CN105907786A - Dual-promoter universal plasmid for expressing T4 ligase of domestic silkworm middle silk gland bioreactor as well as application and method of dual-promoter universal plasmid - Google Patents
Dual-promoter universal plasmid for expressing T4 ligase of domestic silkworm middle silk gland bioreactor as well as application and method of dual-promoter universal plasmid Download PDFInfo
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- CN105907786A CN105907786A CN201610296590.6A CN201610296590A CN105907786A CN 105907786 A CN105907786 A CN 105907786A CN 201610296590 A CN201610296590 A CN 201610296590A CN 105907786 A CN105907786 A CN 105907786A
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 88
- 239000013612 plasmid Substances 0.000 title claims abstract description 67
- 108090000364 Ligases Proteins 0.000 title claims abstract description 65
- 102000003960 Ligases Human genes 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 21
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- 102000004190 Enzymes Human genes 0.000 claims description 5
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- 108010020764 Transposases Proteins 0.000 claims description 5
- 230000000762 glandular Effects 0.000 claims description 5
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- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 4
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Abstract
The invention discloses a dual-promoter universal plasmid for expressing T4 ligase of a domestic silkworm middle silk gland bioreactor as well as application and a method of the dual-promoter universal plasmid. The plasmid takes piggy Bac transposons as a foundation and carries an Amp resistance gene; the plasmid comprises function expression frames of a T4 ligase gene used as an exogenous gene and a green fluorescence EGFP (Enhanced Green Fluorescent Protein) gene used as a marker gene; the plasmid is constructed by utilizing a molecular biology method and two special restriction enzyme cutting sites containing ApaI and NheI are formed between DDDDK and a fibroin light-chain gene polyA; the universal plasmid is subjected to dual-enzyme digestion by adopting the ApaI and the NheI; after the universal plasmid is connected with the T4 ligase gene, the universal plasmid and an auxiliary plasmid are commonly injected into a domestic silkworm fertilized ovum; the green fluorescence protein gene and the T4 ligase gene are transferred into a domestic silkworm genome through utilizing properties of the transposons and are stably inherited and expressed to obtain transgenic domestic silkworms. The transgenic domestic silkworms are screened with the help of a fluorescence marker gene and domestic silkworm silk gland cells are used for specifically synthesizing and secreting T4 ligase protein.
Description
Technical field
The present invention relates to a kind of plasmid and application thereof and method, especially relate in the middle part of a kind of silkworm for expressing T4 ligase
The universal plasmid of sericterium bioreactor double-promoter and application thereof and method.
Background technology
PiggyBac transposon is initially to separate from the genome of cabbage looper (Trichoplusia ni) TN-368 cell strain
Arrive, be the DNA transposon that the transposition activity having now been found that is the highest.PiggyBac transposon system is a kind of non-viral load
Body, transposition efficiency is higher.Compared with sleeping beauty, piggyBac bearer capabilities is relatively big, portability 18kb, Ke Yishi
Will not put in position after now polygenic coexpression, and swivel base fragment is cut and leave trace (footprint), genome can be real
Now accurately repair after excision, in the application of reversible gene, there is important function.
The research of the transgenic bombyx mori sericterium bioreactor of piggyBac transposon mediation has been carried out the more than ten years, the section of countries in the world
Scholar is devoted to the expression of exogenous gene, has built up with fibroin albumen light chain promoter (Fib-L Promoter), fibroin egg
White heavy chain promoter (Fib-H Promoter) and sericin 1 promoter (Ser1Promoter) expression foreign protein turn base
Because of domestic natural silk gland bioreactor.But make a general survey of the result of study of silkworm biological reactor both at home and abroad for over ten years, transgenic bombyx mori silk
The exogenous protein expression efficiency of gland bioreactor is the lowest, is no matter the exogenous gene expressed of sericin promoters driven, or silk
The exogenous gene that element promoters driven is expressed, the expression of thumping majority experiment does not all have reaching about the 1% of cocoon shell weight, far
It is far from reaching the desired efficient expression as expressing fibroin of scientists.
