CN1440982A - Construction process of recombinant DNA expressing fibroblast growth factor (FGF) inside animal mammary gland - Google Patents
Construction process of recombinant DNA expressing fibroblast growth factor (FGF) inside animal mammary gland Download PDFInfo
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- CN1440982A CN1440982A CN 02114900 CN02114900A CN1440982A CN 1440982 A CN1440982 A CN 1440982A CN 02114900 CN02114900 CN 02114900 CN 02114900 A CN02114900 A CN 02114900A CN 1440982 A CN1440982 A CN 1440982A
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Abstract
The present invention relates to molecular biological method of obtaining fibroblast growth factor (FGF) from milk. DNA segment containing animal milk protein gene promoter and/or enhancer and complet or incomplete FGF DNA segment are connected directly or indirectly via T7RNA polymerase and T7 promoter coupling system and through essential modification and loading proper DNA vector, recombinant DNA is constructed. After cloning, screening, proliferation, uprification, mixing liposome and other matter, the recombinant DNA is injected into animal's mammary gland vessel to enter the secretion cell. Or, by means of conventional bioreactor establishing method, mammary gland bioreaction is established and the recombinant DNA is expressed during secreting milk.
Description
The present invention relates to a kind ofly make up recombinant DNA in animal milk, to obtain the method for fibroblast growth factor (FGF) with Protocols in Molecular Biology.
Fibroblast growth factor is that a kind of cell is regulated albumen, and it participates in the important physical regulating and controlling effect in vital movement.It can promote the various kinds of cell Growth and Differentiation, promotes the growth of elastomer cell and strengthens its function, promotes nerve growth and nerve fiber regeneration, is a kind of nerve cell trophic factors.Induce microvascular formation, growth and differentiation, microcirculation improvement.It can make, and epidermic cell upgrades, aged cells comes off, and makes skin be in sliding tender state.
No matter fibroblast growth factor is directly to extract from tissue, still utilizes microbial project production, inevitably all has the weak points such as complex process, cost is high and yield poorly.
The purpose of present method is to overcome the deficiency of above-mentioned prior art, provides a kind of and help fast, obtains from animal milk simply, effectively the method for fibroblast growth factor.
In order to realize the present invention's purpose, at first by the construction method of the recombinant DNA in animal's mammary gland, being expressed as fibroblast growth factor of the present invention, utilize molecular biological technology acquisition to include the complete or imperfect fragment of dna fragmentation and the fibroblast growth factor DNA of animal milk protein gene promoter and/or enhancer, by both directly or indirectly linking to each other, through the suitable dna vector of packing into after the modification of some necessity, consist of one or several recombinant DNAs, mix in the breast duct that injects afterwards animal with the suitable processing of recombinant DNA and with some chemical substances, enter mammary gland secretory cell by breast duct.Or import animal reproduction cell to set up the mammary expression bioreactor by other method of setting up bioreactor, in the lactescent while of mammary gland secretory cell, secrete fibroblast growth factor also relatively largely, therefore can in the milk of animal, obtain fibroblast growth factor.
Specifically be at first to obtain the complete or imperfect fragment of fibroblast growth factor DNA of animal or plant and include the promotor of animal milk protein gene and/or the dna fragmentation of enhanser by molecular biological method, complete or the imperfect fragment of fibroblast growth factor DNA is connected the downstream of the dna fragmentation that includes animal milk protein gene promoter and/or enhanser, after doing the dna modification of a little necessity again, on the appropriate carriers of packing into, constitute recombinant DNA.Perhaps will include the promotor of animal milk protein gene and/or the dna fragmentation of enhanser links to each other with t7 rna polymerase DNA, the T7 promotor links to each other with the complete or imperfect fragment of fibroblast growth factor DNA, above two junction fragments are done the dna modification of a little necessity, on the carrier or more than one carrier of packing into suitable, constitute recombinant DNA, pass through the method for conventional molecular cloning again, a large amount of amplifications and this recombinant DNA of purifying, this recombinant DNA is done (perhaps not doing) certain enzyme cut processing, after some chemical substance (as liposome etc.) is mixed, draw this mixture by appliances such as syringes, inject in the breast duct by the animal teat pipe, make this recombinant DNA enter mammary gland secretory cell along lactiferous ducts.Or organize pipeline to import mammary glandular cell by other, and when animal lactation, extrude milk with conventional method, in milk, can obtain relatively large fibroblast growth factor.Or recombinant DNA imported animal sexual cell to set up the bio-reactor of mammary expression by other method of setting up bio-reactor.
