CN106399363B - Double-promoter universal plasmid for middle silk gland bioreactor of silkworm as well as construction method and application thereof - Google Patents
Double-promoter universal plasmid for middle silk gland bioreactor of silkworm as well as construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a dual-promoter universal plasmid for a middle silk gland bioreactor of silkworms, a construction method and application thereof. Obtaining a sericin 1 gene promoter, an 18s rRNA gene promoter, a His6 sequence, DDDDK, a fibroin light chain protein signal peptide, a fibroin light chain gene 3 'terminal sequence, a silkworm actin gene A3promoter, an EGFP gene and a SV40 3' terminal sequence, then sequentially connecting to construct a plasmid, wherein a first expression frame comprises an A3promoter, an EGFP gene and an SV40 sequence, a second expression frame comprises a sericin 1 gene promoter, an 18s rRNA gene promoter, a fibroin light chain protein signal peptide, a His6 sequence, DDDDK and a fibroin light chain gene 3 'terminal sequence, and ApaI and NheI enzyme cutting sites are contained between the DDDDK and the fibroin light chain gene 3' terminal sequence. The plasmid reduces the workload of constructing the donor plasmid, improves the working efficiency of the silkworm silk gland bioreactor, and improves the expression quantity of the foreign protein.
Description
Technical Field
The invention relates to a plasmid and a construction method and application thereof, in particular to a dual-promoter universal plasmid for a middle silk gland bioreactor of silkworms and a construction method and application thereof.
Background
The piggyBac transposon was originally isolated from the genome of Trichoplusia ni TN-368 cell line and is the DNA transposon with the highest transposition activity found so far. The piggyBac transposition system is a non-viral vector and has high transposition efficiency. Compared with sleeping beauty, the piggyBac vector has larger capacity, can carry 18kb, can realize the co-expression of multiple genes, can not leave a blot (football) at an in-situ point after a transposition fragment is excised, can realize the accurate repair of the excised genome, and has an important role in the application of reversible genes.
Researches on transgenic silkworm silk gland bioreactors mediated by piggyBac transposons have been carried out for over a decade, scientists in various countries around the world are dedicated to the expression of exogenous genes, and transgenic silkworm silk gland bioreactors for expressing exogenous proteins by silk fibroin light chain promoters (Fib-L Promoter), silk fibroin heavy chain promoters (Fib-H Promoter) and sericin 1 promoters (Ser1Promoter) have been established.
Since the piggyBac vector mostly contains two expression frames, and has more elements and the same restriction enzyme cutting sites, when expressing different exogenous genes, the construction of plasmids from the head and the assembly of structures such as a promoter, a signal peptide, an exogenous gene, polyA and the like are needed each time, which wastes time and labor and has low working efficiency.
On the other hand, through the research results of domestic and foreign silkworm bioreactors over a decade, the foreign protein expression efficiency of transgenic silkworm silk gland bioreactors is very low, and the expression level of most experiments is not about 1% of the cocoon shell amount, regardless of the foreign gene driven by a sericin promoter or the foreign gene driven by a fibroin promoter, and far from reaching the high-efficiency expression level expected by scientists and like expressing silk protein.
Disclosure of Invention
In order to solve the problems in the background art, the invention aims to provide a dual-promoter universal plasmid for a middle silk gland bioreactor of silkworms and a construction method and application thereof, and particularly relates to a universal plasmid which does not contain a target gene but has all other necessary elements, which is constructed by utilizing a conventional plasmid construction biotechnology method.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
a dual-promoter universal plasmid for a middle silk gland bioreactor of silkworms:
the plasmid is pBac [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA-promoter-FLSP-His6-DDDDK-FLPA ], is based on piggyBac transposon and has Amp resistance genes, and comprises two transposon arms PBL and PBR of the piggyBac transposon and two functional expression frames between the two transposon arms.
