression vector, carrier for expression of eukaryon adopts any those skilled in the art to be usually used in animal genetic engineering, and with can be at the carrier for expression of eukaryon of the drug resistance gene (as neomycin gene etc.) of eukaryotic cell expression, as carrier for expression of eukaryon pCMVp-NEO-BAN, carrier for expression of eukaryon pEGFP, carrier for expression of eukaryon pEGFT-Actin, carrier for expression of eukaryon pcDNA
TM3.1/myc His or carrier for expression of eukaryon pcDNA6/His
TMEtc.; The effect of drug resistance gene is the transgenic cell that can be used for screening as nuclear donor cell.
But a kind of method of self secreting the zytase Porcine reconstructed embryos of cultivating, the method comprises the steps:
Be prepared into above-mentioned zytase recombinant expression vector By Transfecting Porcine fetal fibroblast and after screening the nuclear donor cell that turns xylanase gene;
Preparation stoning porcine oocytes;
Form reconstructed embryo in the stoning porcine oocytes that the nuclear donor cell implantation step 1 that step 1 is prepared prepares;
After above-mentioned reconstruct ovum is activated processing, the reconstructed embryo that obtains activating.
In above-mentioned steps 1, nuclear donor cell can be used any one porcine somatic cell, but the inventor more is conducive to improve with porcine fetus fibroblasts the efficient for preparing transgenic pig with cloning by the rear discovery of research.
In above-mentioned steps 2, nuclear donor cell is injected the stoning porcine oocytes, its method for implanting adopts the method for microinjection.
In above-mentioned steps 2, the reconstruct ovum being activated processing, is to adopt electric Activiation method.
But a kind of method of cultivation of transgenic pig of self secretion zytase, this method of cultivation is after the reconstructed embryo with above-mentioned activation carries out vitro culture, normotrophic reconstructed embryo is operated by embryo transfer move in the replace-conceive Gilt Uterus, through the pregnancy of replace-conceive sow, but the transgenic pig of minute puerperium acquisition self secretion zytase.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention successfully imports the Exogenous-xylanase Additive gene by genetic engineering technique the genome of pig, and express under controlling element is controlled, thus make pig can be in autodigestion road secretion zytase be eliminated pig feed the anti-oxidant action of xylan;
2. compare with the anti-oxidant action of eliminating xylan by interpolation zytase in pig feed, the present invention not only can significantly improve the efficient to xylan degrading in pig feed, improve pig to digestion and the assimilated efficiency of feed nutrition material, improve the growth performance and the discharging that reduces pollutent in ight soil of pig, can also significantly reduce the feed cost of pig, improve the profit of raising pigs.
Description of drawings
Fig. 1 is the RT-PCR amplification electrophorogram of xylanase from aspergillus niger gene xynB;
Wherein, M is DNA Marker, and 1 and 2 is all the RT-PCR amplified production of xylanase from aspergillus niger gene xynB;
Fig. 2 turns xylanase gene porcine fetus fibroblasts PCR the 5th generation to identify electrophorogram;
Wherein, M is DNA Marker, 1,2,3 be the 5th generation the cellular genome pcr amplification product, 4 is blank;
Fig. 3 identifies electrophorogram for the PCR that turns xylanase gene (XynB) pig;
Wherein, M is DL2000 Marker, and 1～5 is transgenic pig, and N is the negative control take the ear tissue DNA of a non-transgenic pig as the pcr amplification template, and P is take with the transgenosis plasmid of the XnyB gene positive control as the pcr amplification template;
Fig. 4 is for turning the Southern Blot detected result electrophorogram of xylanase gene (XynB) pig;
wherein, M is the Marker of DIG mark, 1～5 turns xylanase gene (XynB) pig for the PCR test positive, NC is used for the ear tissue DNA of a non-transgenic pig the negative control of Southern hybridization, 1C is used for the transgenosis plasmid with the XnyB gene positive control of 1 copy of Southern hybridization, 2C is used for the transgenosis plasmid with the XnyB gene positive control of 2 copies of Southern hybridization, 5C is used for the transgenosis plasmid with the XnyB gene positive control of 5 copies of Southern hybridization, 10C is used for the transgenosis plasmid with the XnyB gene positive control of 10 copies of Southern hybridization,
Fig. 5 is that RT-PCR analyzes the electrophorogram that xylanase gene XynB expresses in turning the various tissues of xylanase gene pig;
Wherein, M is DNA Marker, and Sm is glandula submandibularis, and Pa is the parotid gland, and Sl is sublingual gland, and Li is liver, and Ki is kidney, and He is heart, and Sp is spleen, and Lu is lung, and In is small intestine, and St is stomach, the negative contrast of N (water).
