CN1247898A - Construction method of recombinant DNA for expressing SOD in animal's mammary gland - Google Patents
Construction method of recombinant DNA for expressing SOD in animal's mammary gland Download PDFInfo
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- CN1247898A CN1247898A CN 98113340 CN98113340A CN1247898A CN 1247898 A CN1247898 A CN 1247898A CN 98113340 CN98113340 CN 98113340 CN 98113340 A CN98113340 A CN 98113340A CN 1247898 A CN1247898 A CN 1247898A
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Abstract
The present invention relates to molecular biology technology to obtain SOD in milk. The DNA fragment and SOD DNA complete or incomplete fragment containing gene promoter and/or enhancer of animal milk protein are connected directly or indirectly through T7 RNA polymeterase and T7 promoter coupling system and loaded into proper DNA vector after essential modification to constitute recombinant DNA, which, after cloning, screeing and amplification and purifying, is mixed with liposome and other matter and injected directly into animal's mammary gland duct and secretion cell or is expressed through established mammary gland positioning and expressing biological reactor during secreting milk. While milk is obtained normally, the expression product, SOD, of recombinant DNA is obtained.
Description
The present invention relates to a kind of with the method for Protocols in Molecular Biology structure recombinant DNA with acquisition SOD in animal milk.
SOD claims superoxide dismutase again, is a kind of enzyme with the effect of degraded free radical unique in the body.
SOD is widely used in anti-ageing at present, and is anticancer, anti-inflammatory, regulate immunity etc., as in senile dementia, senile cataract, hypertension, coronary heart disease, arteriosclerosis, rheumatoid arthritis, lupus erythematosus, certain prevention and therapeutic action are all arranged in the kinds of tumors etc., and good beauty treatment, skin care, senile-resistant efficacy are arranged.The SOD product is mainly derived from biological cell at present, from histocytes such as the red corpuscle of animals such as pig, ox, the heart, brain, extract, perhaps utilize gene clone technology in culturing cell, to express, and then from supernatant, extract, above technology all has 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 SOD.
In order to realize the present invention's purpose, at first by the construction process that is used in animal's mammary gland, expressing the recombinant DNA of SOD of the present invention, utilize molecular biological technology acquisition to include the complete or imperfect fragment of the dna fragmentation and the SOD DNA of animal milk protein gene promoter and/or enhanser, by both directly or indirectly linking to each other, through the suitable dna vector of packing into after the modification of some necessity, constitute one or several recombinant DNAs, mix in the breast duct that injects animal in the back with the suitable processing of recombinant DNA and with some chemical substances, enter mammary gland secretory cell by breast duct.Or import animal sexual cell to set up the mammary expression bio-reactor by other method of setting up bio-reactor, and in the lactescent while of mammary gland secretory cell, secrete SOD also relatively largely, therefore can in the milk of animal, obtain SOD.
Specifically be at first to obtain the complete or imperfect fragment of SOD 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 SOD 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 SOD 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, make this recombinant DNA enter mammary gland secretory cell along lactiferous ducts by the animal teat pipe, when the animal lactation, extrude milk, in milk, can obtain relatively large SOD with ordinary method.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 people MnSODcDNA that gets total length 1Kb with contain ox β-caseincDNA promotor, that be about 800bp and be connected, the pCDA3 carrier of packing into builds up recombinant DNA, behind colony screening, amplification, extraction and purifying, interact with liposome, be mixed with the liquid that concentration is 0.1mg/ml 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 SOD.
Embodiment 2: will contain promotor, ox β-casein the cDNA that is about 800bp is connected with t7 rna polymerase DNA, in addition the T7 promotor is linked to each other with the people MnSOD cDNA of total length 1Kb, through being respectively charged into the carrier of two pCDNA3 behind the necessary dna modification, through colony screening, amplification, behind extraction and the purifying, interact with liposome, be mixed with the liquid that concentration is 0.1mg/ml 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 animal enters lactation period, contain promotor, ox β-casein the cDNA that is about 800bp will induce the synthetic of t7 rna polymerase, and the latter will induce the synthetic of SOD by acting on the T7 promotor, therefore can obtain active SOD from milk.
