Recombinant magnetospirillum gryphiswaldense and applications thereof

A magnetic spirochete, recombinant plasmid technology, applied in the direction of recombinant DNA technology, bacteria, introduction of foreign genetic material using vectors, etc., can solve the problems of not exerting the advantages of magnetosomes, loss of antibody activity, etc.

Inactive Publication Date: 2015-01-14
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

From the analysis of the above experiments, it can be seen that these experiments mainly use the amino groups of the antibody to connect to the magnetosomes, and these amino groups are more distributed at the nitrogen terminal, which is also the region (Fab) where the antibody binds to the antigen. Magnetosome coupling can no longer bind the antigen, resulting in loss of antibody activity
At the same time, in the above method, the coupling of magnetosomes and antibodies still requires expensive bifunctional reagents, and the advantages of magnetosomes as biological materials have not been brought into play.

Method used

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  • Recombinant magnetospirillum gryphiswaldense and applications thereof
  • Recombinant magnetospirillum gryphiswaldense and applications thereof
  • Recombinant magnetospirillum gryphiswaldense and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Construction of mamC and mamF mutants of M. gryphiswaldense MSR-1

[0034] By constructing recombinant plasmids, using the method of PCR amplification respectively, the upstream and downstream fragments of about 1 kb of the mamC gene (respectively denoted as S and X fragments, the S fragment is shown in SEQ ID No.1, and the X fragment is shown in SEQ ID No. .2), these two fragments and the gentamicin (Gm) resistance gene fragment of 0.8kb were directionally cloned into the multiple cloning site of the suicide vector pUX19 to construct the recombinant suicide plasmid pUX19-MamC-S-Gm -X, and then replace the mamC gene fragment with the resistance gene by homologous double crossover. Specific steps are as follows:

[0035] 1. Use primers mamC-S-F-XbaⅠ / mamC-S-R-KpnⅠ and mamC-X-F-KpnⅠ / mamC-X-R-PstⅠ to amplify the upstream and downstream regions of the mamC gene (viewing the upstream and downstream regions in the direction of the whole genome), and electrophoresis separation...

Embodiment 2

[0045] The acquisition of embodiment 2spa gene, bccp gene and the construction of recombinant monomer msa gene

[0046] Cultivate Staphylococcus aureus (Staphylococcus aureus) ATCC6538, extract its whole genome as a template, and use primers Spa-LF (as shown in SEQ ID No.9) and Spa-RB (as shown in SEQ ID No.10) to amplify Get the full-length spa gene. Wherein, primer Spa-LF comprises Linker sequence (Gly 4 Ser) 3 , Primer Spa-RB contains BamHI restriction site.

[0047] Genomic DNA of Magnetospira wild-type strain MSR-1 was extracted as above, and used as a template to amplify with primers BCCP-F (as shown in SEQ ID No.11) and BCCP-R (as shown in SEQ ID No.12). The biotin carboxyl carrier protein gene bccp was added. Wherein, primer BCCP-F comprises Linker sequence (Gly 4 Ser) 3 , Primer BCCP-R contains XbaI restriction site.

[0048] According to the sequence of the streptavidin gene (sa) of wild-type Streptomyces avidinii, we code it for valine at position 55, threoni...

Embodiment 3

[0049] Construction of each fusion protein gene of embodiment 3

[0050] The construction of the expression plasmid pMamC-MSA containing MamC and monomer streptavidin fusion protein gene, the specific steps are as follows:

[0051] 1. Use the wild-type MSR-1 genome as a template, use mamC-exp1-KpnI as an upstream primer, and mamC-exp2 as a downstream primer to amplify the mamC gene. The gel was cut and recovered, then connected to pMD18-T Simple Vector, and transformed into E.coli DH5α competent cells. After PCR verification and correct sequencing, the resulting recombinant plasmid was named pT-MamC-exp.

[0052] 2. Use pT-MamC-exp and recombinant monomeric streptavidin fragment MSA as templates simultaneously, use mamC-exp1-KpnI as upstream primer, and MSA-EcoRI as downstream primer, perform fusion PCR to obtain mamC gene and msa The fusion fragment of the gene was electrophoresed on agarose gel, recovered by gel cutting, then connected to pMD18-T Simple Vector, transformed ...

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Abstract

The invention provides a recombinant magnetospirillum gryphiswaldense and a construction method thereof. The construction method comprises the following steps: firstly, constructing a magnetotactic bacterium mutant strain loosing magnetosome membrane protein gene, then performing gene fusion to the magnetosome membrane protein gene and a functional protein gene, connecting onto pMD18-T Simple Vector, converting into E. coli DH5alpha competent cells, and constructing to obtain recombinant plasmid. The recombinant magnetospirillum gryphiswaldense constructed by adopting the method can be used for synthesizing magnetosome with the surface displayed with functional protein, the magnetosome can be easily connected with an antibody, even nucleic acid, saccharides, lipids and non-antibody proteins and other functional proteins to form a magnetic complex with specific functions, and the complex not only can be used for magnetic separation, but also can be assembled into a magnetic material or a member with a specific structure.

Description

technical field [0001] The invention relates to a magnetospira, in particular to a recombinant magnetospira for synthesizing functionalized magnetosomes and its application. Background technique [0002] Magnetotactic bacteria (MTB) are a polyphyletic group of aquatic prokaryotes, which have spherical, egg-shaped, rod-shaped, arc-shaped and spiral shapes, and can be unicellular or multicellular. Generally have terminal or twin flagella. Phylogenetically belonging to Proteobacteria (Proteobacteria) α-Proteobacteria (Alphaproteobacteria), δ-Proteobacteria (Deltaproteobacteria), γ-Proteobacteria (Gammaproteobacteria) and Nitrospirae (Nitrospirae). [0003] Magnetotactic bacteria can absorb a large amount of iron from the environment to form a special organelle, which contains a single magnetic domain of iron tetroxide (Fe 3 o 4 ) or iron tetrasulfide (Fe 3 S 4 ) crystals, which are coated with unit membranes composed of lipids and proteins, called magnetosomes. Different ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N1/21C12R1/01
Inventor 田杰生胡俊英徐俊王旭李丽刘凌子钱欣欣姜伟李颖李季伦
Owner CHINA AGRI UNIV
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