Embodiment
Below in conjunction with specific embodiment, the present invention is done further and describe, but specific embodiment is not done any restriction to the present invention.
It is the Exogenous-xylanase Additive gene that specific embodiment adopts xylanase from aspergillus niger gene XynB.
One, the clone of xylanase from aspergillus niger gene XynB
Referring to the xylanase from aspergillus niger gene xynB sequence of delivering in Genbank (Genbank accession number: DQ174549) design primer xynBF and xynBR amplification mature polypeptide coding sequence.
The nucleotide sequence of described xynBF is as shown in SEQ ID NO:1, and the nucleotide sequence of described xynBR is as shown in SEQ ID NO:2.
Extract the total RNA of aspergillus niger and carry out RT-PCR amplification, RT-PCR response procedures: 50 ℃ of 30 min, 94 ℃ of 2 min with it as template; 94 ℃ of 30 s, 52 ℃ of 30 s, 35 circulations of 72 ℃ of 1 min, last 72 ℃ are extended 10 min.
The RT-PCR product detects with 1% agarose gel electrophoresis, reclaiming PCR product fragment is connected with the pMD18-T carrier, connect product conversion DH5 α and carry out positive bacterium colony evaluation, the single white colony of picking from the flat board of cultivating, be inoculated in the LB liquid nutrient medium that 2mL contains penbritin, 12～16 h are cultivated in 37 ℃ of 220 r/min jolting, directly get bacterium liquid and carry out the PCR evaluation as template, reaction system and program are carried out with reference to Dongsheng TaqMix kit specification sheets, the PCR product detects with 1% agarose gel electrophoresis, and result as shown in Figure 1.
As can be seen from Figure 1, the pcr amplification product band conforms to the 567bp length of expection between 500bp and 750bp.The clone of selection PCR test positive is sent to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carries out positive and negative both direction order-checking, and sequencing result is as shown in SEQ ID NO:5.With aspergillus niger xylan gene order (the Genbank accession number: DQ174549) relatively, find the similarity of both tools 99.5%, prove that it is aspergillus niger xylan gene that this institute obtains gene of delivering in sequencing result and Genbank.
Two, the structure of parotid gland tissues specifically expressing zytase transgene carrier
Parotid secretion albumen (parotid secretory protein, PSP) be a kind of glycoprotein of higher, the tissue specific expression of expression amount in saliva, find by research, pig PSP gene is tissue specific expression, mainly express at the parotid gland, separately also have low abundance to express at glandula submandibularis.
The controlling element of specific embodiment adopts parotid secretion albumen (PSP) promotor.
Xylanase from aspergillus niger gene XynB and pig parotid secretion albumen PSP gene signal peptide that step 1 amplification is obtained splice, and fragment is merged in redesign primer pPSPxynB1 and pPSPxynB2 amplification, adds restriction enzyme at two ends
AscThe I restriction enzyme site is used
AscThe I enzyme is cut the PCR product, endonuclease bamhi is reclaimed, to reclaim at last fragment is connected with recombinant plasmid pPSPBGPNeo, connect product and transform DH5 α, cut with enzyme by bacterium liquid PCR equally and identify and order-checking, obtain parotid gland specifically expressing zytase transgene carrier pPSPBGP-xynB, its length is 800bp.
The sequence of above-mentioned pig parotid secretion albumen PSP gene signal peptide is seen article " Yin HF; Zhao ZH; Fan BL; Liu ZL, Lu W, Liu YF; Li N. cDNA cloning; genomic structure, chromosomal mapping, and expression analysis of parotid secretory protein in pig. Genomics. 2004 Jan that Yin etc. delivers; 83 (1): 9-18. ".
