CN100386437C - Method for constructing idiosyncratic carrier of galactophore of transgenic animal in high expression level - Google Patents
Method for constructing idiosyncratic carrier of galactophore of transgenic animal in high expression level Download PDFInfo
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- CN100386437C CN100386437C CNB2006100404369A CN200610040436A CN100386437C CN 100386437 C CN100386437 C CN 100386437C CN B2006100404369 A CNB2006100404369 A CN B2006100404369A CN 200610040436 A CN200610040436 A CN 200610040436A CN 100386437 C CN100386437 C CN 100386437C
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Abstract
The present invention relates to a constructing method for specificity vectors of the mammary glands of a transgenic animal with a high expression level, which belongs to the technical field of biological engineering. The technology is applied to the preparation of bioreactors for the mammary glands of transgenic animals. Part of non-coding sequences of cytomegaloviruses are used as enhancer sequences, and the enhancer sequences of the cytomegaloviruses are constructed between regulatory sequences of animal lactoprotein and functional genes; consequently, expression vectors for the specificity of the mammary glands is formed. In the present invention, enhancer sequences of eukaryotic genes are used as enhancer sequences (approximately 500 bp) of cytomegaloviruses, the regulatory sequences of animal lactoprotein can be casein non-coding regions or lactoglobulin non-coding regions, and the functional genes can be cDNA or genomic DNA; the expression vectors for the specificity of the mammary glands are formed by all the elements together so that the expression level and the expression rate of bioreactors for the mammary glands of transgenic animals can increased.
Description
Technical field
The invention belongs to technical field of bioengineering, this technology is applied to prepare mammary gland bioreactor of transgenic animals.
Background technology
Mammary gland bioreactor of transgenic animals is to utilize the animal's mammary gland organ to produce biologics or biological products, has very high commercial application value, but efficient is lower in the galactophore biological reactor preparation, expression rate in galactophore of transgenic animal and expression level are at random with uncertain, and most expression level is low.At present, general mammary specific expression gene is made up of milk-protein controlling element and functional gene two portions, though this gene component has regulating and controlling effect to the specificity that starts the functional gene expression and express, expression level and expression rate are very low.Its overall gene structure is: 5 '-end milk-protein regulating and controlling sequence → functional gene → 3 '-end milk-protein regulating and controlling sequence.Though this member also has the expression example, expression rate is 1-10% only; Expression product content is about 0.1-3mg/ml in the milk.The low preparation cost that increases mammary gland bioreactor of transgenic animals of expression rate; Expression level is crossed to hang down have been increased the cost of later development again and has reduced the economic benefit of producing medicine with galactophore biological reactor.Therefore, the expert who studies galactophore biological reactor is both at home and abroad seeking the method that improves expression level.
At present, the specific expressed subject matter that will solve of animal's mammary gland remains expression level, and the simple milk-protein regulating and controlling sequence of using is difficult to reach high level in the member.According to report in the past, this fusion gene member is used for the animal transgenosis, and the expression product content in animal milk is generally below 2mg/ml.
Summary of the invention
The present invention seeks to invent a kind of construction process that in animal's mammary gland, can improve the galactophore of transgenic animal specific expression vector of expression level.
The present invention is an enhancer sequence with cytomegalovirus (CMV) non-coding sequence, the cytomegalovirus enhancer sequence is structured between animal milk protein regulating and controlling sequence and the functional gene, form mammary gland specific expression vector, thereby can improve mammary specific expression level and expression rate.
It is cytomegalovirus enhancer sequence (about 500bp) that the present invention uses the eukaryotic gene enhancer sequence; The animal milk protein regulating and controlling sequence can be casein non-coding region or lactoglobulin non-coding region; Functional gene can be cDNA or genomic dna, and these elements are formed mammary gland specific expression vector together, to improve mammary gland bioreactor of transgenic animals expression level and expression rate.
The application of the CMV sequence in gene constructed is crucial, does not still have the similar application precedent at home and abroad.Prove that by experiment the present invention can improve mammary specific expression amount 1～100,000 times, improves 2～5 times of expression rates.The present invention can significantly improve the output of the biological device of galactophore of transgenic animal, has very big using value, and especially the raising of expression level and expression specificity can reduce the input that later development utilizes, and the 100-1000 that increases economic efficiency doubly.