One functional expression frame is a green fluorescent protein gene expression frame started by an A3Promoter, namely A3 Promoter-EGFP-SV 40, and the other functional expression frame is an expression frame comprising a bombyx mori sericin 1 gene Promoter, an 18s rRNA Promoter, a Fibroin light chain gene signal peptide, hexa-histidine, an enterokinase enzyme cutting site and a bombyx mori Fibroin light chain gene 3' terminal sequence, namely Ser1Promoter-18s rRNA Promoter-fibrin L chain signal peptide-His 6-DDDDDDK-fibrin L chain protein PolyA.
The DDDDDDK-FLPA contains two specific enzyme cutting sites of ApaI and NheI, and the two enzyme cutting sites are used for specifically cutting and connecting plasmids with exogenous genes to form functional plasmids.
Secondly, a construction method of a double-promoter universal plasmid of a middle silk gland bioreactor of silkworms, which comprises the following steps:
1) constructing a plasmid piggy-14413 containing [ sericin 1 gene promoter 575-renilla luciferase gene-SV 40] - [ sericin 1 gene promoter 4023-firefly luciferase gene-SV 40] - [ A3 gene promoter-EGFP-SV 40] ([ Ser 575-Rluc-SV40] - [ Ser 4023-Fluc-SV40] - [ A3-GFP-SV40]) by a molecular biological method, taking the plasmid piggy-14413 as a template, obtaining the base sequence of the plasmid piggy-14413 as SEQ ID NO.1, taking ggtaccGTTGGCGGTCTTTGGATCG sequence 1 as SEQ ID NO.2 and gaattccttaagGCACACACACTACATACCATGTATTTG sequence 2 as SEQ ID NO.3 as primers, and obtaining a sericin gene promoter with the fragment length of 583bp by PCR amplification, wherein the base sequence is as SEQ ID NO. 4;
2) carrying out double digestion on the plasmid piggy-5257 by using EcoRI and KpnI, wherein the base sequence of the plasmid piggy-5257 is shown as SEQ ID NO.5, and obtaining a fragment which is 4549bp in length, contains 18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase digestion site sequence-bombyx mori fibroin protein light chain 3' sequence (18s rRNA-FLSP-His 6-DDDDK-FLPA) and Amp resistance gene;
connecting the sericin 1 gene promoter obtained in the step 1) with a fragment containing 18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3 'sequence and Amp resistance gene to obtain a plasmid piggy-5140 containing sericin 1 gene promoter-18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3' sequence (Ser 1-18s rRNA-FLSP-His 6-DDDDK-FLPA);
3) carrying out enzyme digestion on the piggy-5140 plasmid obtained in the step 2) by using AflII, BglII and ScaI to obtain a 2493bp fragment containing a sericin 1 gene promoter-18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme digestion site sequence-bombyx mori fibroin protein light chain 3' sequence (Ser1-FLSP-His 6-DDDDK-FLPA);
4) carrying out double digestion on plasmid piggy-6212 containing an A3 gene promoter-green fluorescent protein gene-SV 40(A3-GFP-SV40) and an Amp resistance gene by using AflII and BglII, wherein the base sequence of the plasmid piggy-6212 is shown as SEQ ID NO.6, and obtaining a fragment which is 5870bp in length and contains the A3 gene promoter-green fluorescent protein gene-SV 40(A3-GFP-SV 40);
5) connecting the fragment containing sericin 1 gene promoter-18s rRNA gene promoter-silk fibroin light chain signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3 ' sequence obtained in the step 3) with the fragment containing A3 gene promoter-EGFP-SV 40 obtained in the step 4) to obtain a bombyx mori silk fibroin light chain 3 ' sequence ([ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA gene promoter-silk fibroin light chain signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3 ' sequence (plasmid Piggy-8363) of the bombyx mori silk gland bioreactor dual-promoter, which is used as the plasmid of the invention, the base sequence is shown as SEQ ID NO. 7.
And thirdly, the application of the plasmid in constructing the donor plasmid for expressing various exogenous genes.
