CN106399363A - Bombyx mori middle silkgland bioreactor dual-promoter universal plasmid as well as construction method and application thereof - Google Patents
Bombyx mori middle silkgland bioreactor dual-promoter universal plasmid as well as construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a bombyx mori middle silkgland bioreactor dual-promoter universal plasmid as well as a construction method and an application thereof. The construction method comprises the following steps: acquiring a sericin 1 gene promoter, a 18s rRNA gene promoter, a His 6 sequence, DDDDK, a fibroin light-chain protein signal peptide, a fibroin light-chain gene 3' end sequence, a bombyx mori actin gene A3 promoter, an EGFP gene and an SV40 3' end sequence, and then conducting sequential linking so as to construct the plasmid, wherein a first expression cassette incoludes the A3 promoter, the EGFP gene and the SV40 sequence; a second expression cassette includes the sericin 1 gene promoter, the 18s rRNA gene promoter, the fibroin light-chain protein signal peptide, the His 6 sequence, the DDDDK and the fibroin light-chain gene 3' end sequence; and two restriction enzyme sites, namely ApaI and NheI, exist between the DDDDK and the fibroin light-chain gene 3' end sequence. With the application of the plasmid provided by the invention, the workload of constructing the donor plasmid is reduced, the working efficiency of a bombyx mori silkgland bioreactor is improved and the expression amount of foreign protein is improved.
Description
Technical field
The present invention relates to a kind of plasmid and its construction method and application, especially relate in the middle part of a kind of silkworm
The universal plasmid of sericterium bioreactor double-promoter and its construction method and application.
Background technology
PiggyBac transposon is initially the base from cabbage looper (Trichoplusia ni) TN-368 cell line
Because separately obtain in group, it is the transposition activity highest DNA transposons having now been found that.PiggyBac turns
Base system is a kind of non-virus carrier, and transposition efficiency is higher.Compared with sleeping beauty, piggyBac
Bearer capabilities are larger, can carry 18kb, it is possible to achieve polygenic coexpression, and swivel base fragment be removed after not
Trace (footprint) can be left by point in the original location, genome is accurately repaired, in reversible base after can realizing excision
In the application of cause, there is important function.
The more than ten years have been carried out in the research of the transgenic bombyx mori sericterium bioreactor of piggyBac transposon mediation,
The scientist of countries in the world is devoted to the expression of foreign gene, has built up with fibroin albumen light chain promoter
(Fib-L Promoter), fibroin albumen heavy chain promoter (Fib-H Promoter) and sericin 1 start
Sub (Ser1Promoter) expresses the transgenic bombyx mori sericterium bioreactor of foreign protein.
Contain two expression cassettes because piggyBac carrier is most, there is more element and identical digestion position
Point, so, when expressing different foreign genes every time, need from the beginning to build plasmid, assembling promoter, letter
Number structure such as peptide, foreign gene, polyA, wastes time and energy, operating efficiency is low.
On the other hand, but make a general survey of the result of study of silkworm biological reactor both at home and abroad for over ten years, transgenosis man
The exogenous protein expression efficiency of silk gland bioreactor is all very low, no matter being the expression of silk gum promoters driven
Foreign gene, or the foreign gene of fibroin promoters driven expression, the expression of thumping majority experiment does not all have
Have and reaching the 1% about of cocoon shell weight, be far from reaching the desired fibroin as expression of scientists
High efficient expression level.
Content of the invention
In order to solve problem present in background technology, it is an object of the invention to proposing in the middle part of a kind of silkworm
The universal plasmid of sericterium bioreactor double-promoter and its construction method and application, specifically utilize conventional structure
The biological technique method building plasmid builds one kind without genes of interest, but has the logical of other all required elements
Use type plasmid.
In order to achieve the above object, the step of the technical solution used in the present invention is as follows：
First, the universal plasmid of a kind of silkworm middle division of silkgland bioreactor double-promoter：
Described plasmid is pBac [A3-EGFP-SV40]-[Ser1promoter-18s rRNA
- promoter-FLSP-His 6-DDDDK-FLPA], it is and to carry Amp based on piggyBac transposon
Resistant gene, including two swivel base arm PBL and PBR of piggyBac transposon, and two swivel base arms it
Between two functional expression frames.