Animal milk protein regulating and controlling sequence of the present invention can comprise the regulating and controlling sequence of the 2-12kb in the 4-10kb of sheep beta-casein upstream and downstream; Also can comprise goat beta-lactoglobulin upstream 3-10kb and downstream 2-10kb; These regulating and controlling sequences all can merge with enhancer sequence to be used, and makes up mammary gland specific expression vector.
Described enhancer sequence is the non-coding sequence (about 500bp) of CMV.Having invented these controlling elements must use jointly with the cmv enhancer sequence, as long as enhancer sequence is put in position, will improve mammary gland expression levels and expression rate.
The present invention includes following concrete steps:
1) the cytomegalovirus enhancer sequence derives from virus or commercial applications plasmid vector (as pcDNA), gets wherein fragment;
2) cytomegalovirus enhancer sequence gene fragment is structured between animal milk protein regulating and controlling sequence and the functional gene, forms expression vector;
3) expression vector that makes up is increased in general plasmid vector (as pBR-322), and remove the plasmid vector part, reclaim animal's mammary gland specific expression vector fragment with corresponding restriction endonuclease (deciding) according to the restriction enzyme site in the plasmid.
Transgenic animal are made in the open close microinjection excessively of these carrier-pellets or other transgenic method, will in these transgenic animal milk, obtain and the corresponding to expression product of functional gene.
In addition, above-mentioned steps 3) in, plasmid vector can be selected different plasmid vectors according to the size of each element.
The present invention uses cmv enhancer sequence (518bp) and comes adjusting function expression of gene level, comprises the 4-10kb of beta-casein upstream and the 3-12kb in downstream for simultaneously Heshan sheep milk-protein regulating and controlling sequence; Beta-lactoglobulin upstream 3-10kb and downstream 2-10kb.These regulating and controlling sequences and cmv enhancer sequence merge uses the mammary gland specific expression vector of structure.
Embodiment
One, gene constructed process:
1, the cytomegalovirus enhancer sequence derives from the pcDNA carrier, gets wherein fragment, and this gene fragment has following sequence signature:
cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc?cccgcccatt 60
gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc?attgacgtca?120
atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt?atcatatgcc?180
aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt?atgcccagta?240
catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca?tcgctattac?300
catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg?actcacgggg?360
atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc?aaaatcaacg?420
ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg?gtaggcgtgt?480
acggtgggag?gtctatataa?gcagagctct?caattata
2, the cytomegalovirus enhancer sequence is structured between the 3-12kb (non-coding area sequence) and human lactoferrin gene (cDNA) in the 4-10kb of goat beta-casein upstream and downstream, forms mammary gland specific expression vector.
Or the cytomegalovirus enhancer sequence is structured between goat beta-lactoglobulin upstream 3-10kb and downstream 2-10kb (non-coding area sequence) and the human lactoferrin gene (cDNA), also can form mammary gland specific expression vector.
3, the carrier of Gou Jianing increases in plasmid pBR-322, and cuts the plasmid part with restriction endonuclease Sal1 and Not1, reclaims carrier segments.
Two, verification the verifying results:
The method of these dna fragmentation earthing microinjections is imported mouse fertilized egg, obtain former generation 1 of transgenic mice (mother), this mouse grows up and produces its milk of back collection, separate the acquisition whey and prepare transgenic mice with the gene component that these several elements make up, obtain to integrate 1 of female mouse, through the ELISA detection of expression, its lactoferrin content reaches 20-50mg/ml, and (people just Ruzhong content is 6-8mg/ml, the later stage Ruzhong is 2-4mg/ml), reach 5 times of people's colostrum.
It also is transgenic mice that the back godmother mouse of this transgenic mice breeding has part, the expression level in offspring's transgenic mouse Ruzhong and former generation the mouse basically identical.