Summary of the invention
In order to solve problem present in background technology, it is an object of the invention to propose a kind of for expressing T4 ligase
The silkworm middle division of silkgland universal plasmid of bioreactor double-promoter and application thereof and method, utilize transgenic bombyx mori technology by T4
In ligase channel genes domestic silkworm gene group, and specifically expressing in domestic silkworm silk glandular cell, it is developed to synthesis secretion single
The silkworm of T4 ligase.
In order to achieve the above object, the step of the technical solution used in the present invention is as follows:
One, a kind of silkworm middle division of silkgland universal plasmid of bioreactor double-promoter for expressing T4 ligase:
Described plasmid is piggy-9887 plasmid, and its base sequence such as SEQ ID NO.1, is based on piggyBac transposon
And with Amp resistant gene, including two swivel base arm PBL and PBR of piggyBac transposon, and two swivel base arms it
Between two merits including the T4 ligase gene as exogenous gene and the green fluorescence EGFP gene as marker gene
Can expression cassette.
One functional expression frame is the Green Fluorescent Protein Gene Expression frame that A3 promoter starts, i.e. A3Promoter
EGFP-SV40, another functional expression frame is to comprise domestic silkworm silk glue protein 1 gene promoter, 18s rRNA promoter, silk
Fibroin light chain gene signal peptide, hexahistine, enterokinase cleavage site DDDDK, T4 ligase gene and domestic silkworm silk
The expression cassette of fibroin light chain gene 3 ' end, i.e. Ser1promoter-18s rRNA promoter-Fibroin L chain signal
peptide-His 6-DDDDK-T4Ligase-Fibroin L chain PolyA。
Described piggy-9887 plasmid is to utilize Ser1promoter and 18s rRNA promoter double-promoter to drive T4 even
Connect the expression of enzyme gene.
Two, the preparation side of a kind of silkworm middle division of silkgland universal plasmid of bioreactor double-promoter for expressing T4 ligase
Method, comprises the following steps:
1) with the T4 ligase-5 with ApaI restriction enzyme site sequence ' terminal sequence and the T4 with NheI restriction enzyme site sequence
Ligase-3 ' terminal sequence is primer, with containing A3 gene promoter-green fluorescence protein gene-SV40-sericin gene promoter-T4
The piggy-10522 plasmid of ligase gene-SV40 ([A3-EGFP-SV40]-[Ser promoter-T4Ligase-SV40]) is
Template, the base sequence of piggy-10522 plasmid such as SEQ ID NO.2, the T4 of a length of 1536bp is obtained by PCR amplification
Ligase gene, its base sequence such as SEQ ID NO.3；
2) with ApaI and NheI double digestion piggy-8363 plasmid, the base sequence of piggy-8363 plasmid such as SEQ ID NO.4,
Obtain a length of 8361bp comprises [A3-EGFP-SV40]-[Ser1promoter-18s rRNA promoter-FLSP-His
6-DDDDK-FLPA] and the gene order of Amp resistant gene；
3) Connection Step 1) the T4 ligase gene that obtains and step 2) gene order that obtains, is contained
[A3-EGFP-SV40]-[Ser1promoter-18s rRNA promoter-FLSP-His 6-DDDDK-T4Ligase-FLPA] and
The PiggyBac-9887 plasmid of Amp resistant gene, its base sequence such as SEQ ID NO.1.
Three, the application that universal plasmid of the present invention is expressed at T4 ligase.
