CN115433736A - Gateway prokaryotic vector system for efficiently expressing and purifying small-label active fusion protein - Google Patents
Gateway prokaryotic vector system for efficiently expressing and purifying small-label active fusion protein Download PDFInfo
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- 239000013598 vector Substances 0.000 title claims abstract description 49
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 9
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 239000012634 fragment Substances 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 21
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- UNFWWIHTNXNPBV-WXKVUWSESA-N spectinomycin Chemical compound O([C@@H]1[C@@H](NC)[C@@H](O)[C@H]([C@@H]([C@H]1O1)O)NC)[C@]2(O)[C@H]1O[C@H](C)CC2=O UNFWWIHTNXNPBV-WXKVUWSESA-N 0.000 claims abstract description 17
- 229960000268 spectinomycin Drugs 0.000 claims abstract description 17
- 239000013604 expression vector Substances 0.000 claims abstract description 15
- 229930027917 kanamycin Natural products 0.000 claims abstract description 14
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims abstract description 14
- 229960000318 kanamycin Drugs 0.000 claims abstract description 14
- 229930182823 kanamycin A Natural products 0.000 claims abstract description 14
- 238000005215 recombination Methods 0.000 claims abstract description 9
- 230000006798 recombination Effects 0.000 claims abstract description 9
- 239000001963 growth medium Substances 0.000 claims description 21
- 239000013612 plasmid Substances 0.000 claims description 14
- 238000013461 design Methods 0.000 claims description 12
- 238000012163 sequencing technique Methods 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 9
- 238000010369 molecular cloning Methods 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 239000012880 LB liquid culture medium Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 238000007689 inspection Methods 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 6
- 101150102092 ccdB gene Proteins 0.000 claims description 4
- 101150113191 cmr gene Proteins 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 108700026244 Open Reading Frames Proteins 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 abstract description 6
- 230000014509 gene expression Effects 0.000 abstract description 5
- 230000009465 prokaryotic expression Effects 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 2
- 235000018102 proteins Nutrition 0.000 description 14
- 238000012216 screening Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention relates to a Gateway prokaryotic vector system for efficiently expressing and purifying small-label active fusion protein, which comprises the following steps in a specific development process: 1) Taking a PET30a traditional vector as an initial skeleton, inserting the attR1-CmR-ccdB-attR2 fragment obtained by amplification into the initial skeleton through enzyme digestion reaction and recombination ligation reaction to obtain 6xHis-GW with kanamycin resistance K -6xHis vector; 2) With 6XHis-GW K The-6 xHis vector is used as a framework, and the spectinomycin resistance gene segment obtained by amplification is inserted into 6xHis-GW through enzyme digestion reaction and recombination connection reaction K -6XHis backbone, resulting in 6XHis-GW with spectinomycin resistance S -6xHis vector; 3) 6xHis-GW S The-6 xHis and the ENTR entry vector are matched for use through LR reaction, and a final prokaryotic expression vector can be constructed simply and quickly. The invention has the advantages that: can be used for high-efficiency accurate expression of single protein and has good stabilityThe contrast is strong, false positive does not exist, the purification work of active protein can be effectively simplified and promoted, and convenience is provided for the functional research of protein.
Description
Technical Field
The invention relates to a Gateway prokaryotic vector system for quickly and efficiently expressing active protein.
