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pEX-MBP recombinant plasmid for DisA expression

A recombinant plasmid and plasmid technology, applied in the field of genetic engineering, can solve the problems of high cost, high protein preparation cost, poor solubility of DisA protein, etc., and achieve the effect of high purity, low cost and good applicability

Inactive Publication Date: 2015-01-21
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The system is induced by arabinose (Khlebnikov A, Risa O, Skaug T. et al.; Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture, J Bacteriol, 2000, 182(24): 7029~7034), the first problem is that arabinose cannot diffuse freely through the cell membrane, and secondly, although the price of arabinose is cheaper than IPTG, if a large amount of protein is to be expressed, the cost will still be high
[0007] In the prior art, the traditional T7 expression system is still used for the expression of DisA protein, that is, the DisA gene is transferred into the E. coli BL21 expression strain through the pET28α(+) vector Among them, the DisA protein prepared by this method has poor solubility, and IPTG is used as an inducer when inducing expression, which has the disadvantages of high protein preparation cost and inability to produce in large quantities. Therefore, it is urgent to develop an expression method suitable for large-scale expression of DisA protein. system

Method used

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  • pEX-MBP recombinant plasmid for DisA expression
  • pEX-MBP recombinant plasmid for DisA expression
  • pEX-MBP recombinant plasmid for DisA expression

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Experimental program
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Embodiment 1

[0038] The pEX-MBP recombinant plasmid used for DisA protein expression, which is transformed on the basis of pBbB3a-GFP, first according to the Quik Change Site-Directed Mutagenesis Kit (Agilent Technologies, 200518) kit instructions, pBbB3a-GFP Mutate the SspI points at 279 and 5099 above, and mutate AAT / ATT to TAT / ATT. After the mutation, the vector is tentatively named pBbB3a-GFP-M, and then pBbB3a-GFP-M is digested with NdeI and BamHI. The His6-MBP-TEV-LIC sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the synthesized sequence was ligated into the pMD19-T plasmid vector. When ligated with pBbB3a-GFP-M, the pMD19-T-His6-MBP-TEV- Double enzyme digestion of LIC plasmid, and then ligation with pBbB3a-GFP-M after double enzyme digestion can obtain the pEX-MBP recombinant plasmid used for the expression of DisA protein in the present invention, named pEX-MBP, wherein His6 represents 6 histidines Label, MBP (Maltose-binding protein) indicates maltase-b...

Embodiment 2

[0072] The pEX-MBP recombinant plasmid used for the expression of DisA protein prepared in Example 1 is used to construct pEX-MBP-DisA, and the construction process is as follows: Figure 4 As shown, and then induce the expression of DisA protein, the specific process is briefly described as follows:

[0073]First PCR clones the DisA gene, then connects the DisA gene to the pEX-MBP recombinant plasmid prepared in Example 1 by a non-enzymatic ligation method, constructs the pEX-MBP-DisA recombinant plasmid expression vector, and transforms the vector into Escherichia coli BL21 Induced expression was carried out, and sodium propionate was used as an inducer during the induced expression. After the expression was completed, the DisA protein could be obtained by crushing, centrifuging, and purifying.

[0074] The detailed preparation steps are briefly introduced as follows:

[0075] (1) PCR cloning of the DisA gene

[0076] ① Design and synthesis of primers

[0077] According t...

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Abstract

The invention belongs to the technical field of genetic engineering, and relates to a pEX-MBP recombinant plasmid for DisA expression and preparation thereof. The base sequence of the pEX-MBP recombinant plasmid is shown in a sequence table. The recombinant plasmid is prepared by transforming on the basis of pBbB3a-GFP. When the recombinant plasmid is used for preparing a soluble DisA protein, sodium propionate is taken as an inducer. The pEX-MBP recombinant plasmid provided by the invention is specifically applied to the expression and purification of a DisA protein, and compared with a traditional recombinant plasmid expression body constructed by using a pET28 alpha (+) vector, the solubility of the expressed DisA protein is high; and a constructed expression system takes sodium propionate as an inducer which is low in cost and has no toxicity to bacteria, therefore, the expression system is suitable for expressing a DisA protein, and meanwhile, the expressed protein can specifically cut off relevant tag proteins, and the purity of an obtained DisA protein is high.

Description

Technical field [0001] The invention is a genetic engineering technology field involving a DISA (DNA Integrity Scanning Protein A, DNA damage repair protein) expression. Background technique [0002] DISA (DNA Integrity Scanning Protein A) is the abbreviation of DNA damage repair protein. It can find the body's DNA damage and repair it to maintain the integrity of the DNA.Studies have found that it has the activity of DiaDenylate Cyclase (DAC), which can catalyze two ATPs to generate cyclistotura nucleotide C-DI-AMP.Not only is it closely related to the regulation of the cell form and the synthesis of the cell wall, but it is also related to the pathogenicity of pathogenic bacteria and the resistance of bacteria. Therefore, C-DI-AMP also has good practical value in immunology. [0003] At present, in the protein expression system of E. coli, the T7 expression system is widely used (Kang Y, Son MS, Hoang TT.; One Step Engineering of T7-EXPRESSIONS for Protein Production: Increasin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N9/88
Inventor 杨国宇郭准王江王月影郭豫杰李和平朱河水钟凯鲁维飞韩立强耿娟
Owner HENAN AGRICULTURAL UNIVERSITY