Four, a kind of silkworm middle division of silkgland bioreactor double-promoter universal plasmid expression T4 for expressing T4 ligase is even
The method connecing enzyme, step is as follows:
Use molecular biology method to build and comprise [A3-EGFP-SV40]-[Ser1promoter-18s rRNA
Promoter-FLSP-DDDDK-T4Ligase-FLPA] piggy-9887 plasmid (its base sequence such as SEQ ID NO.1)
As in silkworm middle division of silkgland express T4 linker because of carrier, this plasmid includes work based on piggyBac transposon
For exogenous gene T4 linker because of and as the green fluorescence EGFP gene expression cassette of marker gene；
(1) method using microinjection, by described piggy-9887 plasmid and the auxiliary that can provide piggyBac transposase
Plasmid in the concentration ratio than 1:1 import after silkworm lays eggs 6 little time within germ cell in, utilize in piggy-9887 plasmid
T4 ligase gene is inserted in domestic silkworm gene group by piggyBac transposon；
(2) raising after egg-incubation to adult, then continue generation with the non-transgenic silkworm copulation production of hybrid seeds, this is on behalf of G1 generation,
G1 generation second day one age, by fluorescence stereomicroscope observation filter out body colour express green fluorescence EGFP marker gene turn base
Because of silkworm, raise to adult continuous with the non-transgenic silkworm copulation production of hybrid seeds for becoming G2 generation again；
(3) G2 uses one batch rearing for silkworm, filters out and expresses green fluorescence EGFP marker gene under fluorophor stereomicroscope
Silkworm, uses and makes G3 generation with the mutual copulation of Bombycis mori of moth district；
(4) G3 uses one batch rearing for silkworm, expresses the mutual copulation of Bombycis mori of green fluorescence EGFP marker gene, system with moth district
Become G4 generation；
(5) start from G4 generation and through continuous 3 generations use the single moth district of green fluorescent protein phenotype to raise, one batch rearing and same moth
The same procedure of district's Bombycis mori copulation carries out selecting and copulation, is bred as green fluorescence gene and T4 ligase gene pure, sericterium thin
Born of the same parents can the transgenic bombyx mori of synthesis secretion T4 ligase；
(6) by domestic natural silk gland cell synthesis secretion T4 connect, and with family silkworms spin silk cocoon behavior enter Bombyx bombycis.
Described piggy-9887 plasmid is to utilize Ser1promoter and 18s rRNA promoter double-promoter to drive T4 to connect
The expression of enzyme gene.
The T4 ligase gene of described step (6) is at domestic silkworm silk glandular cell specifically expressing, at silk fibroin protein light chain signal peptide
Effect under be secreted into sericterium lumen of gland, and be secreted into Bombyx bombycis with the behavior of weaving silk.
The present invention is first to build the piggy-9887 carrier (as shown in Figure 1) of silkworm synthesis secretion T4 ligase gene, then profit
By microinjection transgenic bombyx mori technology by this plasmid and the helper plasmid (as shown in Figure 2) that piggyBac transposase can be provided
Import in silkworm germ cell together, rely on the transposition features of piggyBac transposon, make green fluorescence protein gene and T4 even
Connect enzyme channel genes in silkworm genome, and obtain stable heredity and expression, thus formulate into one can be thin at domestic natural silk gland
The transgenic bombyx mori of born of the same parents' specificity synthesis secretion T4 ligase, selfing makes T4 ligase gene pure, and incubation can secrete T4 even
Connect the transgenic bombyx mori of enzyme, then utilize this kind of silkworm synthesis secretion T4 ligase.
The invention have the advantages that:
The present invention is by fluorized marking genescreen transgenic bombyx mori, and this transgenic bombyx mori can at domestic silkworm silk glandular cell specifically
Synthesis secretion T4 ligase, T4 ligase has functional activity.The present invention develops a kind of novel T4 ligase and produces work
Skill, lays a good foundation for a large amount of T4 ligases that produce.
Accompanying drawing explanation
Fig. 1 is the piggy-9887 plasmid construct figure of the present invention.
Fig. 2 is the helper plasmid structure chart that can provide piggyBac transposase.
Detailed description of the invention
The invention will be further described with embodiment below in conjunction with the accompanying drawings.
Embodiments of the invention are as follows:
A) plasmid of the present invention is prepared:
Connect with the T4 ligase 5 ' terminal sequence with ApaI restriction enzyme site sequence and the T4 with NheI restriction enzyme site sequence
Enzyme 3 ' terminal sequence is primer, to connect containing A3 gene promoter-green fluorescence protein gene-SV40-sericin gene promoter-T4
Enzyme gene-SV40 ([A3-EGFP-SV40]-[Ser promoter-T4Ligase-SV40]) piggy-10522 plasmid (its base
Sequence such as SEQ ID NO.2) it is template, it is thus achieved that T4 ligase gene (its base sequence such as SEQ ID of a length of 1536bp
NO.3), and this sequence 5 ' end is containing ApaI restriction enzyme site, and 3 ' ends are containing NheI restriction enzyme site.