Background
The Gateway system is a set of efficient and accurate molecular cloning technology. The system only needs two simple LR or BP reactions, can ensure that target sequences are randomly, efficiently and accurately converted between an expression vector (Destination vector) and an entry vector (ENTR vector), and can ensure that the side sequences of each target sequence are highly consistent. The active function of the protein is the embodiment of vitality. The study of the structure, function and activity of proteins has been one of the basic goals of biological research. Purification of a functional protein under non-denaturing conditions, and testing of various biological properties, is the main approach to achieve this basic goal. However, the existing protein prokaryotic expression vector system usually adopts an enzyme digestion connection system which is low in efficiency and outdated, is time-consuming and labor-consuming, has a high false positive ratio during screening and cloning, cannot ensure that each expressed small-label fusion protein has a completely consistent flanking connection sequence, and has low contrast. Therefore, a new vector system capable of utilizing Gateway molecular cloning technology is hoped to be developed, so that the method helps us express a large amount of active target proteins more efficiently and accurately, avoids the problem of false positive in the molecular cloning process, solves the problem of inconsistent flanking connection sequences among different fusion proteins, improves the contrast, and makes a previous work for purifying active functional proteins.
Disclosure of Invention
The invention aims to provide a Gateway prokaryotic vector system for efficiently expressing and purifying small-tag active fusion protein so as to solve the problems in the background technology. In addition, the invention is a high-efficiency prokaryotic expression system, and after patent approval, the invention is combined with a eukaryotic expression system (patent application No. 202110577823.0) invented separately by the research group to be developed into a kit set capable of being commercialized. The kit set can be used for expression and purification of all plant source active proteins and expression and purification of most animal source/human source active proteins.
In order to solve the technical problems, the invention develops an empty target expression vector (Destination vector) which can be matched with an entry vector (ENTR vector): 6xHis-GW S 6xHis, a Gateway prokaryotic vector system for efficiently expressing and purifying the small tag active fusion protein, and the specific development scheme is as follows:
1) Using a PET30a carrier as a substrate, carrying out double enzyme digestion on the carrier by using restriction enzymes BamHI and HindIII to obtain an enzyme digestion product, and purifying a main fragment after enzyme digestion;
2) Designing a Gateway locus sequence with recombination sites to amplify a forward primer PET30a-GWF (5-;
3) Detecting the PCR product, and purifying the PCR product with the correct fragment length;
4) Mixing the purified PET30a main segment and the purified PCR product according to a certain proportion, and adding recombinase
Preparing a reaction system by using MultiS and buffer, and reacting at constant temperature of 37 ℃ for half an hour;
5) Introducing the reacted recombinant product into escherichia coli DB3.1 competent cells, adding a proper amount of SOC liquid culture medium, incubating for 1 hour at 37 ℃ with a shaker at 200rpm/min, smearing a proper amount of the growth solution on a solid LB culture medium plate containing kanamycin, and putting the solid LB culture medium plate into a 37 ℃ growth box for overnight culture;
6) Design a forward primer T7F (5' -TTAATACGACTCACTATAG-
3') and a reverse primer CmRR1 (5;
7) After the proper size of clone grows on the culture dish plate, selecting part of the monoclonal antibody to be dissolved in 20ul of water, taking 1.5ul as a sample template, preparing a 20ul PCR reaction system by utilizing a pair of designed detection primers, detecting the clones, and if the PCR can amplify the fragment with the expected size, indicating that the recombination reaction is possible to succeed;
8) Selecting a monoclonal antibody capable of amplifying a fragment with an expected size, inoculating the monoclonal antibody into 5ml of LB liquid culture medium containing kanamycin, and culturing overnight by using a 37-degree shaker at 200 rpm/min;
9) Extracting monoclonal plasmids by using the kit;