The nucleotide sequence of above-mentioned primer pPSPxynB1 is as shown in SEQ ID NO:3, and the nucleotide sequence of above-mentioned primer pPSPxynB2 is as shown in SEQ ID NO:4.
above-mentioned carrier for expression of eukaryon pPSPBGPNeo gets according to the pPAB vector construction, its building process is seen article " the Yin HF, Fan BL, Yang B, Liu YF, Luo J, Tian XH that Yin etc. delivers, Li N. Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control. J Anim Sci. 2006 Mar, 84 (3): 513-9. ".
Three, turn the preparation of xylanase gene nuclear donor cell
The present embodiment is to be prepared into the nuclear donor cell that turns xylanase gene after the parotid gland specifically expressing zytase transgene carrier pPSPBGP-xynB By Transfecting Porcine fetal fibroblast that step 2 is obtained, and concrete steps are as follows:
At first adopt the explanation of those skilled in the art's routine operation and related kit, the pig fetus that obtains take drug induced abortion is as material, separation and Culture obtains the pig fetus and becomes the fiber primary cell, and the cultivation of going down to posterity, and selecting third generation porcine fetus fibroblasts is to carry out follow-up work.
Adopt restriction enzyme
NotThe I enzyme is cut the parotid gland specifically expressing zytase transgene carrier pPSPBGP-xynB that step 2 obtains, after DNA fragmentation with test kit recovery and about 16 kb of purifying, be dissolved in autoclaving water, obtain linearizing parotid gland specifically expressing zytase transgene carrier pPSPBGP-xynB.
Above-mentioned linearizing parotid gland specifically expressing zytase transgene carrier pPSPBGP-xynB is cultivated the porcine fetus fibroblasts system of the third generation by liposome transfection, cultivated 48 hours after transfection, when cell during near 70% degree of converging, cultivate with the type culture liquid that contains 500 μ g/mL G418 medicines, screen the 16th day, begin to occur unicellular in culture dish, change the maintain base that contains 250 μ g/ml G418 medicines keep screening this moment, converges until the 30th day cell grows to approximately 70% again.
The cell of screening was gone down to posterity for the 5th generation, the extraction cell genomic dna is template, adopt its nucleotide sequence of primer 1(as shown in SEQ ID NO:6 with primer) and primer 2 (its nucleotide sequence is as shown in SEQ ID NO:7) carry out pcr amplification, pcr amplification program: 94 ℃, 3min; 94 ℃, 30S; 55 ℃, 30S; 72 ℃, 1min; 35cycles; 72 ℃, 10min, amplification are as shown in Figure 2.
As can be seen from Figure 2, the amplified production band conforms to expection length between 750bp and 1000bp.Amplified production is sent to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and carries out positive and negative both direction order-checking, and sequencing result shows that the sequence of amplified production is identical with expection fragment sequence 100% in carrier pPSPBGP-xynB.The cell that this result proof screening obtains can carry out follow-up test as nuclear donor cell for turning the xylanase gene cell.
But four self secrete the cultivation of zytase Porcine reconstructed embryos
(1) preparation of enucleation oocyte
Collect ovary from the slaughterhouse, after cleaning with the physiological saline of 37 ℃, using 10ml syringe extraction diameter with No. 18 syringe needles is ovocyte in 2～6mm ovarian follicle.Pick out under stereomicroscope that tenuigenin is even, ovarian cumulus is fine and close and wrap up cumulus cell more than 2 layers-ovocyte complex body (COCs), after the washing of M199 maturation culture solution, change over to balance is good in incubator M199 maturation culture solution drip interior or four orifice plates in, at 39 ℃, 5%CO
2After ripe cultivation 42h, adopt those skilled in the art's routine operation to carry out the stoning processing to this porcine oocytes in the incubator of saturated humidity, obtain the stoning porcine oocytes.
(2) preparation of reconstructed embryo and activation
The employing microinjection turns the xylanase gene nuclear donor cell along the stoning otch injection ovum week gap of the stoning porcine oocytes of above-mentioned preparation with an aforementioned preparation, make the cytolemma of nuclear donor cell and the plasma membrane close contact of stoning porcine oocytes, complete embryo's restructuring procedure, obtain reconstructed embryo.