With ApaI and NheI double digestion silkworm middle division of silkgland bioreactor double-promoter universal plasmid piggy-8363 (its alkali
Basic sequence such as SEQ ID NO.4), contained [A3-EGFP-SV40]-[Ser1promoter-18s rRNA
Promoter-FLSP-His 6-DDDDK-FLPA] and the sequence of 8361bp of Amp resistant gene.
Connect two purpose fragments of above-mentioned acquisition, contained [A3-EGFP-SV40]-[Ser1promoter-18s rRNA pro
Moter-FLSP-His 6-DDDDK-T4Ligase-FLPA] and piggyBac-9887 plasmid (its alkali of Amp resistant gene
Basic sequence such as SEQ ID NO.1).
B) T4 ligase is expressed:
By the piggy-9887 plasmid (Fig. 1) of above-mentioned structure and helper plasmid (Fig. 2) that piggyBac transposase can be provided
Mixing by 1:1 ratio, the total concentration of 2 kinds of plasmids is 0.4 μ g/ μ l, and plasmid is dissolved in the phosphate buffer of pH=7,0.5mM
In, then use micro-injection method to import in the germ cell within silkworm lays eggs latter 6 hours, importing cumulative volume is 10 μ l.Will
The silkworm seed of microinjection 25 DEG C, raise to adult under 85% damp condition, pass on non-transgenic silkworm hybrid, be for G1
Generation.In G1 second day one age of generation of transgenic experiments, observe obtain by fluorescence microscope (Olympus, SZX12, Japan)
Take 5, the transgenic bombyx mori moth district expressing EGFP marker gene.Silkworm raising is passed on to adult with non-transgenic silkworm hybrid,
It is for G2.One batch rearing is all used for later transgenic bombyx mori from G2, in an age by fluorescence stereomicroscope observation,
Select express EGFP marker gene transgenic bombyx mori, raise to adult, with the copulation of moth district, so cultivate obtain G3 generation,
G4 generation, T4 ligase gene pure..
G4 for time, take each 1 article of the silkworm in the 3rd day 5 ages in 3 Ge E districts at random, extract posterior silkgland cells genomic DNA
For template, use Inverse PCR to expand piggy-9887 plasmid Insert Fragment in silkworm genome, amplified fragments is entered
Row clone, order-checking and chromosome mapping analysis, result shows, T4 ligase gene is inserted respectively into No. 14 chromosome
At 9822566, at No. 10 chromosome 15294275 and at No. 6 chromosome 5434916, insertion is all genetic interval
District.Result proves that transposon has been inserted in the genome of each transgenic bombyx mori family.
Table 1 transgenic positive silkworm exogenous gene insertion point analysis result
The moth district starting to select EGFP gene type to isozygoty from G5 generation raises, and uses with the Bombycis mori copulation of moth district, is bred as EGFP gene
Isozygoty, posterior silkgland cells can the transgenic bombyx mori new varieties of synthesis secretion T4 ligase.
The Cocoon fibroin of 3 familys extracting said determination insertion point is material, uses SDS-PAGE electrophoresis and His6 to resist
The Western blot technical Analysis of body demonstrates transgenic bombyx mori can express T4 ligase, and can be with family's silkworms spin silk behavior
Enter Bombyx bombycis.
Relatively this is with the transgenic bombyx mori T4 ligase expression of double-promoter and only 1 sericin 1 gene promoter
Transgenic bombyx mori T4 ligase expression, the expression of double-promoter significantly improves 1.2 times.
In summary it can be seen, utilize the inventive method that the silkworm middle division of silkgland universal plasmid of bioreactor double-promoter can be used fast
Speed, easily structure transgenic bombyx mori exogenous gene expression plasmid, can efficiently synthesize T4 ligase at domestic silkworm silk glandular cell,
T4 ligase can be entered lumen of gland, and discharge silkworm body further by sericterium secretion as silkworm silk.This character can stablize table
Reach and heredity.Use this method can produce T4 ligase in a large number, it is possible to increase silkworm and mulberry economic benefit, improve silkworm raiser's income.
