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Construction of bacillus subtilis integrated vector capable of being cloned by using homologous recombination and application thereof

A Bacillus subtilis, homologous recombination technology, applied in the biological field, can solve the problems of not being widely used, inconvenient cloning of foreign genes, and difficult formation of inclusion bodies, etc.

Inactive Publication Date: 2011-01-19
南宁邦尔克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2 There is no obvious codon preference, and the expression product is not easy to form inclusion bodies
However, the multiple cloning site of pMLK83 has only 5 single restriction enzyme sites (Sal I, HindIII, BamH I, SpeI and SacII) that can be used for cloning, and pMLK83 is a relatively large vector with a length of up to 8.8kb, which brings some inconvenience to the cloning of foreign genes into pMLK83
[0003] Recombinant cloning methods, although simple and efficient, have not been widely used

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200]Construction of integration vector pMLK83-BN:

[0201] 1. Synthesize two primers Puc-BN1-L: TCGACGCCGATTC ATTAATGCAG CTGGCACGACAGGTTTCCCG ACGGATCC TTTTCTCCTT ACGCATCTGT GCGGTATTTC ACACCGCATA GC and Puc-BN1-R: TATGCGGTGT GAAATACCGC ACAGATGCGT AAGGAGAAACT GGATCCGTCGGGAAACCT GTCATGCG

[0202] 2. The above two sections of primers Puc-BN1-L and Puc-BN1-R anneal to form a double-stranded DNA, which is then ligated to the pMLK83 vector cut with SalI and SacII. Transform Escherichia coli DH5α competent cells;

[0203] 3. Screen the correct transformants to obtain pMLK83-BN.

Embodiment 2

[0205] Build of puc19Not:

[0206] This example transforms the puc19 vector to construct puc19Not, and adds a NotI restriction endonuclease site to the NdeI restriction endonuclease site of the puc19 vector. This vector can be used to clone genes or promoters that contain NdeI restriction endonuclease sites in their sequences. This vector is constructed as follows:

[0207] 1. Synthesize a primer P1:

[0208] GGAATTCCATATGGCGGCCGCCACCGCTATGCGGTGTGAAATACC;

[0209] 2. A DNA fragment of about 330 bp was amplified by using primer P1 and commonly used sequencing primer M13R(-48) (AGCGGATAACAATTTCACACAGGA) with puc19 as a template. The amplification conditions of PCR are: 94°C for 2min; 94°C for 2min, 60°C for 30s, 72°C for 25s, 20 cycles; 72°C for 5min. Then the amplified fragments were digested with NdeI and KpnI, and about 270bp were recovered from the gel. The DNA fragment was connected to the puc19 vector digested with NdeI and KpnI. Transform Escherichia coli DH5α compet...

Embodiment 3

[0212] The pMLK83-BN vector was used to test the gene activation effect of the P43 promoter in Bacillus subtilis:

[0213] pMLK83-BN has a β-glucuronidase reporter gene. When pMLK83-BN itself was integrated into Bacillus subtilis, this gene was not, or minimally, expressed because there was no promoter preceding the β-glucuronidase reporter gene. However, when a promoter is placed in front of the β-glucuronidase reporter gene, the expression of the β-glucuronidase reporter gene can be initiated. This method can be used to monitor the effect of different promoters. The following example uses the P43 promoter to illustrate the application of the pMLK83-BN vector to test the gene activation effect of the promoter in Bacillus subtilis.

[0214] 1. Clone the P43 promoter into the multiple cloning site of the vector puc19Not:

[0215] Design the upstream primer 5'ATTGCTGGACGCTTATGGAC 3' and the downstream primer 5'CGGGATCCATTCCTCTCTCTTACCTATAAT 3', and amplify about 480bp DNA fra...

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Abstract

The invention discloses construction of a bacillus subtilis integrated vector capable of being cloned by using homologous recombination and application thereof. The construction of the bacillus subtilis integrated vector capable of being cloned by using homologous recombination and the application thereof belong to the technical field of biology. The vector pMLK-BN can transfer an exogenous gene or a promoter cloned on a common cloning vector puc18, puc19 or puc57 to self by a homologous recombination method so as to integrally express exogenous protein in bacillus subtilis or test the promoting effect of the promoter. Compared with a common connecting cloning method, the cloning method of homologous recombination is simple and efficient; and the pMLK-BN can be effectively used as a choice for cloning the exogenous gene or the promoter to the bacillus subtilis to perform integrated expression.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the construction and application of a vector capable of cloning by homologous recombination and integrating and expressing exogenous DNA fragments in Bacillus subtilis. Background technique [0002] Bacillus subtilis is non-pathogenic, does not produce toxins and thermosensitive proteins, is a food-safe strain, and is classified as a food-grade microorganism. Moreover, as an expression system, Bacillus subtilis has the following advantages: 1. It has a strong protein secretion function. It does not need to break cells to extract proteins. It only needs to simply process the fermentation supernatant to obtain a relatively pure target protein. A variety of exogenous proteins have been secreted and expressed in Bacillus subtilis. 2 There is no obvious codon preference, and the expression product is not easy to form inclusion bodies. 3 The fermentation conditions are simple. Therefore...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/66C12N15/63
Inventor 李晓明韦航廖东庆蒙健宗张云光林珊宇李丛陈发忠梁莲华
Owner 南宁邦尔克生物技术有限责任公司
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