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Single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof

A Bacillus subtilis, shuttle expression vector technology, applied in the field of microorganisms and bioengineering, can solve the problems of low transformation efficiency, low transformation efficiency, and low transformation efficiency of the Bacillus subtilis expression system

Inactive Publication Date: 2014-12-24
WUHAN RUIHENGDA BIOLOGICAL ENG
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Problems solved by technology

[0008] (4) The transformation efficiency of the Bacillus subtilis expression system is low, generally less than 1×103 CFU / μg DNA, which is much lower than that of Escherichia coli (generally 1×106-1×109 CFU / μg DNA)
Among them, pUB110 and pWB980 are non-shuttle plasmids, which can only be replicated in Bacillus subtilis, and it is difficult to extract the plasmids. After the digested vector fragments are ligated with foreign genes, the transformation will be very difficult due to the low concentration of the plasmids; currently commercialized The vector and the Escherichia coli-Bacillus subtilis shuttle expression vector constructed by researchers are generally more than 5kb, such as pHT01 and pHT43 are about 8000bp
In order to facilitate the screening of positive clones in Escherichia coli and Bacillus subtilis, these Escherichia coli-Bacillus subtilis shuttle expression vectors usually contain two kinds of resistance gene fragments (use ampicillin resistance in Escherichia coli, use kalamycin resistance in subtilis) or chloramphenicol resistance) and two replicon fragments, resulting in a larger plasmid, low copy number, and difficulty in extraction and genetic transformation
Generally, the size of the plasmid is inversely proportional to the transformation frequency. Plasmids with small molecular weights are easy to transform and have high transformation efficiency; while plasmids with large molecular weights are relatively low in transformation efficiency [Biotechnology Bulletin, 2011(5): 227-230; Appl Microbiol Biotechnol , 2008,78(1):181-188.], the MCS has fewer restriction enzyme sites available for use, and the size of the foreign gene that can be inserted will also be limited

Method used

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  • Single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof
  • Single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof
  • Single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof

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Experimental program
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Embodiment 1

[0069] [Example 1] Cloning of kanamycin resistance gene and construction of promoter probe vector

[0070] Extract pET-28a(+) vector plasmid and pUC57 plasmid. Use the pET-28a(+) plasmid DNA as a template, and use the synthetic P1 / P2 (Table 1) as primers to amplify the Kan gene fragment on the pET-28a(+) vector, which does not contain a promoter. EcoR V and Pci I double-digest Kan gene fragment 1 ( figure 1 ), PCR product recovery product; EcoR V and Pci I double enzyme digestion pUC57 plasmid reclaims 2200bp large fragment ( figure 1 ), connected with the Kan gene restriction fragment, transformed E. coli DH5α, and prepared DH5α supercompetent by Inoue method, the method refers to "Molecular Cloning Experiment Guide (3rd Edition)", and spread it on LB solid containing 100 μg / mL ampicillin Screened on the medium plate, verified by PCR and sequencing (pUC57 vector general sequencing primers M13F and P3), and finally the vector with successful sequencing was named pUCKan.

[...

Embodiment 2

[0072] [Example 2] Cloning of promoter active fragment

[0073] Genomic DNA of Bacillus subtilis (Bacillus subtilis) BS168 and Bacillus licheniformis (Bacillus licheniformis) ATCC14580 was extracted by CTAB method (cetyltrimethylammonium bromide) as templates for gene amplification [Acta Microbiology Sinica, 2006, 46( 1): 7-12.]; The genomic DNA of Bacillus subtilis BS168 and Bacillus licheniformis ATCC14580 was partially digested with Sau3A I, so that the digested fragments were concentrated between 0.5-5kb. Using the pHYKan vector as the promoter probe carrier, the promoter was screened from the genome of Bacillus subtilis [Acta Microbiology, 2004, 44 (4): 457-460; Journal of Fuzhou University (Natural Science Edition), 2013, 41 (3) :391-396.]. The specific method is: use BamH I to completely digest the pHYKan vector, dephosphorylate it, and then connect it with the above-mentioned genomic DNA partial digestion fragment at an appropriate ratio, transform Escherichia coli DH...

Embodiment 3

[0076] [Example 3] The whole plasmid PCR removes the ampicillin Amp resistance gene fragment in the vector

[0077] Primer P22 / P23 (Table 1) was phosphorylated with T4Polynucleotide Kinase from Takara Company. The 20 μL reaction system is: P22 / P23 primer mixture (final concentration of each primer 12.5 μM) 4 μL; ATP (10 mM); 10×Reaction buffer 2 μL; 10×T4 Polynucleotide Kinase (10U / μL) 2 μL; dH 2 O 9.6 μL.

[0078] Extract pUCKanEB1, pUCKanEB2, pUCKanEB3, pUCKanEB4, pUCKanEB5, pUCKanEB6 and pUCKanEB7 plasmid DNAs, use them as templates, and use phosphorylated primers P22 / P23 to amplify. The connection conditions were connected at 22°C for 2 hours, transformed into Escherichia coli DH5α, spread on the LB solid medium plate containing 50 μg / mL kalamycin for screening, verified by PCR and sequencing (sequencing primer P24, Table 1), and finally The successfully sequenced vectors were named pKanEB8, pKanEB9, pKanEB10, pKanEB11, pKanEB12, pKanEB13 and pKanEB14.

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Abstract

The invention discloses a single-resistance escherichia coli-bacillus subtilis shuttle expression vector and application thereof. The bacillus subtilis shuttle expression vector provided by the invention comprises the following elements: a bacillus subtilis formed strong promoter P43, a sequence which encodes signal peptide, multiple cloning sites, an antibiotics resistance gene (kalamycin or chloramphenicol), a bifunctional promoter sequence which can start resistance gene expression of kalamycin or chloramphenicol in escherichia coli and bacillus subtilis, and copy homing sequences of bacillus subtilis and escherichia coli. The expression vector disclosed by the invention not only can be stably copied in escherichia coli, but also can be stably copied in bacillus subtilis, and expresses homologous and heterologous proteins. All vectors just contain one resistance gene, the plasmid of which is less than 400bp, so that gene operation and plasmid conversion are easy to carry out, and the conversion ratio is high. Besides the expression vector of an exogenous gene, the vector can be further used for screening promoters and constructing a gene knock-out vector.

Description

technical field [0001] The invention belongs to the field of microorganisms and bioengineering. In particular, it relates to an Escherichia coli-Bacillus subtilis shuttle expression vector and an application thereof. Background technique [0002] In the expression research of industrial enzymes and industrial fermentation production, the recombinant expression of enzymes, active proteins and single-chain antibodies with microbial expression systems has broad application prospects. Escherichia coli expression system and yeast expression system are currently widely used recombinant expression systems. Many commercial companies and scientific research institutions at home and abroad have developed a variety of expression vectors and expression host bacteria, and the E. coli expression system and yeast expression system The in-depth and systematic transformation of the system has made great progress in the expression of recombinant proteins in these two expression systems. How...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12N9/50C12R1/19C12R1/125
Inventor 汪小锋李青山张金艳马飞刘艳红魏雄
Owner WUHAN RUIHENGDA BIOLOGICAL ENG
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