One function expression cassette is the Green Fluorescent Protein Gene Expression frame that A3 promoter starts, i.e. A3
Promoter EGFP-SV40, another functional expression frame be comprise silkworm sericin 1 gene promoter,
18s rRNA promoter, fibroin albumen light chain gene signal peptide, hexahistine, enterokinase cleavage site and
The expression cassette of silk fibroin protein light chain gene 3 ' end sequence, i.e. Ser1Promoter-18s rRNA
promoter-Fibroin L chain signal peptide-His6-DDDDK-Fibroin L chain PolyA.
Two restriction enzyme sites containing narrow spectrum ApaI and NheI between described DDDDK-FLPA, two
Individual restriction enzyme site is used for cutting to connect by plasmid specificity forming functional plasmid after foreign gene.
2nd, the construction method of the universal plasmid of a kind of silkworm middle division of silkgland bioreactor double-promoter, the party
The step of method is as follows：
1) built by molecular biology method and comprise [sericin 1 gene promoter 575- Renilla luciferase
Gene-SV40]-[sericin 1 gene promoter 4023- Fluc gene-SV40]-[A3 gene
Promoter-EGFP-SV40] ([Ser 575-Rluc-SV40]-[Ser 4023-Fluc-SV40]-[A3-GFP-SV40])
Plasmid piggy-14413, with plasmid piggy-14413 as template, the base sequence of plasmid piggy-14413
As SEQ ID NO.1, with the such as ggtaccGTTGGCGGTCTTTGGATCG sequence of SEQ ID NO.2
1 and as SEQ ID NO.3 gaattccttaagGCACACACACTACATACCATGTATTTG sequence
Row 2 are primer, expand the silk gum gene promoter obtaining a length of 583bp of fragment, its base sequence using PCR
Row such as SEQ ID NO.4；
2) EcoR I and Kpn I double digestion plasmid piggy-5257, the base sequence of plasmid piggy-5257 are used
Row such as SEQ ID NO.5, obtaining length is 4549bp, comprises 18s rRNA gene promoter-fibroin light chain
Protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence (18s
RRNA-FLSP-His 6-DDDDK-FLPA) and Amp resistant gene fragment；
Connection Step 1) sericin 1 gene promoter that obtains with comprise 18s rRNA gene promoter-
Fibroin light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
Row and the fragment of Amp resistant gene, obtain comprising sericin 1 gene promoter -18s rRNA gene opening
Mover-fibroin light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain
The plasmid piggy-5140 of 3 ' sequences (Ser 1-18s rRNA-FLSP-His 6-DDDDK-FLPA)；
3) with AflII and BglII, ScaI digestion step 2) the piggy-5140 plasmid that obtains, obtain length
The sericin 1 gene promoter -18s rRNA gene promoter-fibroin light chain protein that comprises for 2493bp is believed
Number peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
The fragment of (Ser1-FLSP-His 6-DDDDK-FLPA)；
4) comprise A3 gene promoter-green fluorescence protein gene-SV40 with AflII and BglII double digestion
(A3-GFP-SV40) and Amp resistant gene plasmid piggy-6212, the base of plasmid piggy-6212
Sequence such as SEQ ID NO.6, obtain length be 5870bp comprise A3 gene promoter-green fluorescence egg
The fragment of white gene-SV40 (A3-GFP-SV40)；
5) Connection Step 3) obtain comprise sericin 1 gene promoter -18s rRNA gene promoter -
Fibroin light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
Fragment and the step 4 of row) fragment comprising A3 gene promoter-EGFP-SV40 that obtains, comprised
A3 gene promoter-EGFP-SV40- silk gum gene promoter -18s rRNA gene promoter-fibroin light chain egg
White signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
([A3-EGFP-SV40]-[Ser1promoter-18s rRNA-promoter-FLSP-His
6-DDDDK-FLPA]) the universal plasmid piggy-8363 of silkworm middle division of silkgland bioreactor double-promoter
Plasmid as plasmid of the present invention, its base sequence such as SEQ ID NO.7.
3rd, described plasmid is used for the application of the donor plasmid of the various foreign gene of construction expression.