Adopt electric activation method to merge/activate processing to above-mentioned reconstructed embryo.
But five self secrete the preparation of the transgenic pig of zytase
(1) reconstructed embryo vitro culture
With the reconstructed embryo after the activation of above-mentioned preparation in embryo medium, in 39 ℃, 5%CO
2Cultivate in the incubator of saturated humidity.
When above-mentioned embryo medium adopts those skilled in the art to carry out In vitro culture, the conventional formulation of embryo medium used gets final product.
(2) embryo transfer
The acceptor sow is Wen Shi water platform seed farm high-quality sow.
Adopt the common oviduct transplantation method, reconstructed embryo after the activation of above-mentioned preparation is moved in the acceptor Gilt Uterus with surgical method, the 10th day injection pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) 1000IU after transplanting, injection human chorionic gonadotrophin (HCG) 800IU kept gestation in the 13rd day.
After Farrowing, piggy is identified see whether it can self secrete zytase, and concrete operations are as follows:
One, PCR identifies
The ear tissue of the new born piggy of clip uses the extraction agent box Tissue DNA Kit that organizes of Omega company to carry out the DNA extracting, concrete operation step reference reagent box specification sheets.
Take above-mentioned DNA as template, adopt its nucleotide sequence of primer 1(as shown in SEQ ID NO:8) and primer 2 (its nucleotide sequence is as shown in SEQ ID NO:9) carry out pcr amplification, pcr amplification program: 94 ℃, 3min; 94 ℃, 30S; 57 ℃, 30S; 72 ℃, 45S; 35cycles; 72 ℃, 10min, amplification are as shown in Figure 3.
As can be seen from Figure 3, amplified production band (1～5) all between 500bp and 750bp, meets the 621bp length of expection.Pcr amplification product (1～5) is delivered to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd carry out positive and negative both direction order-checking, sequencing result shows that amplified production comprises the XynB gene, and expect in its sequence and transgene carrier pPSPBGP-xynB that (the expection fragment here is the sequence that has comprised xynB gene and the one section 54bp on carrier of 567bp to fragment sequence, overall length is 621bp) 100% identical, but the new born piggy that proves thus the present embodiment is the transgenic pig of self secretion xylanase gene (xynB).
Two, Southern Blot identifies
Aforementioned PCR tests, and but the new born piggy of preliminary evaluation secretes the transgenic pig of zytase for self, also needs to hybridize further by Southern and determines.
Those skilled in the art's routine operation is all adopted in the operation of Southern Blot, specific as follows shown in:
The DNA extracting: method is identified with PCR.
Enzyme is cut: select
XbaThe I enzyme comes enzyme to cut the DNA genome of above-mentioned extracting, and concrete enzyme is cut operation reference reagent box specification sheets.
Enzyme is cut product after test kit reclaims purifying, adopt the routine operation of Southern Blot and carry out Southern Blot in accordance with the specification sheets requirement of related kit, its result as shown in Figure 4.
As can be seen from Figure 4,1～5 Southern hybridization band is between 4361bp and 2322bp, and the hybridization band length meets the 3007bp of expection.This result further proves 1～5 for turning xylanase gene (xynB) pig.In addition, because the positive control of the intensity of these 5 transgenic pig Southern hybridization bands and 1 copy is suitable, estimate that the genome of these 5 transgenic pigs all only inserts the XynB transgenosis of 1 copy.
Three, RT-PCR analyzes
This step is analyzed xylanase gene XynB in the situation of the various tissue expressions of transgenic pig by RT-PCR.
Get and above-mentionedly be verified as one that turns in the xylanase gene pig, extract respectively the DNA of its glandula submandibularis tissue, parotid gland tissues, sublingual gland tissue, liver organization, renal tissue, heart tissue, spleen tissue, lung tissue, stomach-tissue and small intestine, the related kit specification sheets is seen in concrete operations.
Then above-mentioned each DNA is carried out reverse transcription PCR, the related kit specification sheets is seen in concrete operations, and its result as shown in Figure 5.
