Double-promoter universal plasmid for silkworm middle silk gland bioreactor and its construction method and application
A bioreactor and dual-promoter technology, applied in biochemical equipment and methods, microorganisms, and the use of vectors to introduce foreign genetic material, etc., can solve the problem of low work efficiency, failure to achieve high expression levels, low expression efficiency of foreign proteins, etc. problem, to achieve the effect of improving the expression
Inactive Publication Date: 2019-12-20
ZHEJIANG UNIV
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Problems solved by technology
[0004] Since most of the piggyBac vectors contain two expression cassettes with more elements and the same restriction site, each time a different foreign gene is expressed, it is necessary to construct a plasmid from scratch and assemble the promoter, signal peptide and foreign gene , polyA and other structures, time-consuming and labor-intensive, low work efficiency
[0005] On the other hand, looking at the research results of silkworm bioreactors at home and abroad for more than ten years, the expression efficiency of foreign proteins in transgenic silkworm silk gland bioreactors is very low. It is still an exogenous gene driven by a silk fibroin promoter. The expression level in most experiments does not reach about 1% of the cocoon layer, which is far from the high-efficiency expression level expected by scientists like the expression of silk protein.
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[0024] The present invention will be further described below in conjunction with drawings and embodiments.
[0025] Embodiments of the present invention are as follows:
[0026] (1) Primers were designed to obtain the sericin 1 gene promoter (SEQ ID NO.4) using the piggy-14413 plasmid (SEQ ID NO.1) as a template. And add EcoRI enzyme cutting site at the 5' end of the promoter, and add KpnI enzyme cutting site at the 3' end. The PCR product was double-digested with EcoRI and KpnI to obtain a 593bp fragment, namely the sericin 1 gene promoter, and then recovered by gel; the piggy-5257 plasmid (SEQ ID NO.5) was double-digested with EcoRI and KpnI to obtain a 4549bp fragment The fragments were recovered by gel; the obtained two fragments were ligated, and the ligated plasmid was named piggy-5140 plasmid.
[0027] (2) Digest the piggy-5140 plasmid with AflII, BglII and ScaI to obtain a fragment of 2493bp in length; use AflII and BglII to digest the piggy-6212 plasmid (SEQ ID NO.6...
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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 [0001] The invention relates to a plasmid and its construction method and application, in particular to a dual-promoter universal plasmid for silkworm middle silk gland bioreactor and its construction method and application. Background technique [0002] The piggyBac transposon was originally isolated from the genome of Trichoplusia ni TN-368 cell line, and it is the DNA transposon with the highest transposition activity found so far. The piggyBac transposition system is a non-viral vector with high transposition efficiency. Compared with sleeping beauty, the piggyBac vector has a larger capacity and can carry 18kb. It can realize the co-expression of multiple genes, and the transposition fragment will not leave a footprint in the original site after being excised. The genome can be accurately excised. Repair plays an important role in the application of reversible genes. [0003] PiggyBac transposon-mediated transgenic silkworm silk gland bioreactor has be...
Claims
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IPC IPC(8): C12N15/85C12N15/65C12N15/66
CPCC12N15/65C12N15/66C12N15/85C12N2800/105C12N2800/90
Inventor 钟伯雄张玉玉叶露鹏
Owner ZHEJIANG UNIV