The invention firstly designs a primer, and obtains a sequence of a sericin 1 gene promoter by a PCR method; then respectively using restriction enzyme to enzyme-cut corresponding fragments, after electrophoresis to obtain a target band, cutting gel and recycling, using T4ligase to connect the corresponding fragments, and sequentially connecting the required elements (Ser Promoter-18s rRNA Promoter-fibriin L chain signaling-His 6-DDDDDDK-fibriin L chain PolyA); then, the marker gene and the above-mentioned elements are integrated into the same plasmid to obtain a universal plasmid containing the dual promoter, which is finally shown in FIG. 1.
The invention has the beneficial effects that:
the plasmid can introduce any exogenous gene into the plasmid only through one-step enzyme digestion ligation reaction to construct a functional plasmid capable of expressing the exogenous gene, and can start the expression of the exogenous gene by using a double promoter, so as to improve the expression quantity of the exogenous gene.
Drawings
FIG. 1 is a schematic diagram of the composition of a Ser1 and 18s rRNA double-promoter universal plasmid of the invention.
Detailed Description
The invention is further illustrated by the following figures and examples.
The examples of the invention are as follows:
(1) designing a primer, and obtaining a sericin 1 gene promoter (SEQ ID NO.4) by using a piggy-14413 plasmid (SEQ ID NO.1) as a template. And adding EcoRI enzyme cutting site at the 5 'end of the promoter and KpnI enzyme cutting site at the 3' end. Carrying out double enzyme digestion on the PCR product by using EcoR I and KpnI to obtain a 593bp fragment, namely a sericin 1 gene promoter, and then carrying out gel recovery; carrying out double enzyme digestion on the piggy-5257 plasmid (SEQ ID NO.5) by using EcoR I and KpnI to obtain a 4549bp fragment, and recycling gel; the resulting 2 fragments were ligated, and the ligated plasmid was designated as piggy-5140 plasmid.
(2) Carrying out enzyme digestion on the piggy-5140 plasmid by using AflII, BglII and ScaI to obtain a fragment with the length of 2493 bp; carrying out double enzyme digestion on the piggy-6212 plasmid (SEQ ID NO.6) by using AflII and BglII to obtain a fragment with the length of 5870bp, and connecting the fragments of the two orders to obtain a piggy-8363 plasmid (SEQ ID NO.7), namely the double-promoter universal plasmid of the silk gland bioreactor in the middle of silkworms.
(3) The piggy-8363 plasmid is double digested by ApaI and NheI to obtain a gene sequence which is 8361bp in length and contains [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His6-DDDDK-FLPA and Amp resistance genes. And connecting the T4Ligase gene with an ApaI enzyme digestion cohesive end sequence at the upstream end and an NheI enzyme digestion cohesive end sequence at the downstream end with the fragments to construct a plasmid piggy-9887 for expressing the T4Ligase gene by a silkworm middle silk gland bioreactor, wherein the expression frame sequence contained in the plasmid is [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His 6-DDDDDDK-T4 Ligase-FLPA ].
The results show that the plasmid for expressing the T4ligase gene in the middle silk gland of the silkworm can be obtained only by one step by using the dual-promoter universal plasmid piggyy-8363 of the middle silk gland bioreactor of the silkworm.
The expression quantity of the T4ligase gene of the transgenic silkworm obtained by introducing the double-promoter plasmid into the silkworm genome by adopting a transgenic silkworm technology is 1.2 times higher than that of the T4ligase gene started by a single sericin 1promoter, and the difference of results reaches a significant level.
The method for constructing the donor plasmid of the transgenic silkworm by using the dual-promoter universal plasmid of the middle silk gland bioreactor of the silkworm is simple in operation procedure, high in working efficiency and capable of obviously improving the expression quantity of the exogenous gene.