Use the described construction process that is used in animal's mammary gland, expressing the recombinant DNA of SOD of present method, and interior injection of cooperation breast duct or other the method for setting up bio-reactor, overcome and produced the existing appointed condition of SOD in the past and require shortcomings such as height, technical difficulty is big, cost is high, yield poorly, the production of SOD is carried out under the pure natural state, produce in enormous quantities, cost is lower, therefore has the certain economic exploitation and is worth.
Claims (6)
1. one kind makes up recombinant DNA to obtain the method for SOD 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 enhanser directly or indirectly links to each other with the complete or imperfect fragment of SOD DNA, through the suitable dna vector of packing into after the modification of some necessity, constitute recombinant DNA, set up the method for bio-reactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active SOD.
2. according to claim 1 a kind of with the method for Protocols in Molecular Biology structure recombinant DNA with acquisition SOD in animal milk, it is characterized in that the dna fragmentation that will include animal milk protein gene promoter and/or enhanser directly links to each other with the complete or imperfect fragment of SOD DNA, through the suitable dna vector of packing into after the modification of some necessity, constitute recombinant DNA, set up the method for bio-reactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active SOD.
3. according to claim 1 a kind of with the method for Protocols in Molecular Biology structure recombinant DNA with acquisition SOD in animal milk, it is characterized in that the dna fragmentation that will include animal milk protein gene promoter and/or enhanser links to each other with the coupled system of T7 promotor by t7 rna polymerase indirectly with the complete or imperfect fragment of SODDNA, through the suitable dna vector of packing into after the modification of some necessity, constitute recombinant DNA, set up the method for bio-reactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active SOD.
4. describedly make up recombinant DNA in animal milk, to obtain the method for SOD according to claim 1 or 2 or 3 with Protocols in Molecular Biology, the dna fragmentation that it is characterized in that including ox β-casein DNA promotor and/or enhanser and the complete or imperfect fragment of SOD DNA are indirectly or directly link to each other, through the suitable dna vector of packing into after the modification of some necessity, constitute recombinant DNA, set up the method for bio-reactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active SOD.
5. describedly make up recombinant DNA in animal milk, to obtain the method for SOD according to claim 1 or 2 or 3 with Protocols in Molecular Biology, the dna fragmentation that it is characterized in that including ox α-S1-casein DNA promotor and/or enhanser and the complete or imperfect fragment of SODDNA are indirectly or directly link to each other, through the suitable dna vector of packing into after the modification of some necessity, constitute recombinant DNA, set up the method for bio-reactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active SOD.
6. describedly make up recombinant DNA in animal milk, to obtain the method for SOD according to claim 1 or 2 or 3 with Protocols in Molecular Biology, the dna fragmentation that it is characterized in that including ox lactoglobulin DNA promotor and/or enhanser and the complete or imperfect fragment of SODDNA are indirectly or directly link to each other, through the suitable dna vector of packing into after the modification of some necessity, constitute recombinant DNA, set up the method for bio-reactor with injection in this recombinant DNA cooperation breast duct or other, in milk, obtain active SOD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98113340 CN1247898A (en) | 1998-09-14 | 1998-09-14 | Construction method of recombinant DNA for expressing SOD in animal's mammary gland |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98113340 CN1247898A (en) | 1998-09-14 | 1998-09-14 | Construction method of recombinant DNA for expressing SOD in animal's mammary gland |
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| CN1247898A true CN1247898A (en) | 2000-03-22 |
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| CN 98113340 Pending CN1247898A (en) | 1998-09-14 | 1998-09-14 | Construction method of recombinant DNA for expressing SOD in animal's mammary gland |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105828603A (en) * | 2013-12-24 | 2016-08-03 | Lfb美国股份有限公司 | Transgenic production of heparin |
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1998
- 1998-09-14 CN CN 98113340 patent/CN1247898A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105828603A (en) * | 2013-12-24 | 2016-08-03 | Lfb美国股份有限公司 | Transgenic production of heparin |
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