10 The extracted monoclonal plasmid is subjected to sequencing inspection, and if the sequencing result is completely consistent with the theoretical design, the gateway system element, the ccdB and the CmR genes are successfully added, so that 6xHis-GW is obtained K -6xHis vector;
(use of this 6XHis-GW K And (3) carrying out LR reaction on the-6 xHis vector and an ENTR vector containing a target gene and not resisting kanamycin, and screening by utilizing an LB culture medium plate containing kanamycin to obtain a final expression vector. However, if the ENTR vector containing the target gene fragment itself already has the kanamycin resistance gene, 6XHis-GW cannot be reused K Construction of the final expression vector by LR reaction with-6 XHis, instead of the empty target expression vector 6XHis-GW with spectinomycin resistance that we finally developed S -6xHis。)
At 6XHis-GW K 6xHis-GW based on the preparation of-6 xHis S The 6xHis vector undergoes the following further steps, thereby finally obtaining:
11 With 6XHis-GW K Using a-6 xHis vector as a substrate, carrying out enzyme digestion on the substrate by using a restriction enzyme FspI to obtain an enzyme digestion product, and purifying the fragment after enzyme digestion;
12 Design to synthesize the forward primer SPECRF3 (5-
gacagcaggcatcgatgatgcgcagcacgaacccagtggacata-3 ') and reverse primer SPECRR3 (5 ' -atggcggccccacggtgcgcagtccatgcatgatgatatatctccccaa-3 '), using vector pB2GW7 as a template to amplify the entire spectinomycin resistance gene PCR fragment with a promoter, a terminator and an open reading frame;
13 Detecting the PCR product and purifying the PCR product with correct fragment length;
14 Purified 6XHis-GW K Mixing the enzyme-digested fragment of-6 XHis and the purified PCR product in certain proportion, and adding recombinase
Preparing a reaction system by using MultiS and buffer, and reacting at constant temperature of 37 ℃ for half an hour;
15 Introducing the reacted recombinant product into Escherichia coli DB3.1 competent cells, adding an appropriate amount of SOC liquid culture medium, incubating for 1 hour at a shaker of 37 ℃ at 200rpm/min, smearing an appropriate amount of the growth solution on a solid LB culture medium plate containing spectinomycin, and putting the solid LB culture medium plate into a 37 ℃ growth chamber for overnight culture;
16 Selecting normally growing monoclonals, inoculating the monoclonals into 5ml LB liquid culture medium containing spectinomycin, and culturing overnight by a 37-degree shaking table at 200 rpm/min;
17 Using a kit to extract a monoclonal plasmid;
18 Carrying out sequencing inspection on the extracted monoclonal plasmid, and if the sequencing result is completely consistent with the theoretical design, proving that the product is successfully developed to obtain 6xHis-GW S -6xHis, which is used when preparing a specific protein expression vector by molecular cloning, and the plasmid is stored in a large amount. (by adding 6XHis-GW S The-6 XHis vector and the ENTR vector containing the target gene and not spectinomycin resistance carry out LR reaction, and the LB culture medium plate containing spectinomycin is used for screening, so that the final expression vector can be obtained. )
The invention has the advantages that: the invention can be used for large-scale prokaryotic expression of single protein components under various conditions. Both ends of the protein have histone labels (6 XHis) with small molecular weight and no influence on the function of the protein, and the high-efficiency column-passing purification of the protein is facilitated; by adding the ccdB gene and the CmR gene, the problem of false positive in the molecular cloning process is solved; by adding attR1 and attR2 sequence sites of the gateway system, the problem of inconsistent flanking sequences among different fusion proteins is solved, the contrast is improved, and the gateway has the characteristics of high efficiency and accuracy; by adding spectinomycin resistance gene, 6xHis-GW is enabled S In the-6 XHis vector, there is a second option to use the kanamycin resistance gene, allowing us to freely select almost all commercial ENTR vectors for LR reactions. In conclusion, the invention can greatly promote the work of promoting the purification of active protein and establish the foundation for the functional research of protein.
Drawings
FIG. 1 is 6XHis-GW S Schematic structural diagram of-6 XHis vector.
FIG. 2 is 6XHis-GW K Schematic diagram of-6 XHis vector production flow.
FIG. 3 is a graph showing the relationship between 6XHis-GW and 6XHis-GW K 6xHis-GW is prepared on the basis of-6 xHis S Schematic of the flow scheme for the-6 XHis vector.
Detailed Description
The invention is illustrated below by means of specific examples, without being restricted thereto.