Embodiment 1: the rh-bFGF cDNA that gets total length 0.8Kb with contain promotor, ox β-casein the cDNA that is about 800bp connects, the pCDA3 carrier of packing into builds up recombinant DNA, through colony screening, amplification, behind extraction and the purifying, interact with liposome, be mixed with the liquid that concentration is 0.1mg/m1 with physiological saline, get 200ml, inject in the cow breast duct of empty milk phase, after suitably pushing, liquid will enter mammary gland secretory cell by breast duct, when the cow postpartum milk secretion, can in milk, obtain relatively large fibroblast growth factor.
Use the construction method of the described recombinant DNA in animal's mammary gland, being expressed as fibroblast growth factor of this method, and the interior injection of cooperation breast duct or other the method for setting up bioreactor, overcome and produced the existing appointed condition of fibroblast growth factor in the past and require the shortcomings such as height, technical difficulty is big, cost is high, yield poorly, the production of fibroblast growth factor is carried out under the pure natural state, produce in enormous quantities, cost is lower, therefore has certain economic development value.
Claims (6)
1 one kinds make up recombinant DNA to obtain the method for fibroblast growth factor in animal milk with Protocols in Molecular Biology, it is characterized in that the dna fragmentation that will include animal milk protein gene promoter and/or enhancer directly or indirectly links to each other with the complete or imperfect fragment of fibroblast growth factor DNA, through the suitable dna vector of packing into after the modification of some necessity, consist of recombinant DNA, set up the method for bioreactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active fibroblast growth factor.
2 is according to claim 1 a kind of with the method for Protocols in Molecular Biology structure recombinant DNA with acquisition fibroblast growth factor in animal milk, it is characterized in that the dna fragmentation that will include animal milk protein gene promoter and/or enhancer directly links to each other with the complete or imperfect fragment of fibroblast growth factor DNA, through the suitable dna vector of packing into after the modification of some necessity, consist of recombinant DNA, set up the method for bioreactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active fibroblast growth factor.
3 is according to claim 1 a kind of with the method for Protocols in Molecular Biology structure recombinant DNA with acquisition fibroblast growth factor in animal milk, it is characterized in that the dna fragmentation that will include animal milk protein gene promoter and/or enhancer links to each other with the coupled system of T7 promoter by t7 rna polymerase indirectly with the complete or imperfect fragment of fibroblast growth factor DNA, through the suitable dna vector of packing into after the modification of some necessity, consist of recombinant DNA, set up the method for bioreactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active fibroblast growth factor.
4 describedly make up recombinant DNA to obtain the method for fibroblast growth factor in animal milk with Protocols in Molecular Biology according to claim 1 or 2 or 3, the dna fragmentation that it is characterized in that including ox β-casein DNA promoter and/or enhancer and the complete or imperfect fragment of fibroblast growth factor DNA are indirectly or directly link to each other, through the suitable dna vector of packing into after the modification of some necessity, consist of recombinant DNA, set up the method for bioreactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active fibroblast growth factor.
5 describedly make up recombinant DNA to obtain the method for fibroblast growth factor in animal milk with Protocols in Molecular Biology according to claim 1 or 2 or 3, the dna fragmentation that it is characterized in that including ox α-S1-casein DNA promoter and/or enhancer and the complete or imperfect fragment of fibroblast growth factor DNA are indirectly or directly link to each other, through the suitable dna vector of packing into after the modification of some necessity, consist of recombinant DNA, set up the method for bioreactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active fibroblast growth factor.
6 describedly make up recombinant DNA to obtain the method for fibroblast growth factor in animal milk with Protocols in Molecular Biology according to claim 1 or 2 or 3, the dna fragmentation that it is characterized in that including ox lactoglobulin DNA promoter and/or enhancer and the complete or imperfect fragment of fibroblast growth factor DNA are indirectly or directly link to each other, through the suitable dna vector of packing into after the modification of some necessity, consist of recombinant DNA, set up the method for bioreactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active fibroblast growth factor.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 02114900 CN1440982A (en) | 2002-02-27 | 2002-02-27 | Construction process of recombinant DNA expressing fibroblast growth factor (FGF) inside animal mammary gland |
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| CN 02114900 CN1440982A (en) | 2002-02-27 | 2002-02-27 | Construction process of recombinant DNA expressing fibroblast growth factor (FGF) inside animal mammary gland |
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| CN1440982A true CN1440982A (en) | 2003-09-10 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100386437C (en) * | 2006-05-10 | 2008-05-07 | 扬州大学 | Method for constructing idiosyncratic carrier of galactophore of transgenic animal in high expression level |
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2002
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100386437C (en) * | 2006-05-10 | 2008-05-07 | 扬州大学 | Method for constructing idiosyncratic carrier of galactophore of transgenic animal in high expression level |
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