Claims (3)
1. A dual-promoter universal plasmid for a middle silk gland bioreactor of silkworms is characterized in that:
the plasmid is pBac [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA-promoter-FLSP-His6-DDDDK-FLPA ], is based on piggyBac transposon and is provided with an Amp resistance gene, and comprises two transposon arms PBL and PBR of the piggyBac transposon and two functional expression frames between the two transposon arms;
one functional expression frame is a green fluorescent protein gene expression frame started by an A3Promoter, namely A3 Promoter-EGFP-SV 40, and the other functional expression frame is an expression frame comprising a bombyx mori sericin 1 gene Promoter, an 18s rRNA Promoter, a Fibroin light chain gene signal peptide, hexahistidine, an enterokinase enzyme cutting site and a bombyx mori Fibroin light chain gene 3' terminal sequence, namely Ser1Promoter-18s rRNA Promoter-fibrin L chain signal peptide-His 6-DDDDDDK-fibrin L chain polypeptide PolyA;
the DDDDDDK-FLPA contains two specific enzyme cutting sites of ApaI and NheI, and the two enzyme cutting sites are used for specifically cutting and connecting plasmids with exogenous genes to form functional plasmids.
2. A construction method of a double-promoter universal plasmid of a middle silk gland bioreactor of silkworms is characterized by comprising the following steps:
1) constructing a plasmid piggy-14413 containing [ sericin 1 gene promoter 575-renilla luciferase gene-SV 40] - [ sericin 1 gene promoter 4023-firefly luciferase gene-SV 40] - [ A3 gene promoter-EGFP-SV 40] by a molecular biological method, taking the plasmid piggy-14413 as a template, and taking ggtaccGTTGGCGGTCTTTGGATCG sequence 1 as SEQ ID NO.2 and gaattccttaagGCACACACACTACATACCATGTATTTG sequence 2 as SEQ ID NO.3 as primers to obtain a sericin gene promoter with the fragment length of 583bp, wherein the base sequence of the sericin gene promoter is shown as SEQ ID NO. 4;
2) carrying out double digestion on the plasmid piggy-5257 by using EcoRI and KpnI, wherein the base sequence of the plasmid piggy-5257 is shown as SEQ ID NO.5, and obtaining a fragment which is 4549bp in length, contains 18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase digestion site sequence-bombyx mori fibroin protein light chain 3' sequence and Amp resistance gene;
connecting the sericin 1 gene promoter obtained in the step 1) with a fragment containing 18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3 'sequence and Amp resistance gene to obtain a plasmid piggy-5140 containing sericin 1 gene promoter-18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3' sequence;
3) carrying out enzyme digestion on the piggy-5140 plasmid obtained in the step 2) by using AflII, BglII and ScaI to obtain a 2493bp fragment containing a sericin 1 gene promoter-18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme digestion site sequence-bombyx mori fibroin protein light chain 3' sequence;
4) carrying out double digestion on a plasmid piggy-6212 containing an A3 gene promoter-green fluorescent protein gene-SV 40(A3-GFP-SV40) and an Amp resistance gene by using AflII and BglII, wherein the base sequence of the plasmid piggy-6212 is shown as SEQ ID NO.6, and obtaining a 5870bp fragment containing the A3 gene promoter-green fluorescent protein gene-SV 40;
5) connecting the fragment containing sericin 1 gene promoter-18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3 'sequence obtained in the step 3) with the fragment containing A3 gene promoter-EGFP-SV 40 obtained in the step 4) to obtain the middle bombyx mori silk gland bioreactor dual-promoter universal plasmid piggyy-8363 plasmid containing A3 gene promoter-EGFP-SV 40-sericin 1 gene promoter-18s rRNA gene promoter-fibroin light chain protein signal peptide-hexahistidine-enterokinase enzyme cutting site sequence-bombyx mori silk fibroin light chain 3' sequence.
3. The application of double-promoter universal plasmid in middle silk gland bioreactor of silkworm is characterized by comprising the following steps: the plasmid of claim 1, which is used to construct a donor plasmid expressing various foreign genes.
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| 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 |
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| 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 |
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