Above-mentioned detailed description of the invention be used for illustrate the present invention rather than limit the invention, the present invention spirit and
In scope of the claims, any modifications and changes that the present invention is made, both fall within protection scope of the present invention.
Claims (8)
1., for expressing the silkworm middle division of silkgland universal plasmid of bioreactor double-promoter of T4 ligase, its feature exists
In: described plasmid is piggy-9887 plasmid, is based on piggyBac transposon and with Amp resistant gene, including
Two swivel base arm PBL and PBR of piggyBac transposon, and two between two swivel base arms include as exogenous gene
T4 ligase gene and the functional expression frame of green fluorescence EGFP gene as marker gene.
A kind of silkworm middle division of silkgland bioreactor double-promoter for expressing T4 ligase the most according to claim 1
Universal plasmid, it is characterised in that: a functional expression frame is the Green Fluorescent Protein Gene Expression frame that A3 promoter starts,
I.e. A3 Promoter EGFP-SV40, another functional expression frame is to comprise domestic silkworm silk glue protein 1 gene promoter, 18s
RRNA promoter, fibroin albumen light chain gene signal peptide, hexahistine, enterokinase cleavage site DDDDK, T4 are even
Meet enzyme gene and the expression cassette of silk fibroin protein light chain gene 3 ' end, i.e. Ser1 promoter-18s rRNA
promoter-Fibroin L chain signal peptide-His 6-DDDDK-T4 Ligase-Fibroin L chain PolyA。
A kind of silkworm middle division of silkgland bioreactor double-promoter for expressing T4 ligase the most according to claim 2
Universal plasmid, it is characterised in that: described piggy-9887 plasmid is to utilize Ser1 promoter and 18s rRNA promoter
Double-promoter drives the expression of T4 ligase gene.
4. the arbitrary described a kind of silkworm middle division of silkgland bioreactor double startup for expressing T4 ligase of claims 1 to 3
The preparation method of the universal plasmid of son, it is characterised in that comprise the following steps:
1) with the T4 ligase-5 with ApaI restriction enzyme site sequence ' terminal sequence and the T4 with NheI restriction enzyme site sequence
Ligase-3 ' terminal sequence is primer, with containing A3 gene promoter-green fluorescence protein gene-SV40-sericin gene promoter-T4
The piggy-10522 plasmid of ligase gene-SV40 ([A3-EGFP-SV40]-[Ser promoter-T4 Ligase-SV40]) is
Template, the base sequence of piggy-10522 plasmid such as SEQ ID NO.2, it is thus achieved that the T4 ligase gene of a length of 1536bp,
The base sequence such as SEQ ID NO.3 of T4 ligase gene；
2) with ApaI and NheI double digestion piggy-8363 plasmid, the base sequence of piggy-8363 plasmid such as SEQ ID NO.4,
Obtain a length of 8361bp comprises [A3-EGFP-SV40]-[Ser1 promoter-18s rRNA promoter-FLSP-His
6-DDDDK-FLPA] and the gene order of Amp resistant gene；
3) Connection Step 1) the T4 ligase gene that obtains and step 2) gene order that obtains, is contained
[A3-EGFP-SV40]-[Ser1promoter-18s rRNA promoter-FLSP-His 6-DDDDK-T4 Ligase-FLPA] and
The PiggyBac-9887 plasmid of Amp resistant gene.
5. according to the application of the arbitrary described universal plasmid of claims 1 to 3, it is characterised in that: express for T4 ligase
Application.
