Gene engineering bacterium capable of performing high-efficiency expression on Alpha-elaterin-protein and construction method and application thereof

A technology of genetically engineered bacteria and Momordica charantin, applied in the field of bioengineering, can solve the problems of long cycle, cumbersome purification steps, unfavorable extraction, etc.

Inactive Publication Date: 2013-08-21
WUHAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

The traditional Alpha-mosantrin protein separation technology relies on the synthesis of ammonium sulfate precipitation, ion exchange chromatography, molecular sieve chromatography, affinity chromatography, gel filtration and other technologies, the purification steps are cumbersome, the cycle is long, and the cost is high. And this method is subject to the output of bitter melon seed and the restriction of season
In addition, the cumbersome purification steps easily lead to a large loss of protein, and also affect the enzyme activity of Alpha-mosantrin protein
The improved extraction method includes the combination of HPLC chromatography and ion exchange column chromatography, but the requirements for high-end HPLC equipment are relatively high
In 2007, Jesada Pitipanapong and others used a combination of HPLC pumps, high-temperature ovens, stainless steel extraction tubes, pressure gauges and pressure regulators to extract Momordica charantin at 400°C under high temperature and high pressure. Although a high extraction rate was obtained, it produced A large number of environmental pollutants, and more stringent requirements for instruments, is not conducive to large-scale extraction
Traditional protein extraction methods limit the large-scale application of Alpha-Mostein in scientific research and agriculture

Method used

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  • Gene engineering bacterium capable of performing high-efficiency expression on Alpha-elaterin-protein and construction method and application thereof
  • Gene engineering bacterium capable of performing high-efficiency expression on Alpha-elaterin-protein and construction method and application thereof
  • Gene engineering bacterium capable of performing high-efficiency expression on Alpha-elaterin-protein and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0048] A method for constructing a genetically engineered bacterium that efficiently expresses Alpha-mosantrin protein, the steps of which are as follows:

[0049] (1) Synthesis of primers for Alpha-charantin. Download the α-mosantin cDNA sequence uploaded by Ho et al. in 1991 from the NCBI database ( http: / / www.ncbi.nlm.nih.gov / nuccore / 19527 ), analyze its mature peptide fragments, and use Primer5.0 to design primers for amplifying Alpha-mosantrin: upstream primer MCF: Downstream primer MCR: The boxed ones are the restriction endonucleases EcoR I and Xho I respectively, and the upstream of the restriction sites are three protective bases of the restriction endonucleases, and the underlined and bolded sites represent the first codons and stop codons.

[0050] (2) Obtaining the mature peptide gene fragment of Alpha-charantin gene. 5μL PCR reaction system includes: 50ng genomic DNA, 1.5μL 10×pfu buffer, 0.4μM primer combination, 1.5mM MgCl 2 , 250 μl dNTPs, and 0.5U p...

Embodiment 2

[0055] Induction and purification of Alpha-charantin protein:

[0056] (1) Optimization of culture conditions for protein-induced expression. By setting different culture time (6, 8, 10, 12, 14, 18h), culture temperature after adding inducer (22°C, 25°C, 28°C, 30°C, 35°C, 37°C C), the concentration of the inducer IPTG (0.5, 0.75, 1.0, 1.25, 1.5mM), the OD value during induction (0.5, 0.6, 0.7, 0.8) and other culture conditions gradient, and collect the bacterial solution under different induction conditions. SDS-PAGE analysis of the expression of Alpha-mosantrin protein in bacteria cultured under various conditions to determine the optimal expression conditions. The optimal expression condition of the protein is to add IPTG to a final concentration of 0.75mM when the bacterium concentration, namely OD600, reaches 0.6, and then continue to culture the bacterium for 10h under the culture condition of 28°C.

[0057] (2) Protein separation and purification. Expand the cell cult...

Embodiment 3

[0060] The application of a protein produced by a genetically engineered bacterium highly expressing Alpha-mosantrin protein in antibacterial, the process is:

[0061] (1) Resuscitate the fungal species Fusarium solani (F.solani) and Fusarium oxysporum (F.oxysporum) on PDA slant medium for 48 hours;

[0062] (2) pick robust mycelium pieces from the edge of the slant culture medium to inoculate on the PDA plate culture medium and carry out subculture;

[0063] (3) When the plaque area occupies 50% of the plate, place filter paper sheets (1 cm in diameter) containing 0 μg, 0.1 μg, 0.2 μg, and 0.4 μg of Alpha-mosantrin protein at a position of 0.5 cm from the outer edge of the plaque, After 24 hours, the antibacterial phenomenon was observed, and it was found that the antibacterial zone gradually increased with the increase of protein concentration;

[0064] (4) Cultivate F.solani and F.oxysporum fungi on PDA medium for 5-7 days, then collect spores with sterile water and adjust...

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Abstract

The invention discloses a gene engineering bacterium capable of performing high-efficiency expression on an Alpha-elaterin-protein and a construction method and application thereof. A recombined strain, CCTCC NO.M2013174, can be obtained by converting a recombination plasmid pET-28a-aMC to Rosetta(DE3)pLys of Escherichiacoli. The strain can resist Kanamycin and Chloromycetin and can well grow in an LB liquid culture medium; and under a condition of inducing of a low-concentration isopropyl-beta-d-thiogalactoside (IPTG) inducing agent, a large amount of active proteins can be synthesized and accumulated in a cytoplasm. The gene engineering bacterium is high in repeatability, high in operability, low in cost and suitable for industrial production and application of the Alpha-elaterin-protein. The yield of the Alpha-elaterin-protein expressed by the engineering strain is 115 mg / L; the yield of the Alpha-elaterin-protein which is purified at downstream is 85 mg / L; the purity of the Alpha-elaterin-protein is 94.7 percent; and the recycling rate of the Alpha-elaterin-protein is 73.9 percent.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to a genetically engineered bacterium for efficiently expressing Alpha-mosantrin protein, and also relates to a method for constructing a genetically engineered bacterium for efficiently expressing Alpha-mosantrin protein and the genetically engineered bacterium use. It is not only suitable for the construction of various ribosome-inactivating protein engineering bacteria, but also conducive to the simplified and large-scale production of active ribosome-inactivating proteins. This method also laid a theoretical foundation for the antibacterial application of Alpha-mosantrin and other ribosome inactivating proteins in vitro. Background technique [0002] During the growth process, plants will be infected by various pathogens such as bacteria, fungi, and viruses in the environment. Fungal and bacterial diseases are the most serious types of plant diseases, ac...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P21/02C07K14/415C07K1/22A01N47/44A01P1/00A01P3/00C12R1/19
Inventor 丁毅王书珍
Owner WUHAN UNIV
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