Examples
Gateway system prokaryotic expression vector-6 xHis-GW for fast and high-efficiency expression of active protein S -6xHis. The specific manufacturing method comprises the following steps:
1) Using a PET30a vector as a substrate, carrying out double enzyme digestion on the vector by using restriction enzymes BamHI and HindIII to obtain an enzyme digestion product, and purifying a main fragment after enzyme digestion;
2) Designing a Gateway locus sequence with recombination sites to amplify a forward primer PET30a-GWF (5-;
3) Detecting the PCR product, and purifying the PCR product with the correct fragment length;
4) Mixing the purified PET30a main fragment and the purified PCR product according to a certain proportion, and adding recombinase
Preparing a reaction system by using MultiS and buffer, and reacting at constant temperature of 37 ℃ for half an hour;
5) Introducing the recombinant product after reaction into Escherichia coli DB3.1 competent cells, adding an appropriate amount of SOC liquid culture medium, incubating for 1 hour at 37 ℃ with a shaker at 200rpm/min, smearing an appropriate amount of the growth liquid on a solid LB culture medium plate containing kanamycin, and placing the solid LB culture medium plate in a 37 ℃ growth chamber for overnight culture;
6) Design a forward primer T7F (5' -TTAATACGACTCACTATAG-
3') and a reverse primer CmRR1 (5;
7) After the proper size of clone grows on the plate of the culture dish, selecting part of the monoclonal antibody to be dissolved in 20ul of water, taking 1.5ul of the monoclonal antibody as a sample template, preparing a 20ul PCR reaction system by utilizing a pair of designed detection primers, detecting the clones, and if the PCR can amplify fragments with the expected size, indicating that the recombination reaction is possibly successful;
8) Selecting a monoclonal antibody capable of amplifying a fragment with an expected size, inoculating the monoclonal antibody into 5ml of LB liquid culture medium containing kanamycin, and culturing overnight by using a 37-degree shaker at 200 rpm/min;
9) Extracting monoclonal plasmids by using the kit;
10 The extracted monoclonal plasmid is subjected to sequencing inspection, and if the sequencing result is completely consistent with the theoretical design, the gateway system element, the ccdB and the CmR genes are successfully added, so that 6xHis-GW is obtained K -6xHis vector;
(use of this 6XHis-GW K -6xHis vector, LR reaction with ENTR vector containing target gene and not kanamycin-resistant, screening with LB medium plate containing kanamycin, and finally obtaining the expression vector. However, if the ENTR vector containing the target gene fragment itself already has the kanamycin resistance gene, 6XHis-GW cannot be reused K Construction of the final expression vector by LR reaction with-6 XHis, instead of the empty target expression vector 6XHis-GW with spectinomycin resistance that we finally developed S -6xHis。)
At 6XHis-GW K 6xHis-GW based on the preparation of-6 xHis S The-6 xHis vector undergoes the following further steps, thereby finally obtaining:
11 With 6XHis-GW K Using a-6 xHis vector as a substrate, carrying out enzyme digestion on the substrate by using a restriction enzyme FspI to obtain an enzyme digestion product, and purifying the fragment after enzyme digestion;
12 Design to synthesize the forward primer SPECRF3 (5-
gacagcaggcatcgatgatgcgcagcacgaacccagtggacata-3 ') and reverse primer SPECRR3 (5 ' -atggcggccccacggtgcgcagtccatgcatgatgatatatctccccaa-3 '), using vector pB2GW7 as a template to amplify the entire spectinomycin resistance gene PCR fragment with a promoter, a terminator and an open reading frame;
13 Detecting the PCR product and purifying the PCR product of the correct fragment length;
14 Purified 6XHis-GW K Mixing the enzyme-digested fragment of-6 XHis and the purified PCR product in certain proportion, and adding recombinase
Preparing a reaction system by using MultiS and buffer, and reacting for half an hour at a constant temperature of 37 ℃;
15 Introducing the reacted recombinant product into Escherichia coli DB3.1 competent cells, adding an appropriate amount of SOC liquid culture medium, incubating for 1 hour at a shaker of 37 ℃ at 200rpm/min, smearing an appropriate amount of the growth solution on a solid LB culture medium plate containing spectinomycin, and putting the solid LB culture medium plate into a 37 ℃ growth chamber for overnight culture;
16 Selecting normally growing monoclonals, inoculating the monoclonals into 5ml LB liquid culture medium containing spectinomycin, and culturing overnight by a 37-degree shaking table at 200 rpm/min;
17 Using a kit to extract monoclonal plasmids;
18 Carrying out sequencing inspection on the extracted monoclonal plasmid, and if the sequencing result is completely consistent with the theoretical design, proving that the product is successfully developed to obtain 6xHis-GW S 6XHis, which is retained in large quantities and is used when preparing a specific protein expression vector by molecular cloning.