6. one kind connects for expressing the silkworm middle division of silkgland bioreactor double-promoter universal plasmid expression T4 of T4 ligase
The method connecing enzyme, it is characterised in that the step of the method is as follows:
(1) method using microinjection, by described piggy-9887 plasmid and the auxiliary that can provide piggyBac transposase
Plasmid in the concentration ratio than 1:1 import after silkworm lays eggs 6 little time within germ cell in, utilize in piggy-9887 plasmid
T4 ligase gene is inserted in domestic silkworm gene group by piggyBac transposon；
(2) raising after egg-incubation to adult, then continue generation with the non-transgenic silkworm copulation production of hybrid seeds, this is on behalf of G1 generation,
G1 generation second day one age, by fluorescence stereomicroscope observation filter out body colour express green fluorescence EGFP marker gene turn base
Because of silkworm, raise to adult continuous with the non-transgenic silkworm copulation production of hybrid seeds for becoming G2 generation again；
(3) G2 uses one batch rearing for silkworm, filters out and expresses green fluorescence EGFP marker gene under fluorophor stereomicroscope
Silkworm, uses and makes G3 generation with the mutual copulation of Bombycis mori of moth district；
(4) G3 uses one batch rearing for silkworm, expresses the mutual copulation of Bombycis mori of green fluorescence EGFP marker gene, system with moth district
Become G4 generation；
(5) start from G4 generation and through continuous 3 generations use the single moth district of green fluorescent protein phenotype to raise, one batch rearing and same moth
The same procedure of district's Bombycis mori copulation carries out selecting and copulation, is bred as green fluorescence gene and T4 ligase gene pure, sericterium thin
Born of the same parents can the transgenic bombyx mori of synthesis secretion T4 ligase；
(6) by domestic natural silk gland cell synthesis secretion T4 connect, and with family silkworms spin silk cocoon behavior enter Bombyx bombycis.
A kind of silkworm middle division of silkgland bioreactor double-promoter for expressing T4 ligase the most according to claim 6
The method of universal plasmid expression T4 ligase, it is characterised in that: described piggy-9887 plasmid is to utilize Ser1 promoter
With the expression that 18s rRNA promoter double-promoter drives T4 ligase gene.
A kind of silkworm middle division of silkgland bioreactor double-promoter for expressing T4 ligase the most according to claim 6
The method of universal plasmid expression T4 ligase, it is characterised in that: the T4 ligase gene of described step (6) is at domestic silkworm silk
Glandular cell specifically expressing, is secreted into sericterium lumen of gland under the effect of silk fibroin protein light chain signal peptide, and with behavior secretion of weaving silk
To Bombyx bombycis.
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| Publication number | Priority date | Publication date | Assignee | Title |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100112651A1 (en) * | 2005-01-14 | 2010-05-06 | Archer-Daniels-Midland Company | Compositions and methods for manipulating carbon flux in cells |
| CN102226202A (en) * | 2011-05-13 | 2011-10-26 | 浙江大学 | Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm |
| CN104278032A (en) * | 2014-02-14 | 2015-01-14 | 上海美百瑞生物医药技术有限公司 | Novel double promoter structural unit |
| CN104593413A (en) * | 2014-12-31 | 2015-05-06 | 浙江大学 | Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland |
| CN104846011A (en) * | 2015-03-18 | 2015-08-19 | 浙江大学 | Method for synthesizing royal jelly main protein 1 by using bombyx mori posterior silkgland |
-
2016
- 2016-05-04 CN CN201610296590.6A patent/CN105907786B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100112651A1 (en) * | 2005-01-14 | 2010-05-06 | Archer-Daniels-Midland Company | Compositions and methods for manipulating carbon flux in cells |
| CN102226202A (en) * | 2011-05-13 | 2011-10-26 | 浙江大学 | Method for synthetizing secretion lysozyme by middle silkgland cell of silkworm |
| CN104278032A (en) * | 2014-02-14 | 2015-01-14 | 上海美百瑞生物医药技术有限公司 | Novel double promoter structural unit |
| CN104593413A (en) * | 2014-12-31 | 2015-05-06 | 浙江大学 | Method for synthesizing secreted human serum albumin employing bombyx mori posterior silk gland |
| CN104846011A (en) * | 2015-03-18 | 2015-08-19 | 浙江大学 | Method for synthesizing royal jelly main protein 1 by using bombyx mori posterior silkgland |
Non-Patent Citations (1)
| Title |
|---|
| 章倩倩等: "CMV与SP双启动子增强外源基因在小鼠骨骼肌中的表达效率", 《中国生物化学与分子生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114480500A (en) * | 2022-03-04 | 2022-05-13 | 西南大学 | Construction method of transgenic sericin cocoon bioreactor |
| CN114480500B (en) * | 2022-03-04 | 2023-09-08 | 西南大学 | Construction method of transgenic sericin cocoon bioreactor |
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