Dual-promoter inducible secretable shuttle plasmid and construction method thereof

A dual-promoter, shuttle plasmid technology, applied in the field of bioengineering, can solve problems such as high cost, inability to large-scale production, and small amount of bactericidal peptides

Inactive Publication Date: 2011-01-19
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a dual-promoter inducible secretable shuttle plasmid and its construction method. The dual-promoter inducible secretory shuttle plasmid and its construction method should solve the problem of vectors in the prior art. The amount of cecropin expressed is small, or the technical problem is that the cost is too high to be mass-produced

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  • Dual-promoter inducible secretable shuttle plasmid and construction method thereof
  • Dual-promoter inducible secretable shuttle plasmid and construction method thereof
  • Dual-promoter inducible secretable shuttle plasmid and construction method thereof

Examples

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Effect test

Embodiment 1

[0042] Example 1 Construction of dual-promoter inducible secretable shuttle plasmid

[0043] Construction of double promoter inducible secretory Escherichia coli and Bacillus subtilis shuttle plasmids such as figure 1 shown. Escherichia coli pET-28a is a Novagen commercial expression plasmid. NdeI and XhoI double-digested pET-28a, and inserted the second functional peptide of bovine lactoferrin containing NdeI and XhoI restriction sites, thereby deleting NheI in the multiple cloning site. The deletion of NheI is to introduce the cleavage site NheI of signal peptidase in the promoter signal peptide functional fragment constructed subsequently, and NheI is also the site where the gene is inserted in the correct direction; the inserted 0.3kb bovine lactoferrin gene can be inserted in the IPTG or lactose Induced expression was performed under induction.

[0044] The DNA sequence of the cloned bovine lactoferrin is shown in SEQ ID NO: 1, and the amino acid sequence is shown in ...

Embodiment 2

[0068] The thus constructed Escherichia coli-Bacillus subtilis shuttle plasmid can be used as a common expression vector, and a promoter, a signal peptide and a terminator sequence are cloned on the shuttle plasmid. This promoter is suitable for gene expression in prokaryotes. Transformation of embodiment 2 shuttle plasmids in Bacillus sphaericus

[0069] The transformation of the plasmid in Bacillus sphaericus basically adopts the protoplast method [Chang S S, Cohen S N. MolGen Genet, 1979, 168: 111-115]. VY medium (2.5% veal extract powder, 1% yeast powder) was used as common medium, and DM3, PAB, SMM, SMMP medium were used as medium for protoplast transformation. Bacillus subtilis was activated, and its single colony was inserted into 3mL VY liquid medium, and cultured overnight at 37°C with shaking. Transfer 1% to fresh culture medium, shake culture at 37°C for 2.5h, that is, the bacteria grow to the middle and late stages of logarithmic growth. The concentration of sod...

Embodiment 3

[0071] Transformation of embodiment 3 shuttle plasmids in Bacillus subtilis

[0072] The transformation of Bacillus subtilis adopts the competent method. In Bacillus subtilis spore-forming medium (0.1% peptone, 0.07% yeast powder, 0.1% KH 2 PO 4 , 0.1% Glucose, 0.02% MgSO 4 , 0.02% (NH 4 ) 2 SO 4 ) plates were picked and inoculated in SPI (1.4% K 2 HPO 4 , 0.6% KH 2 PO 4 , 0.028% MgSO 4 , 0.2% (NH 4 ) SO 4 , 0.1% yeast powder, 0.5% glucose, 0.6% Na 3 Citrate, 0.02% casamino acid) culture medium, shake culture at 110rpm / min at 30°C overnight, connect fresh SPI with 1 / 25 volume of seed, shake culture at 250rpm / min at 37°C for 4-5h, OD 600 ≈2.0, then transfer to SPII with 1 / 10 of the inoculum volume of the culture medium (that is, add 0.0005mol / L CaCl to SPI 2 , 0.0025mol / L MgCl 2 ) in the culture medium, shake culture at 110rpm / min at 37°C for 1.5h, collect the bacteria by centrifugation at 5000rpm / min, leave 1 / 10 of the supernatant of the volume of the bacteria f...

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Abstract

The invention belongs to the field of biological genetic engineering, and provides a dual-promoter inducible secretable shuttle plasmid. The shuttle plasmid consists of an enzyme cutting Escherichia coli pET-28a plasmid and an enzyme cutting bacillus subtilis pE194 plasmid, wherein a lysozyme-antibacterial peptide functional fragment and an antibacterial peptide gene are inserted into the enzyme cutting Escherichia coli pET-28a plasmid. The invention also provides a method for constructing the dual-promoter inducible secretable shuttle plasmid. The shuttle plasmid is transferred to a prokaryote to express proteins such as toxalbumin, cecropin and the like in a fusion mode without toxicity to a host. The shuttle plasmid constructed by the method can be induced by IPTG or lactose to improve gene expression level; the shuttle plasmid is also suitable for exocrine expression, and a target protein for the exocrine expression is convenient to process and is not polluted by endotoxin; and because the shuttle plasmid has dual functional promoters, different genes can be inserted in appropriate correct directions for gene expression.

Description

Technical field: [0001] The present invention relates to the field of bioengineering, in particular to a shuttle plasmid, in particular to a dual-promoter inducible and secretable shuttle plasmid and a construction method thereof. Background technique: [0002] In the field of bioengineering, in order to effectively express cloned genes, it is often necessary to construct expression vectors. Escherichia coli expression system is the most commonly used one in genetic engineering research. Although it cannot perform post-translational processing like the eukaryotic system, the genetic background of Escherichia coli is relatively clear, easy to operate, short in growth cycle, and economically safe, so it is still the most widely used and irreplaceable system. It has become an indispensable and important tool in research and practical application. Some important exogenous genes have been highly expressed in Escherichia coli, accounting for 30% or even 40% of the total bacteria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N15/66A61K48/00A61P31/04A61P31/10
CPCY02A50/30
Inventor 乐科易张培德
Owner FUDAN UNIV
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