The present invention first designs primer, obtains the sequence of sericin 1 gene promoter with the method for PCR；
Again respectively with the corresponding fragment of digestion with restriction enzyme, after electrophoresis obtains purpose band, cut glue reclaim, use
Corresponding fragment is connected by T4 ligase, makes required element (Ser Promoter-18s rRNA
Promoter-Fibroin L chain signal peptide-His6-DDDDK-Fibroin L chain PolyA) according to
Secondary connection；Then marker gene and said elements are incorporated into same plasmid, obtain containing double-promoter
Universal plasmid, finally as shown in Figure 1.
The invention has the beneficial effects as follows：
Plasmid of the present invention can be for any one foreign gene, as soon as only will need to appoint through step digestion coupled reaction
A kind of what foreign gene imports plasmid, is built into the plasmid that functional is capable of expression alien gene, and permissible
Start exogenous gene expression using double-promoter it is intended to improve the expression of foreign gene.
Brief description
Fig. 1 is the composition schematic diagram of the universal plasmid of Ser1 and 18s rRNA double-promoter of the present invention.
Specific embodiment
The invention will be further described with reference to the accompanying drawings and examples.
Embodiments of the invention are as follows：
(1) design primer, with piggy-14413 plasmid (SEQ ID NO.1) as template, obtain sericin
1 gene promoter (SEQ ID NO.4).And the restriction enzyme site at promoter 5 ' end plus EcoR I, 3 '
End plus the restriction enzyme site of KpnI.With EcoR I and KpnI double digestion PCR primer, obtain the piece of 593bp
Section, i.e. sericin 1 gene promoter, then glue reclaim；With EcoR I and KpnI double digestion piggy-5257
Plasmid (SEQ ID NO.5), obtains the fragment of 4549bp, glue reclaim；By 2 obtained fragments even
Connect, the plasmid after connection is named as piggy-5140 plasmid.
(2) use AflII and BglII, ScaI digestion piggy-5140 plasmid, obtain the fragment that length is 2493bp；
With AflII and BglII double digestion piggy-6212 plasmid (SEQ ID NO.6), obtaining length is 5870bp
Fragment, connect above-mentioned two purpose fragments, obtain piggy-8363 plasmid (SEQ ID NO.7), i.e. silkworm
The universal plasmid of middle division of silkgland bioreactor double-promoter.
(3) use ApaI and NheI double digestion piggy-8363 plasmid, obtaining length is comprising of 8361bp
[A3-EGFP-SV40]-[Ser1promoter-18s rRNA promoter-FLSP-His6-DDDDK-FLPA
Gene order with Amp resistant gene.Upstream end is carried ApaI digestion cohesive end sequence, downstream
T4 with NheI digestion cohesive end sequence is connected enzyme gene and is connected with above-mentioned fragment, is built in silkworm
Portion's sericterium bioreactor expresses the plasmid piggy-9887 that T4 connects enzyme gene, the expression cassette that this plasmid comprises
Sequence is [A3-EGFP-SV40]-[Ser1promoter-18s rRNA promoter-FLSP-His
6-DDDDK-T4Ligase-FLPA].
The above results illustrate, using the universal plasmid of silkworm middle division of silkgland bioreactor double-promoter
Piggy-8363, only the step silkworm middle division of silkgland that just can obtain being in express the plasmid that T4 connects enzyme gene.
This double-promoter plasmid is imported by domestic silkworm gene group, the transgenosis man of acquisition using transgenic bombyx mori technology
The T4 ligase gene expression amount of silkworm, connects enzyme gene than the T4 being started with single sericin 1 promoter
Expression is high 1.2 times, and result difference reaches the level of signifiance.
Illustrate to turn base using the universal plasmid construction of silkworm middle division of silkgland bioreactor double-promoter of the present invention
Because of the donor plasmid of silkworm, operation sequence is simple, high working efficiency, and the expression of foreign gene also has
Significantly improve.
Claims (5)
1. a kind of universal plasmid of silkworm middle division of silkgland bioreactor double-promoter it is characterised in that：
Described plasmid is pBac [A3-EGFP-SV40]-[Ser1 promoter-18s rRNA
- promoter-FLSP-His 6-DDDDK-FLPA], it is and to carry Amp based on piggyBac transposon
Resistant gene, including two swivel base arm PBL and PBR of piggyBac transposon, and two swivel base arms it
Between two functional expression frames.
