CN105907786B - Double-promoter universal plasmid for expressing T4ligase of bombyx mori middle silk gland bioreactor and application and method thereof - Google Patents
Double-promoter universal plasmid for expressing T4ligase of bombyx mori middle silk gland bioreactor and application and method thereof Download PDFInfo
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Abstract
The invention discloses a dual-promoter universal plasmid of a bombyx mori middle silk gland bioreactor for expressing T4ligase and application and a method thereof. The plasmid is based on piggyBac transposon, is provided with Amp resistance gene, comprises a T4ligase gene as an exogenous gene and a functional expression frame of a green fluorescent EGFP gene as a marker gene, is constructed by a molecular biology method, contains two enzyme cutting sites with ApaI and NheI specificity between DDDDK and a fibroin light chain gene polyA, adopts ApaI and NheI double enzyme cutting universal plasmid, is connected with a T4ligase gene, is injected into a silkworm fertilized egg together with an auxiliary plasmid, leads the green fluorescent protein gene and the T4ligase gene into a silkworm genome by utilizing transposon characteristics, and is stably inherited and expressed to obtain the transgenic silkworm. The invention screens transgenic silkworms by means of fluorescent marker genes, and utilizes the silkgland cells of the silkworms to specifically synthesize and secrete T4ligase protein.
Description
Technical Field
The invention relates to a plasmid and application and a method thereof, in particular to a dual-promoter universal plasmid of a bombyx mori middle silk gland bioreactor for expressing T4ligase and application and a method 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 foreign genes, and transgenic silkworm silk gland bioreactors for expressing foreign proteins by silk fibroin light chain promoters (Fib-LPromoters), silk fibroin heavy chain promoters (Fib-H promoters) and sericin 1 promoters (Ser1 promoters) have been established. However, through the research results of domestic and foreign silkworm bioreactors over a decade, the foreign protein expression efficiency of the transgenic silkworm silk gland bioreactor is very low, and the expression amount of most experiments is not about 1% of the cocoon shell amount, regardless of the foreign gene driven by the sericin promoter or the foreign gene driven by the 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 of a silkworm middle silk gland bioreactor for expressing T4ligase, and application and a method thereof, wherein a T4ligase gene is introduced into a silkworm genome by using a transgenic silkworm technology and is specifically expressed in silkworm silk gland cells, so that silkworms capable of synthesizing and secreting single T4ligase are developed.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
a dual-promoter universal plasmid of a domestic silkworm middle silk gland bioreactor for expressing T4 ligase:
the plasmid is a piggy-9887 plasmid, the base sequence of the plasmid is shown as SEQ ID No.1, the plasmid is based on a piggyBac transposon and is provided with an Amp resistance gene, two transposon arms PBL and PBR of the piggyBac transposon are included, and two functional expression frames which comprise a T4ligase gene serving as an exogenous gene and a green fluorescent EGFP gene serving as a marker gene are arranged 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 silk Fibroin light chain gene signal peptide, hexahistidine, an enterokinase enzyme digestion site DDDDDDK, a T4Ligase gene and the 3' tail end of a bombyx mori silk Fibroin light chain gene, namely Ser1Promoter-18s rRNA Promoter-fibrin signal peptide-His 6-DDDDDDDDK-T4 ligand-fibrin L chain PolyA.
The piggy-9887 plasmid utilizes a Ser1promoter and an 18s rRNA promoter to drive the expression of a T4ligase gene.
Secondly, a preparation method of a double-promoter universal plasmid of a domestic silkworm middle silk gland bioreactor for expressing T4ligase, which comprises the following steps:
1) taking a T4 Ligase-5 'end sequence with an ApaI enzyme cutting site sequence and a T4 Ligase-3' end sequence with an NheI enzyme cutting site sequence as primers, taking a piggy-10522 plasmid containing an A3 gene promoter-green fluorescent protein gene-SV 40-sericin gene promoter-T4Ligase gene-SV 40([ A3-EGFP-SV40] - [ Ser promoter-T4Ligase-SV40]) as a template, obtaining a T4Ligase gene with the length of 1536bp by PCR amplification, wherein the base sequence is SEQ ID No. 3;
2) carrying out double digestion on the piggy-8363 plasmid by ApaI and NheI, wherein the base sequence of the piggy-8363 plasmid is shown as SEQ ID NO.4, and obtaining a gene sequence which is 8361bp in length and contains [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His6-DDDDK-FLPA ] and an Amp resistance gene;
3) connecting the T4Ligase gene obtained in the step 1) with the gene sequence obtained in the step 2) to obtain a PiggyBac-9887 plasmid containing [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His 6-DDDDK-T4Ligase-FLPA ] and Amp resistance genes, wherein the base sequence of the PiggyBac-9887 plasmid is shown as SEQ ID NO. 1.
