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Recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacteria and application thereof

A technology of pet28a-ag43 and recombinant engineering bacteria, which is applied in the field of genetic engineering, can solve the problems of limiting the application of cell surface display technology, and achieve the effects of rapid response, improved enzyme protein expression activity, and thorough reaction

Inactive Publication Date: 2018-10-19
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the application of cell surface display technology in production is greatly limited due to the lack of general commercially available plasmids.

Method used

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  • Recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacteria and application thereof
  • Recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacteria and application thereof
  • Recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacteria and application thereof

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preparation example Construction

[0039] The present invention provides a method for preparing the recombinant plasmid described in the above scheme, comprising the steps of:

[0040]1) Using EcoRI and HindIII to perform double enzyme digestion on the genes encoding plasmid pET28a and Ag43, respectively, to obtain pET28a after digestion and the gene encoding Ag43 after digestion;

[0041] 2) Ligate the digested pET28a and the digested Ag43 coding gene in step 1) to obtain the recombinant plasmid pET28a-Ag43.

[0042] In the present invention, EcoRI and HindIII are used to carry out double enzyme digestion on the coding genes of plasmid pET28a and Ag43 respectively, so as to obtain pET28a after digestion and Ag43 after digestion. In the present invention, the preparation method of the Ag43-encoding gene preferably includes the following steps:

[0043] a. amplifying and culturing Escherichia coli K-12 MG1655 to obtain Escherichia coli K-12 MG1655 cells;

[0044] b. extracting the total DNA of the Escherichia ...

Embodiment 1

[0068] Escherichia coli K-12 MG1655 was amplified and cultured in LB medium at 37°C for 16 hours to obtain Escherichia coli K-12MG1655 cells. The obtained Escherichia coli K-12 MG1655 cells were centrifuged at a speed of 4000 rpm for 10 min, and the precipitate was collected, which was Escherichia coli. The total DNA of Escherichia coli K-12MG1655 was extracted by liquid nitrogen grinding method at a speed of 300 rpm for 5-10 min. Using the total DNA of Escherichia coli K-12 MG1655 as a template, the Ag43 coding gene was amplified using primers Ag43-EcoRI-F and Ag43-HindIII-R (amplification conditions: 94°C, 4min; 94°C, 1min; 56°C , 30s; 72°C, 1kb / min; 29 cycles; 72°C, 10min. The amplification system is: genomic DNA or plasmid DNA 10-100ng, front-end primer (10μM) 2-3μL, back-end primer (10μM)

[0069] 2~3μL, dNTP mixture (10μM) 1~3μL, 10×Ex Taq buffer (Mg 2+ ) 5 μL, Ex Taq polymerase 0.5 μL, the total system is 50 μL. The gene encoding Ag43 was obtained.

[0070] EcoRI an...

Embodiment 2

[0073] Use continuous overlap extension PCR to excise 53-137aa or 53-699aa of the Ag43 gene in the recombinant plasmid pET28a-Ag43 obtained in Example 1, target gene fragment 100ng, 10×QuickCut buffer 5 μL, QuickCut enzyme I 1-2 μL, QuickCut Enzyme II 1~2μL, the total system is 50μL. Insert the multiple cloning site MC for ligation; the reaction system is 3:1 mass ratio of enzyme-digested target gene fragment to enzyme-digested plasmid DNA, 10×T4 DNA ligation buffer 2-3 μL, T4 DNA ligase 2-3 μL, total system 20 μL. The surface display plasmids pET28a-Ag43-138 and pET28a-Ag43-700 were obtained.

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Abstract

The invention provides recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacteria and application thereof and belongs to the technical field of gene engineering. The recombinant plasmid pET28a-Ag43 is inserted into EcoRI and HindIII multiple cloning sites of plasmid pET28a from an auto-transport protein Ag43 encoding gene, and the auto-transport protein Ag43 encoding gene has a nucleotide sequence shown by SEQ ID No.1 in a sequence table. A heterologous protein gene segment can be quickly connected to the multiple cloning sites of the recombinant plasmid in a double enzyme digestion mode to construct the recombinant engineering bacteria, so that heterologous protein can be displayed and expressed on the surface of a cell, and wall breaking for releasing endoproteinase is avoided; meanwhile, zymoprotein is fixed to the surface of the cell, and convenience is brought to recycling the zymoprotein.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant plasmid pET28a-Ag43, a surface display plasmid, recombinant engineering bacteria and applications thereof. Background technique [0002] Cell surface display technology refers to the fusion expression of ankyrin and foreign protein, and the ability of ankyrin to bind to the outer membrane of the cell to carry the autotransporter to the surface of the cell. On the one hand, it overcomes the barrier caused by the cell membrane. The inaccessibility of substances and enzyme proteins improves the reaction efficiency of substrates and enzymes. On the other hand, cell surface display greatly simplifies the process of enzyme protein purification and recycling. Therefore, cell surface display can be widely used in the construction of protein screening libraries, antibody vaccine development, whole-cell catalysis and environmental repair. [0003] Howe...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/31C12N1/21C12R1/19
CPCC07K14/245C12N15/70
Inventor 敬科举朱先峰郭燕蓝凌雪萍
Owner XIAMEN UNIV
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