Escherichia coli-bacillus subtilis shuttle expression vector and application thereof
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A Bacillus subtilis, shuttle expression vector technology, applied in the field of genetic engineering, can solve the problems of easily lost plasmids and poor stability of plasmids
Active Publication Date: 2012-06-27
山东黄三角生物技术产业研究院有限公司
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[0007] (Ⅲ) The stability of the plasmid is poor. Compared with the E. coli expression system, Bacillus subtilis cells are prone to lose the plasmid during replication
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Embodiment 1
[0031] The design of embodiment 1 expression regulation element
[0032] Select the B. subtilis P43 promoter, the signal peptide (CSN) from B. subtilis chitosanase, use the terminator sequence from F1 phage, and the RBS sequence designed according to the B. subtilis ribosome sequence, and analyze the B in various proteins by comparison. .subtilis signal peptidase cleavage site characteristics Design signal peptidase cleavage sites, select five kinds of enzyme cleavage sites to form MCS. See SEQ ID No.2 for the full sequence of the set of expression regulatory elements, and see the composition model of the sequence figure 1 .
Embodiment 2
[0033] Construction of embodiment 2 expression vector
[0034] 1. Synthesis of expression regulatory elements
[0035] Since the expression control element does not continuously come from a template, and its partial sequence is completely artificially designed, artificial overlapping PCR technology is used. First, the genomic DNA of B. subtilis was extracted, and the P43 promoter (denoted as A, figure 2 ).
[0036] Then chemically synthesized 7 oligonucleotide sequences of 34-65bp length (see Table 1), by overlapping PCR2 program (using zqxrsmS1, zqxrsmS2, zqxrsmS3, zqxrsmAs1, zqxrsmAs2, zqxrsmAs3, zqxrsmAs4 as templates, zqxrsmS1 and zqxrsmAs4 as primers, Tm =50°C), to obtain the RSB-CSN-MCS-6His tag-TAATGA fragment (denoted as B, image 3 ).
[0037] Finally, through the PCR3 program (using A and B as templates, using zqxP43S and zqxrsmAs4 as primers, Tm=68°C), the P43 / CSN ( Figure 4 ) fragment. Cut the gel to recover the P43 / CSN DNA and connect it to the pMD18T vect...
Embodiment 3
[0043] Example 3 Analysis of the enzyme cleavage site of the expression control element
[0044] It was confirmed by enzyme digestion analysis and sequencing analysis that NdeI, SalI, NotI, XhoI, and BamHI in the vector MCS can be used as the insertion site of the gene. After NdeI, there should be a CSN signal peptide coding sequence, and before BamHI, there should be a 6His coding sequence.
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Abstract
The invention discloses an Escherichia coli-bacillus subtilis shuttle expression vector and application thereof, and belongs to the field of genetic engineering. The Escherichia coli-bacillus subtilis shuttle expression vector comprises a bacillus subtilis promoter sequence, a bacillus subtilis signal peptide coding sequence, an artificially designed ribosome binding sequence which can be identified by bacillus subtilis, the artificially designed enzyme cutting site of bacillus subtilis signal peptidase, an artificially designed multiple cloning site, a bacteriophage terminator sequence, a bacillus subtilis replicating initial sequence, and an Escherichia coli replicating initial sequence. The vector can be replicated in Escherichia coli and can also be replicated and expressed in the bacillus subtilis, so that vectors for a bacillus subtilis expression system are enriched; and the vector uses a B.subtilis deacetylated chitinase signal peptide for the first time, the signal peptide has high capability of guiding to secrete extracellular protein, a reporter gene is aspergillus nidulans pectate lyase gene, and the extracellular expression level reaches 600U / mL which is higher than that when the pectate lyase gene uses other vectors.
Description
technical field [0001] The invention designs an Escherichia coli-Bacillus subtilis shuttle expression vector and its application, belonging to the field of genetic engineering. Background technique [0002] In the research on the expression of enzymes used in the food industry and industrial fermentation production, the recombinant expression of food enzymes with microbial expression systems has broad application prospects. At present, the E. coli expression system and yeast expression system have convenient sources. Many research institutions have developed a variety of expression vectors and expression host bacteria over the years, and have modified the E. coli expression system and yeast expression system in multiple directions. However, many problems in the E. coli expression system still exist, such as the formation of inclusion bodies and the toxicity of E. coli expression host bacteria. Yeast expression system The expression system has unique advantages because it ca...
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