And thirdly, the universal plasmid is applied to the expression of T4 ligase.
The method for expressing the T4ligase by the double-promoter universal plasmid of the silkworm middle silk gland bioreactor for expressing the T4ligase comprises the following steps:
constructing a piggy-9887 plasmid (the base sequence of which is shown as SEQ ID NO.1) containing [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-DDDDK-T4Ligase-FLPA ] as a vector for expressing a T4 connecting gene in the middle silk gland of the silkworm by adopting a molecular biological method, wherein the plasmid contains a T4 connecting gene as an exogenous gene and a green fluorescent EGFP gene expression frame as a marker gene on the basis of piggyBac transposon;
(1) and (2) carrying out microinjection on the piggyy-9887 plasmid and an auxiliary plasmid capable of providing piggyBac transposase according to the concentration ratio of 1:1, introducing the gene into fertilized eggs of the silkworms within 6 hours after the silkworms lay eggs, and inserting the T4ligase gene into the silkworm genome by using piggyBac transposons in piggy-9887 plasmids;
(2) breeding the silkworm eggs after hatching to adult, then mating with the non-transgenic silkworm to produce seeds for successive generations, wherein the generation is G1 generation, observing and screening the transgenic silkworm with the body color expressing the green fluorescent EGFP marker gene by a fluorescence stereomicroscope on the second day of the first age of G1 generation, breeding until the adult then mating with the non-transgenic silkworm to produce seeds for successive generations to form G2 generation;
(3) breeding G2 generation silkworms with single moth, screening silkworms expressing green fluorescent EGFP marker genes under a fluoroscope, and mutually mating silkworms in the same moth area to prepare G3 generation silkworms;
(4) breeding G3 generation silkworms with single moth, and mutually mating the silkworms expressing green fluorescent EGFP marker genes in the same moth region to prepare G4 generation silkworms;
(5) starting from the G4 generation, adopting the moth region feeding with pure green fluorescent protein phenotype, single moth breeding and the same method of mating with the moth region silkworm moth for continuous 3 generations to select and mate, breeding green fluorescent gene and T4ligase gene homozygous, and the silk gland cell can synthesize transgenic silkworm secreting T4 ligase;
(6) the silkworm silk gland cell synthesized and secreted T4 is connected and enters silkworm cocoons along with the silkworm spinning and cocooning behaviors.
The piggy-9887 plasmid utilizes a Ser1promoter and an 18s rRNA promoter to drive the expression of a T4ligase gene.
The T4ligase gene in the step (6) is specifically expressed in bombyx mori silk gland cells, secreted to a silk gland cavity under the action of bombyx mori silk fibroin light chain signal peptide and secreted to a silkworm cocoon along with a spinning behavior.
The invention firstly constructs a piggyy-9887 vector (shown in figure 1) of silkworm synthetic secretion T4ligase gene, then introduces the plasmid and an auxiliary plasmid (shown in figure 2) capable of providing piggyBac transposase into a silkworm fertilized egg by utilizing microinjection transgenic silkworm technology, leads a green fluorescent protein gene and a T4ligase gene into a silkworm genome by depending on the transposition characteristic of the piggyBac transposon, obtains stable heredity and expression, thereby creating a transgenic silkworm capable of specifically synthesizing and secreting T4ligase in silkworm silk gland cells, selfing the T4ligase gene to breed the transgenic silkworm capable of secreting T4ligase, and then synthesizing and secreting T4ligase by utilizing the silkworm.
The invention has the beneficial effects that:
the invention screens the transgenic silkworms by means of fluorescent marker genes, the transgenic silkworms can specifically synthesize and secrete T4ligase in the silk gland cells of the silkworms, and the T4ligase has functional activity. The invention develops a novel T4ligase production process, and lays a foundation for mass production of T4 ligase.
Drawings
FIG. 1 is a structural diagram of the piggy-9887 plasmid of the present invention.
Fig. 2 is a diagram of a helper plasmid structure capable of providing piggyBac transposase.
Detailed Description
The invention is further illustrated by the following figures and examples.
The examples of the invention are as follows:
A) preparation of plasmids of the invention:
a T4Ligase 5 'end sequence with an ApaI enzyme cutting site sequence and a T4Ligase 3' end sequence with an NheI enzyme cutting site sequence are used as primers, and a plasmid (a base sequence of which is SEQ ID NO.2) containing an A3 gene promoter-green fluorescent protein gene-SV 40-sericin gene promoter-T4Ligase gene-SV 40([ A3-EGFP-SV40] - [ Ser promoter-T4Ligase-SV40]) piggy-10522 is used as a template to obtain a T4Ligase gene (a base sequence of which is SEQ ID NO.3) with the length of 1536bp, wherein the 5 'end of the sequence contains an ApaI enzyme cutting site, and the 3' end of the sequence contains an NheI enzyme cutting site.