Claims (4)
1. the construction process of the idiosyncratic carrier of galactophore of transgenic animal of high expression level in animal's mammary gland, it is characterized in that with cytomegalovirus part non-coding sequence be enhancer sequence, the cytomegalovirus enhancer sequence is structured between animal milk protein regulating and controlling sequence and the functional gene, forms mammary gland specific expression vector; Described cytomegalovirus enhancer sequence is characterized as:
cgttacataa?cttacggtaa?atggcccgcc?tggctgaccg?cccaacgacc?cccgcccatt?60
gacgtcaata?atgacgtatg?ttcccatagt?aacgccaata?gggactttcc?attgacgtca?120
atgggtggag?tatttacggt?aaactgccca?cttggcagta?catcaagtgt?atcatatgcc?180
aagtacgccc?cctattgacg?tcaatgacgg?taaatggccc?gcctggcatt?atgcccagta?240
catgacctta?tgggactttc?ctacttggca?gtacatctac?gtattagtca?tcgctattac?300
catggtgatg?cggttttggc?agtacatcaa?tgggcgtgga?tagcggtttg?actcacgggg?360
atttccaagt?ctccacccca?ttgacgtcaa?tgggagtttg?ttttggcacc?aaaatcaacg?420
ggactttcca?aaatgtcgta?acaactccgc?cccattgacg?caaatgggcg?gtaggcgtgt?480
acggtgggag?gtctatataa?gcagagctct?caattata。
2. according to the construction process of the idiosyncratic carrier of galactophore of transgenic animal of the described high expression level of claim 1, it is characterized in that described animal milk protein regulating and controlling sequence is the 4-10kb of goat beta-casein upstream and the 3-12kb in downstream.
3. according to the construction process of the idiosyncratic carrier of galactophore of transgenic animal of the described high expression level of claim 1, it is characterized in that described animal milk protein regulating and controlling sequence is goat beta-lactoglobulin upstream 3-10kb and downstream 2-10kb.
4. according to the construction process of the idiosyncratic carrier of galactophore of transgenic animal of claim 1 or 2 or 3 described high expression levels, it is characterized in that comprising following concrete steps:
1) intercepting cytomegalovirus enhancer sequence gene fragment from virus gene sequence or commercialization plasmid vector;
2) cytomegalovirus enhancer sequence gene fragment is structured between animal milk protein regulating and controlling sequence and the functional gene, forms expression vector;
3) expression vector that makes up is increased in general plasmid vector, and cut the plasmid part, reclaim animal's mammary gland specific expression vector fragment with corresponding restriction endonuclease.
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| CNB2006100404369A CN100386437C (en) | 2006-05-10 | 2006-05-10 | Method for constructing idiosyncratic carrier of galactophore of transgenic animal in high expression level |
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| CN100386437C true CN100386437C (en) | 2008-05-07 |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2258854A1 (en) * | 2009-05-20 | 2010-12-08 | FH Campus Wien | Eukaryotic host cell comprising an expression enhancer |
| CN102492722B (en) * | 2011-12-06 | 2014-09-10 | 广西大学 | Mammary gland-specific expression buffalo prolactin (PRL) gene vector, construction method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058994C (en) * | 1997-08-15 | 2000-11-29 | 中国农业大学 | High effect expressing carrier for animal mammary gland tissue specificity |
| CN1440982A (en) * | 2002-02-27 | 2003-09-10 | 黄伟民 | Construction process of recombinant DNA expressing fibroblast growth factor (FGF) inside animal mammary gland |
| WO2003106674A2 (en) * | 2002-06-14 | 2003-12-24 | Chromagenics B.V. | Means and methods for regulating gene expression |
| CN1661018A (en) * | 2004-10-11 | 2005-08-31 | 中国人民解放军第四军医大学 | Carrier for expressing promotor of casein gene of milch goats grew up in the entral shanxi plain |
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2006
- 2006-05-10 CN CNB2006100404369A patent/CN100386437C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058994C (en) * | 1997-08-15 | 2000-11-29 | 中国农业大学 | High effect expressing carrier for animal mammary gland tissue specificity |
| CN1440982A (en) * | 2002-02-27 | 2003-09-10 | 黄伟民 | Construction process of recombinant DNA expressing fibroblast growth factor (FGF) inside animal mammary gland |
| WO2003106674A2 (en) * | 2002-06-14 | 2003-12-24 | Chromagenics B.V. | Means and methods for regulating gene expression |
| CN1661018A (en) * | 2004-10-11 | 2005-08-31 | 中国人民解放军第四军医大学 | Carrier for expressing promotor of casein gene of milch goats grew up in the entral shanxi plain |
Non-Patent Citations (4)
| Title |
|---|
| 乳腺特异性基因转基因山羊表达特性的研究. 成勇等.畜牧兽医学报，第30卷第3期. 1999 |
| 乳腺特异性基因转基因山羊表达特性的研究. 成勇等.畜牧兽医学报，第30卷第3期. 1999 * |
| 绵羊乳腺特异表达hALR基因载体的构建. 马玉珍等.内蒙古大学学报（自然科学版），第34卷第6期. 2003 |
| 绵羊乳腺特异表达hALR基因载体的构建. 马玉珍等.内蒙古大学学报（自然科学版），第34卷第6期. 2003 * |
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