2. the universal matter of a kind of silkworm middle division of silkgland bioreactor double-promoter according to claim 1
Grain it is characterised in that：
One function expression cassette is the Green Fluorescent Protein Gene Expression frame that A3 promoter starts, i.e. A3
Promoter EGFP-SV40, another functional expression frame be comprise silkworm sericin 1 gene promoter,
18s rRNA promoter, fibroin albumen light chain gene signal peptide, hexahistine, enterokinase cleavage site and
The expression cassette of silk fibroin protein light chain gene 3 ' end sequence, i.e. Ser1 Promoter-18s rRNA
promoter-Fibroin L chain signal peptide-His 6-DDDDK-Fibroin L chain PolyA.
3. a kind of silkworm middle division of silkgland bioreactor double-promoter according to claim 1 and 2 is general
Type plasmid it is characterised in that：
Two restriction enzyme sites containing narrow spectrum ApaI and NheI between described DDDDK-FLPA, two
Individual restriction enzyme site is used for cutting to connect by plasmid specificity forming functional plasmid after foreign gene.
4. the construction method of the universal plasmid of a kind of silkworm middle division of silkgland bioreactor double-promoter, its feature
The step being the method is as follows：
1) built by molecular biology method and comprise [sericin 1 gene promoter 575- Renilla luciferase
Gene-SV40]-[sericin 1 gene promoter 4023- Fluc gene-SV40]-[A3 gene
Promoter-EGFP-SV40] plasmid piggy-14413, with plasmid piggy-14413 as template, with such as SEQ
The ggtaccGTTGGCGGTCTTTGGATCG sequence 1 of ID NO.2 and as SEQ ID NO.3
GaattccttaagGCACACACACTACATACCATGTATTTG sequence 2 is primer, obtains fragment
The silk gum gene promoter of a length of 583bp, its base sequence such as SEQ ID NO.4；
2) EcoR I and Kpn I double digestion plasmid piggy-5257, the base sequence of plasmid piggy-5257 are used
Row such as SEQ ID NO.5, obtaining length is 4549bp, comprises 18s rRNA gene promoter-fibroin light chain
Protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence and Amp
The fragment of resistant gene；
Connection Step 1) sericin 1 gene promoter that obtains with comprise 18s rRNA gene promoter-
Fibroin light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
Row and the fragment of Amp resistant gene, obtain comprising sericin 1 gene promoter -18s rRNA gene opening
Mover-fibroin light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain
The plasmid piggy-5140 of 3 ' sequences；
3) with AflII and BglII, ScaI digestion step 2) the piggy-5140 plasmid that obtains, obtain length
The sericin 1 gene promoter -18s rRNA gene promoter-fibroin light chain protein that comprises for 2493bp is believed
The fragment of number peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence；
4) comprise A3 gene promoter-green fluorescence protein gene-SV40 with AflII and BglII double digestion
(A3-GFP-SV40) and Amp resistant gene plasmid piggy-6212, the base of plasmid piggy-6212
Sequence such as SEQ ID NO.6, obtain length be 5870bp comprise A3 gene promoter-green fluorescent protein
The fragment of gene-SV40；
5) Connection Step 3) obtain comprise sericin 1 gene promoter -18s rRNA gene promoter -
Fibroin light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
Fragment and the step 4 of row) fragment comprising A3 gene promoter-EGFP-SV40 that obtains, comprised
A3 gene promoter-EGFP-SV40- sericin 1 gene promoter -18s rRNA gene promoter-fibroin
Light chain protein signal peptide-hexahistine-enterokinase cleavage site sequence-silk fibroin protein light chain 3 ' sequence
Silkworm middle division of silkgland bioreactor double-promoter universal plasmid piggy-8363 plasmid.
5. a kind of universal plasmid of silkworm middle division of silkgland bioreactor double-promoter application it is characterised in that：
Plasmid described in claims 1 to 3 is used for the donor plasmid of the various foreign gene of construction expression.
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Cited By (1)
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| CN114480500A (en) * | 2022-03-04 | 2022-05-13 | 西南大学 | Construction method of transgenic sericin cocoon bioreactor |
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| 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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