ApaI and NheI are used for double enzyme digestion of the bombyx mori middle silk gland bioreactor double-promoter universal plasmid piggy-8363 (the base sequence is shown as SEQ ID NO.4) to obtain a sequence of 8361bp containing [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His 6-DDK-FLPA ] and Amp resistance genes.
And connecting the two target fragments to obtain piggyBac-9887 plasmid (the base sequence of which is shown in SEQ ID NO.1) containing [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His 6-DDDDK-T4Ligase-FLPA ] and an Amp resistance gene.
B) Expression of T4 ligase:
the piggyy-9887 plasmid (fig. 1) constructed above and the helper plasmid (fig. 2) capable of providing piggyBac transposase were mixed at a ratio of 1:1, the total concentration of 2 plasmids was 0.4 μ g/μ l, and the plasmids were dissolved in 0.5mM phosphate buffer at pH 7, and then introduced into fertilized eggs within 6 hours after the eggs laying by silkworms using microinjection method, and the total volume was 10 μ l. The silkworm eggs injected in a micro-way are raised to be imagoes under the conditions of 25 ℃ and 85% humidity, and the generation is G1 generation through hybridization and passage with non-transgenic silkworms. On the next day of the first age of the G1 generation of the transgenic experiment, 5 transgenic bombyx mori regions expressing the EGFP marker gene were obtained by observation with a fluorescence microscope (Olympus, SZX12, japan). The silkworm is raised until the adult silkworm and the non-transgenic silkworm are crossed and passaged, and the G2 is obtained. And (3) breeding the transgenic silkworms from the G2 th generation by adopting single moths, observing the silkworms by a fluoroscope microscope at one age, selecting the transgenic silkworms expressing the EGFP marker gene, breeding the silkworms to adults, mating the silkworms with a moth area, and further breeding to obtain the G3 generation, the G4 generation and the T4ligase gene homozygous. .
In G4 generation, 1 each 5-year-old 3-day silkworm of 3 moth regions is randomly selected, rear silk gland cell genome DNA is extracted as a template, an insert fragment of a piggyy-9887 plasmid in the genome of the silkworm is amplified by Inverse PCR, cloning, sequencing and chromosome positioning analysis are carried out on the amplified fragment, and the result shows that T4ligase genes are respectively inserted into a No. 14 chromosome 9822566 position, a No. 10 chromosome 15294275 position and a No. 6 chromosome 5434916 position, and the inserted genes are gene spacer regions. The results demonstrated that the transposon had been inserted into the genome of each transgenic silkworm line.
TABLE 1 analysis of foreign gene insertion sites of transgenic positive silkworms
Selecting a moth area homozygous for the EGFP genotype from the G5 generation for feeding, mating the moths in the same moth area to breed a new transgenic silkworm variety homozygous for the EGFP gene and capable of synthesizing and secreting T4ligase by using rear silk gland cells.
The silkworm cocoon silk proteins of 3 families of the determined insertion sites are extracted as materials, and SDS-PAGE electrophoresis and Western blot analysis of His6 antibody prove that the transgenic silkworms can express T4ligase and can enter the silkworm cocoons along with the silking behavior of the silkworms.
Compared with the expression quantity of the transgenic silkworm T4ligase with the double promoters and the expression quantity of the transgenic silkworm T4ligase with only 1 sericin 1 gene promoter, the expression quantity of the double promoters is obviously improved by 1.2 times.
In conclusion, the method can be used for quickly and conveniently constructing the exogenous gene expression plasmid of the transgenic silkworm by using the dual-promoter universal plasmid of the middle silk gland bioreactor of the silkworm, can efficiently synthesize the T4ligase in the silk gland cells of the silkworm, and can secrete the T4ligase into the gland cavity from the silk gland like silk so as to further spit out the silkworm. This trait has been able to be stably expressed and inherited. The method can be used for producing the T4ligase in large quantity, can improve the economic benefit of the sericulture and increase the income of silkworm farmers.
The foregoing detailed description is intended to illustrate and not limit the invention, which is intended to be within the spirit and scope of the appended claims, and any changes and modifications that fall within the true spirit and scope of the invention are intended to be covered by the following claims.