As can be seen from Figure 5, the XnyB transgenosis is mainly expressed at the parotid gland of transgenic pig, and also has low abundance to express at glandula submandibularis.This result proves the XynB gene not only at the transgenic pig expression in vivo, and is organizing specific expression, meets the requirement of the original design of experiment.
Four, xylanase activity is measured
But self secrete the transgenic pig of zytase for five to above-mentioned PCR proof, and 2 cotemporary non-transgenic pigs (with comparing) all collect saliva, and measure xylan enzyme activity in saliva with reference to national standard " the mensuration spectrophotometry of fodder additives Xylanase activity " (GBT 23874-2009).
Through measuring, in 2 non-transgenic pig salivas, the enzyme of zytase is lived and is 0U/ml, and the average enzyme of 5 transgenic pigs is lived and is 0.15U/ml.Wherein there is the enzyme work of zytase in a transgenic pig saliva to reach 0.60U/ml.
This result shows, the transgenic pig of the inventive method preparation can be secreted zytase really efficiently in saliva.
Can find out by above-mentioned three identification experiments, but the present invention not only can access the transgenic pig of self secretion zytase, and zytase is tissue specific expression efficiently.
SEQUENCE LISTING
＜110〉Wu Zhenfang
＜120〉but the method for cultivation of a kind of zytase recombinant expression vector and a kind of self secretion zytase transgenic pig
<130> 2
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Claims (7)
1. zytase recombinant expression vector, it is characterized in that this recombinant expression vector is with after the xylanase gene of external source and controlling element splicing, be inserted in carrier for expression of eukaryon, build the zytase recombinant expression vector that obtains by the controlling element regulating and expressing.
2. a kind of zytase recombinant expression vector according to claim 1, the xylanase gene that it is characterized in that described external source refers to derive from the xylanase gene in microorganism, yeast or insect.
3. a kind of zytase recombinant expression vector according to claim 2, is characterized in that the xylanase gene of described external source refers to xylanase from aspergillus niger gene XynB.
4. a kind of zytase recombinant expression vector according to claim 1, it is characterized in that described controlling element is the controlling element of oral cavity tissue specifically expressing, the controlling element of parotid gland tissues specifically expressing, the controlling element of the controlling element of stomach-tissue specifically expressing or intestinal tissue specifically expressing.
5. a kind of zytase recombinant expression vector according to claim 1, is characterized in that described carrier for expression of eukaryon is pCMVp-NEO-BAN, pEGFP, pEGFT-Actin, pcDNA
TM3.1/myc His or pcDNA6/His
TM
6. but cultivate the method for self secreting the zytase Porcine reconstructed embryos for one kind, it is characterized in that the method comprises the steps:
Step 1
Be prepared into the zytase recombinant expression vector By Transfecting Porcine fetal fibroblast of the described any one of claim 1 ~ 5 and after screening the nuclear donor cell that turns xylanase gene;
Preparation stoning porcine oocytes;
Step 2
Form reconstructed embryo in the stoning porcine oocytes that the nuclear donor cell implantation step 1 that step 1 is prepared prepares;
After above-mentioned reconstruct ovum is activated processing, the reconstructed embryo that obtains activating.
7. but self secrete the method for cultivation of the transgenic pig of zytase for one kind, it is characterized in that this method of cultivation is after the reconstructed embryo that claim 6 activates is carried out vitro culture, normotrophic reconstructed embryo is operated by embryo transfer move in the replace-conceive Gilt Uterus, through the pregnancy of replace-conceive sow, but the transgenic pig of minute puerperium acquisition self secretion zytase.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103695451A (en) * | 2013-08-07 | 2014-04-02 | 吴珍芳 | Transgenic vector of salivary gland tissue specific expression foreign protein and transgenic pig and construction method thereof |
| CN103695451B (en) * | 2013-08-07 | 2016-04-06 | 华南农业大学 | The transgene carrier of salivary gland tissue specific expression foreign protein and transgenic pig and construction process thereof |
| CN104593417A (en) * | 2015-02-04 | 2015-05-06 | 江苏省农业科学院 | Xylanase intestinal directional expression vector and cell line thereof |
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