(by adding 6XHis-GW S -6XHis vector and Noncystein resistant ENTR containing target geneThe vector is subjected to LR reaction, and a final expression vector can be obtained by screening an LB culture medium plate containing spectinomycin. )
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (1)
1. The Gateway prokaryotic vector system for efficiently expressing and purifying the small-tag active fusion protein is characterized in that the specific preparation method comprises the following steps:
1) Using a PET30a vector as a substrate, carrying out double enzyme digestion on the vector by using restriction enzymes BamHI and HindIII to obtain an enzyme digestion product, and purifying a main fragment after enzyme digestion;
2) Designing a Gateway locus sequence with recombination sites to amplify a forward primer PET30a-GWF (5-;
3) Detecting the PCR product, and purifying the PCR product with the correct fragment length;
4) Mixing the purified PET30a main fragment and the purified PCR product according to a certain proportion, and adding recombinase
Preparing a reaction system by using MultiS and buffer, and reacting at constant temperature of 37 ℃ for half an hour;
5) Introducing the recombinant product after reaction into Escherichia coli DB3.1 competent cells, adding an appropriate amount of SOC liquid culture medium, incubating for 1 hour at 37 ℃ with a shaker at 200rpm/min, smearing an appropriate amount of the growth liquid on a solid LB culture medium plate containing kanamycin, and placing the solid LB culture medium plate in a 37 ℃ growth chamber for overnight culture;
6) Design a forward primer T7F (5' -TTAATACGACTCACTATAG-
3') and a reverse primer CmRR1 (5;
7) After the proper size of clone grows on the culture dish plate, selecting part of the monoclonal antibody to be dissolved in 20ul of water, taking 1.5ul as a sample template, preparing a 20ul PCR reaction system by utilizing a pair of designed detection primers, detecting the clones, and if the PCR can amplify the fragment with the expected size, indicating that the recombination reaction is possible to succeed;
8) Selecting a monoclonal capable of amplifying a fragment with an expected size, inoculating the monoclonal into 5ml of LB liquid culture medium containing kanamycin, and culturing overnight by using a 37-degree shaking table at 200 rpm/min;
9) Extracting monoclonal plasmid by using a kit;
10 Carrying out sequencing inspection on the extracted monoclonal plasmid, and if the sequencing result is completely consistent with the theoretical design, proving that the addition of gateway system elements and ccdB and CmR genes is successful to obtain 6xHis-GW K -6xHis vector;
at 6XHis-GW K 6XHis-GW based on the preparation of-6 XHis S The 6xHis vector undergoes the following further steps, thereby finally obtaining:
11 With 6XHis-GW K Using a-6 xHis carrier as a substrate, carrying out enzyme digestion on the substrate by using restriction enzyme FspI to obtain an enzyme digestion product, and purifying fragments after enzyme digestion;
12 Design and synthesize forward primer SPECRF3 (5 'gacagggcacgatcatgaTGCGCAGGCACGAACCCAGGTGGACATA-3') with recombination site and reverse primer SPECRR3 (5 'atggcgcccacggTGCGCAGTCATGATATATCCCAA-3'), amplify the whole spectinomycin resistance gene PCR fragment with promoter, terminator and open reading frame by taking vector pB2GW7 as a template;
13 Detecting the PCR product and purifying the PCR product of the correct fragment length;