Claims (6)
1. A dual-promoter universal plasmid of a bombyx mori middle silk gland bioreactor for expressing T4ligase is characterized in that: the plasmid is a piggy-9887 plasmid, 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, wherein the functional expression frames comprise a T4ligase gene serving as an exogenous gene and a green fluorescent EGFP gene serving as a marker gene;
one functional expression box is a green fluorescent protein gene expression box started by an A3Promoter, namely A3 Promoter-EGFP-SV 40, and the other functional expression box is an expression box containing a bombyx mori sericin 1 gene Promoter, an 18s rRNA Promoter, a silk Fibroin light chain gene signal peptide, hexahistidine, an enterokinase enzyme digestion site DDDDDDK, a T4Ligase gene and the 3' tail end of a bombyx mori silk Fibroin light chain gene, namely Ser1Promoter-18s rRNA Promoter-fibrin L chain polypeptide-His 6-DDDDDDK-T4 Ligase-fibrin L chain polypeptide PolyA;
the piggy-9887 plasmid utilizes a Ser1promoter and an 18s rRNA promoter to drive the expression of a T4ligase gene.
2. The method for preparing the dual-promoter universal plasmid of the bombyx mori middle silk gland bioreactor for expressing the T4ligase as claimed in claim 1, which is characterized by comprising the following steps:
1) taking a T4 Ligase-5 'end sequence with an ApaI enzyme cutting site sequence and a T4 Ligase-3' end sequence with an NheI enzyme cutting site sequence as primers, taking a piggy-10522 plasmid containing an A3 gene promoter-green fluorescent protein gene-SV 40-sericin gene promoter-T4Ligase gene-SV 40([ A3-EGFP-SV40] - [ Ser promoter-T4Ligase-SV40]) as a template, and taking the base sequence of the piggy-10522 plasmid as SEQ ID NO.2 to obtain a T4Ligase gene with the length of 1536bp, and the base sequence of the T4Ligase gene as SEQ ID NO. 3;
2) carrying out double digestion on the piggy-8363 plasmid by ApaI and NheI, wherein the base sequence of the piggy-8363 plasmid is shown as SEQ ID NO.4, and obtaining a gene sequence which is 8361bp in length and contains [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His6-DDDDK-FLPA ] and an Amp resistance gene;
3) connecting the T4Ligase gene obtained in the step 1) with the gene sequence obtained in the step 2) to obtain a PiggyBac-9887 plasmid containing [ A3-EGFP-SV40] - [ Ser1promoter-18s rRNA promoter-FLSP-His 6-DDDDK-T4Ligase-FLPA ] and an Amp resistance gene.
3. Use of the universal plasmid according to claim 1, wherein: use for the expression of T4 ligase.
4. A method for expressing T4ligase by double-promoter universal plasmids of a bombyx mori middle silk gland bioreactor for expressing T4ligase is characterized by comprising the following steps:
(1) the piggy-9887 plasmid of claim 1 and a helper plasmid capable of providing piggyBac transposase are injected by microinjection in a concentration ratio of 1:1, introducing the gene into fertilized eggs of the silkworms within 6 hours after the silkworms lay eggs, and inserting the T4ligase gene into the silkworm genome by using piggyBac transposons in piggy-9887 plasmids;
(2) breeding the silkworm eggs after hatching to adult, then mating with the non-transgenic silkworm to produce seeds for successive generations, wherein the generation is G1 generation, observing and screening the transgenic silkworm with the body color expressing the green fluorescent EGFP marker gene by a fluorescence stereomicroscope on the second day of the first age of G1 generation, breeding until the adult then mating with the non-transgenic silkworm to produce seeds for successive generations to form G2 generation;
(3) breeding G2 generation silkworms with single moth, screening silkworms expressing green fluorescent EGFP marker genes under a fluoroscope, and mutually mating silkworms in the same moth area to prepare G3 generation silkworms;
(4) breeding G3 generation silkworms with single moth, and mutually mating the silkworms expressing green fluorescent EGFP marker genes in the same moth region to prepare G4 generation silkworms;
(5) starting from the G4 generation, adopting the moth region feeding with pure green fluorescent protein phenotype, single moth breeding and the same method of mating with the moth region silkworm moth for continuous 3 generations to select and mate, breeding green fluorescent gene and T4ligase gene homozygous, and the silk gland cell can synthesize transgenic silkworm secreting T4 ligase;
(6) the silkworm silk gland cell synthesized and secreted T4 is connected and enters silkworm cocoons along with the silkworm spinning and cocooning behaviors.
5. The method for expressing the T4ligase by the double-promoter universal plasmid of the bombyx mori middle silk gland bioreactor for expressing the T4ligase as the claim 4 shows that: the piggy-9887 plasmid utilizes a Ser1promoter and an 18s rRNA promoter to drive the expression of a T4ligase gene.
6. The method for expressing the T4ligase by the double-promoter universal plasmid of the bombyx mori middle silk gland bioreactor for expressing the T4ligase as the claim 4 shows that: the T4ligase gene in the step (6) is specifically expressed in bombyx mori silk gland cells, secreted to a silk gland cavity under the action of bombyx mori silk fibroin light chain signal peptide and secreted to a silkworm cocoon along with a spinning behavior.
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