Bacillus cereus NiR (nitrite reductase), gene and application

A technology of Bacillus cereus and nitrite, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of difficult purification work and low content of target protein

Inactive Publication Date: 2015-11-18
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because there are too many types of proteins in the crude enzyme solution, but

Method used

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  • Bacillus cereus NiR (nitrite reductase), gene and application
  • Bacillus cereus NiR (nitrite reductase), gene and application
  • Bacillus cereus NiR (nitrite reductase), gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The clone expression of NiR gene in bacillus cereus LJ01 comprises the steps:

[0036] according to The DNA sequence in the design of the PCR primers of the NiR gene: the upstream primer is M1: 5'-GATGCCATGGATGAGTTATGAAAAAGTAT-3' (NcoI), such as SEQ ID NO.2, the downstream primer is M2: 5'-ACGCGTCGACCTAAGACGCTATTACTTCT-3' (SalI), Such as SEQIDNO.3, and artificially synthesize this pair of upstream and downstream primers;

[0037] According to the steps of the instructions in the kit (bacteria genomic DNA small purification kit TaKaRaMiniBESTBacterialGenomicDNAExtractionKitVer.2.0, the product of TakaraBiotechnology company, purchased from Guangzhou Ruizhen Biotechnology Co., Ltd.), the extraction of genomic DNA in Bacillus cereus, NiR Cloning of genes, recovery of PCR target fragments.

[0038] Using the extracted Bacillus cereus genomic DNA as a template, PCR method was used to amplify the target gene fragment. The PCR reaction system was: 5 μL 10×buffer; 4 μL dNTP;...

Embodiment 2

[0046] The recombinant E.coliBL21 obtained in Example 1 was used to induce the expression of Bacillus cereus NiR, specifically as follows: the positive clone strain obtained in Example 1 was plated on the LB solid medium (containing 50 μg / mL ampicillin), Cultivate overnight in a 37°C incubator, then pick a single colony from the solid medium (containing 50 μg / mL ampicillin) and inoculate it into a liquid medium (containing 50 μg / mL ampicillin), and culture overnight at 37°C and 180 rpm ; Then inoculate LB liquid medium (containing 50 μg / mL ampicillin) with volume percentage 2% inoculum, cultivate to logarithmic growth phase (OD) under 37 ℃ and 180rpm 600 =0.6), adding IPTG with a final concentration of 1mmol / L for induction, after induction cultivation at 30°C and 180rpm for 4-5h, the culture solution was obtained, the culture solution thalline was collected by centrifugation, and NiR was extracted. The specific steps are as follows:

[0047] Centrifuge the culture solution at...

Embodiment 3

[0050] Utilize the crude protein solution induced and expressed by recombinant Escherichia coli obtained in Example 2 to carry out affinity chromatography purification to obtain high-purity NiR. Equilibrate the Ni column with phosphate buffer. After loading the sample, use the same buffer to elute the impurity protein. After washing to the baseline, use the phosphate buffer containing 500mM imidazole to elute to obtain the recombinant protein. The yield is 1L fermentation 20-30 mg of recombinant protein can be obtained in the solution. The eluted protein was concentrated by polyethylene glycol 20000 and then detected by SDS-PAGE. The results are shown in Figure 6 , where M is the standard, band 1 is the protein eluted with a sample volume of 30mL, and band 2 is the protein eluted with a sample volume of 70mL. Comparing band 1 and band 2, the arrow indicates about 20KDa is the target protein NiR, and the bands 3, 4, and 5 indicate Figure 5 The band indicated by the arrow 3 ...

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Abstract

The invention discloses a Bacillus cereus NiR (nitrite reductase), a gene and an application. The NiR gene in Bacillus cereus LJ01 is cloned and induced for expression in Escherichia coli with the PCR (polymerase chain reaction) technology, cells of blank-plasmid-containing BL21 subjected to IPTG (isopropyl beta-D-thiogalactopyranoside) induction, recombinant-plasmid-containing BL21 subjected to induction without IPTG and recombinant-plasmid-containing BL21 subjected to IPTG induction are subjected to ultrasonic crushing for extraction of crude enzymes, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is performed on the crude enzymes for detection of recombinant protein expression, high-purity recombinant protein is obtained through affinity chromatography purification, and the problem about separation of NiR with higher purity and content from the Bacillus cereus is solved. A large amount of NiR recombinant protein can be used for degrading nitrite in food, non-acid environments and aquatic water.

Description

technical field [0001] The present invention relates to the cloning and expression of a Bacillus cereus nitrite reductase gene and the nitrite reductase (NiR) with high purity obtained through mass expression and high-efficiency affinity chromatography purification method, so as to inhibit the recombinant protein nature research. Background technique [0002] Nitrite is a potential carcinogen. There are two main reasons: (1) Large intake of nitrite is harmful to the human body. The acute poisoning effect of nitrite is to cause Methemoglobin, which can produce common Symptoms of acute nitrite poisoning, mainly manifested as tissue hypoxia, cyanosis, accompanied by changes in the color of lips, nails and skin; (2) Nitrite can also cause chronic harm to humans or animals. Under certain conditions, it can react with amines, the decomposition products of proteins in food, to generate N-nitroso compounds. At present, more than 100 such compounds have been synthesized, and about 8...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/70A23L1/015C02F3/34C02F101/16A23L5/20
Inventor 刘冬梅罗彤晖杨丹霞陈舒然黄智斌
Owner SOUTH CHINA UNIV OF TECH
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