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PPK2 protein and application thereof as 30kd standard substance for polyacrylamide gel electrophoresis

A protein and gene-encoding technology, applied in the field of bioengineering, can solve problems such as unreported, and achieve the effects of non-degradable, long half-life and stable properties

Inactive Publication Date: 2020-06-05
TIANJIN VOCATIONAL INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of PPK2 protein as a standard for polyacrylamide gel electrophoresis

Method used

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  • PPK2 protein and application thereof as 30kd standard substance for polyacrylamide gel electrophoresis
  • PPK2 protein and application thereof as 30kd standard substance for polyacrylamide gel electrophoresis
  • PPK2 protein and application thereof as 30kd standard substance for polyacrylamide gel electrophoresis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Bioinformatics prediction

[0032] After obtaining the amino acid sequence of the PPK2 protein from the NCBI database (WP_AGG33389.1), the secondary structure of the PPK2 protein was predicted using the PSIPRED website (http: / / web.expasy.org / protparam); using the ExPASy website (http: / / bioinf. cs.ucl.ac.uk / psipred / ) to predict basic physicochemical properties of PPK2.

Embodiment 2

[0033] Example 2: Whole plasmid digestion and sequencing

[0034] Recombinant expression plasmid PPK2 / pET30a synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. was digested with restriction endonucleases NdeΙ and HindⅢ to carry out double-enzyme digestion identification of the plasmid, and the results of DNA agarose gel electrophoresis experiments should have A 5250bp band and a 798bp band; at the same time, the PPK2 / pET30a recombinant expression plasmid was handed over to Jinweizhi Biotechnology Co., Ltd. to complete the sequencing of the target gene PPK2. Finally, confirm the accuracy of the recombinant expression vector.

Embodiment 3

[0035] Example 3: PPK2 protein test expression and identification

[0036] The recombinant expression plasmid PPK2 / pET30a constructed above was transformed into Escherichia coli BL21 (DE3) competent cells, and then spread evenly on LB plates (containing 50 μg·mL -1 kanamycin), and then placed in a 37°C incubator overnight. Pick a single clone from the transformed plate and inoculate it into 4ml of LB medium (containing 50μg·mL -1 Kanamycin), cultivated in a constant temperature shaker at 37°C, 220r / min, until OD 600 When the value is 0.5-0.8, add IPTG to the test tube culture solution to make the final concentration 0.2mM·L -1 , and then placed at 16°C, 25°C, and 37°C to induce expression. Centrifuge the induced culture medium at 12,000 rpm for 5 minutes, remove the supernatant, add PBS buffer to resuspend the pellet, and finally add SDS-PAGE loading buffer to heat the sample at 100°C for 10 minutes, then centrifuge to take the supernatant for SDS-PAGE Electrophoresis. Ba...

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Abstract

The invention discloses a PPK2 protein and application thereof as a 30kd standard substance for polyacrylamide gel electrophoresis. Ppk2 genes are inserted into a prokaryotic expression vector pET30aby virtue of NdeI and HindIII, a recombinant expression vector is transferred into an escherichia coli BL21(DE3) strain by virtue of a physical method, a target protein PPK2 is induced to realize trial expression at 37 DEG C, 25 DEG C and 16 DEG C by virtue of IPTG, and an expression product is authenticated and expressed by virtue of SDS-PAGE electrophoresis and Western Blotting; and finally, amplification culture is carried out by virtue of 3.2L of expression bacteria liquid, and the expression product is sequentially separated and purified by virtue of Ni-IDA affinity chromatography, cation-exchange chromatography and gel filtration chromatography. A result shows that a large intestine prokaryotic expression system can be induced to realize stable and efficient expression by virtue of the IPTG with a final concentration of 0.2mM.L<-1> at 16 DEG C, the PPK2 protein has an exclusive charging property, stable performance and a long half-life period, exists in a solution in a monomer manner, is unlikely to degrade and has an application prospect as the 30kd standard substance for the polyacrylamide gel electrophoresis.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a PPK2 protein and its application in polyacrylamide gel electrophoresis 30kd standard product. Background technique [0002] The main function of the protein standard is to indicate the molecular weight corresponding to the protein band on the polyacrylamide gel electrophoresis lane of the protein. It is a positive control. Only when the standard is accurate can the experimental results be convincing. In addition, protein standards can also indicate the success of membrane transfer during Western Blotting or the degree of protein electrophoresis on the gel, so choosing the correct protein standard is one of the necessary conditions for the success of molecular biology experiments. [0003] Polyphosphate kinase 2 (Polyphosphate kinase 2, PPK2) plays an important role in the pathogenicity of many pathogenic bacteria, but no ppk2 gene has been found in humans and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70G01N27/447G01N30/02
CPCC12N9/12C12Y207/04001C12N15/70G01N30/02G01N27/447
Inventor 闫东科宫艳超许凤霞李陇梅吕平李冬
Owner TIANJIN VOCATIONAL INST
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