Construction method of recombinant bacillus subtilis for integration and expression of foreign protein by virtue of Tn7 transposable element
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A Bacillus subtilis, foreign protein technology, applied in the biological field, can solve the problem of retaining antibiotic genes, etc., and achieve the effect of high integration efficiency
Inactive Publication Date: 2015-11-18
NANNING XINKEJIAN BIOTECH
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[0005] The purpose of the present invention is to provide a method for constructing recombinant Bacillus subtilis using Tn7 transposable elements to integrate and express foreign proteins, so as to solve the problem that the integration of homologous recombination still needs to retain the antibiotic gene in the integrated fragment
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Embodiment 1
[0020] This example illustrates the method for constructing a recombinant Bacillus subtilis that uses the Tn7 transposable element to transpose and integrate and express the β-glucanase gene.
[0021] The β-glucanase gene of this example is the β-glucanase gene from Bacillus subtilis. The construction of β-glucanase gene transposition integration recombinant Bacillus subtilis expression strain is as follows:
[0022] 1. Plasmids containing araC (repressor in the arabinose operon), Pbad (araBAD promoter), Escherichia coli replicon pBR322ori and bla (ampicillin resistance gene), such as the size of Invitrogen's 4.1kb plasmid pBAD / His-A, cut pBAD / His-A with BspHI, remove about 1kb bla fragment, and recover 3.1kb fragment.
[0023] 2. Using a plasmid containing the temperature-sensitive replicon pWV01ori / ts of Bacillus subtilis and the chloramphenicol resistance gene cat, such as the plasmid pAW016 preserved by Nanning Xinkejian Biotechnology Co., Ltd. as a template, use the pri...
Embodiment 2
[0035] This example illustrates the construction method of recombinant Bacillus subtilis using Tn7 transposable element to transpose and integrate and express xylanase gene.
[0036] The xylanase gene of this example is the xylanase gene from Bacillus stearothermophilus. The construction of xylanase gene transposition integration recombinant Bacillus subtilis expression strain is as follows:
[0037] 1. Plasmids containing araC (repressor in the arabinose operon), Pbad (araBAD promoter), Escherichia coli replicon pBR322ori and bla (ampicillin resistance gene), such as the size of Invitrogen's 4.1kb plasmid pBAD / His-A, cut pBAD / His-A with BspHI, remove about 1kb bla fragment, and recover 3.1kb fragment.
[0038] 2. Using a plasmid containing the temperature-sensitive replicon pWV01ori / ts of Bacillus subtilis and the chloramphenicol resistance gene cat, such as the plasmid pAW016 preserved by Nanning Xinkejian Biotechnology Co., Ltd. as a template, use the primer AflIII-CmFOR:...
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Abstract
The invention discloses a construction method of recombinant bacillus subtilis for integration and expression of foreign protein by virtue of a Tn7 transposable element; specifically, the construction method comprises the following steps: constructing a transposable integration and expression plasmid containing an antibiotics resistance gene, transposable Tn7 left and right arm boxes, a multiple cloning site and Tn7 transposase TnsABCD segments as well as an escherichia coli replicon pBRe322 ori and a bacillus subtilis temperature-sensitive replicon; interpolating a bacillus subtilis promoter, signal peptide, foreign protein gene and like expression elements into the transposable integration and expression plasmid, and converting to a bacillus subtilis host, so that the foreign protein gene is integrated and expressed on an attTn7 site at downstream 3' end of bacillus subtilis glms gene; and implementing fluctuation temperature culture so that the temperature-sensitive replicon on the plasmid becomes ineffective, thus obtaining recombinant bacillus subtilis that an integration plasmid containing the antibiotics resistance gene and the Tn7 transposase is not reserved anymore. The construction method disclosed by the invention has the advantages that stable foreign gene transposable integration is obtained, and the recombinant bacterium is free from an antibiotics resistance gene and is consistent with food safety requirements.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a recombinant Bacillus subtilis that utilizes Tn7 transposable elements to integrate and express foreign proteins. Background technique [0002] In the production of active protein for food and medicine, the heterologous expression of active protein gene by genetic engineering technology has been proved to be an effective method to improve the yield and purity. As a commonly used genetic engineering host bacteria, Escherichia coli grows rapidly, is easy to cultivate, has low fermentation cost, and has no complex protease system, and the recombinant protein is relatively stable, so it becomes a good host for the production of recombinant protein. However, Escherichia coli is used to produce enzymes for the food industry and there are still some defects, mainly including: 1. Escherichia coli is not recognized as a safe strain; 2. In the high-density fermentati...
Claims
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