14 Purified 6XHis-GW K Mixing the enzyme-digested fragment of-6 XHis and the purified PCR product in certain proportion, and adding recombinase
Preparing a reaction system by using MultiS and buffer, and reacting for half an hour at a constant temperature of 37 ℃;
15 Introducing the reacted recombinant product into Escherichia coli DB3.1 competent cells, adding an appropriate amount of SOC liquid culture medium, incubating for 1 hour at a shaker of 37 ℃ at 200rpm/min, smearing an appropriate amount of the growth solution on a solid LB culture medium plate containing spectinomycin, and putting the solid LB culture medium plate into a 37 ℃ growth chamber for overnight culture;
16 Selecting normally growing monoclonals, inoculating the monoclonals into 5ml LB liquid culture medium containing spectinomycin, and culturing overnight by a 37-degree shaking table at 200 rpm/min;
17 Using a kit to extract monoclonal plasmids;
18 Carrying out sequencing inspection on the extracted monoclonal plasmid, and if the sequencing result is completely consistent with the theoretical design, proving that the product is successfully developed to obtain 6xHis-GW S -6xHis, which is used when preparing a specific protein expression vector by molecular cloning, and the plasmid is stored in a large amount.
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| US20040132133A1 (en) * | 2002-07-08 | 2004-07-08 | Invitrogen Corporation | Methods and compositions for the production, identification and purification of fusion proteins |
| JP2006141320A (en) * | 2004-11-22 | 2006-06-08 | Invitrogen Japan Kk | Method for cloning plural nucleic acid fragments |
| US20060183193A1 (en) * | 2005-01-07 | 2006-08-17 | Horanyi Peter S | Vectors and methods for high throughput co-expression |
| CN103374064A (en) * | 2012-04-24 | 2013-10-30 | 中国农业大学 | Plant root hair development related protein TaRSL4, and coding gene and application thereof |
| CN103374063A (en) * | 2012-04-24 | 2013-10-30 | 中国农业大学 | Plant root hair development related protein TaRHD6, and coding gene and application thereof |
| KR20160087489A (en) * | 2015-01-13 | 2016-07-22 | 경희대학교 산학협력단 | A cambium-specific promoter and uses thereof |
-
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- 2021-12-13 CN CN202111521272.2A patent/CN115433736A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040132133A1 (en) * | 2002-07-08 | 2004-07-08 | Invitrogen Corporation | Methods and compositions for the production, identification and purification of fusion proteins |
| JP2006141320A (en) * | 2004-11-22 | 2006-06-08 | Invitrogen Japan Kk | Method for cloning plural nucleic acid fragments |
| US20060183193A1 (en) * | 2005-01-07 | 2006-08-17 | Horanyi Peter S | Vectors and methods for high throughput co-expression |
| CN103374064A (en) * | 2012-04-24 | 2013-10-30 | 中国农业大学 | Plant root hair development related protein TaRSL4, and coding gene and application thereof |
| CN103374063A (en) * | 2012-04-24 | 2013-10-30 | 中国农业大学 | Plant root hair development related protein TaRHD6, and coding gene and application thereof |
| KR20160087489A (en) * | 2015-01-13 | 2016-07-22 | 경희대학교 산학협력단 | A cambium-specific promoter and uses thereof |
Non-Patent Citations (1)
| Title |
|---|
| WENJUN XIE, ET AL.: "A Split-GFP Gateway Cloning System for Topology Analyses of Membrane Proteins in Plants", PLOS ONE, vol. 12, no. 1, pages 1 - 8 * |
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