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PPK2 protein, and application of PPK2 protein as polyacrylamide gel electrophoresis 35kd standard substance

A protein and gene-encoding technology, applied in the field of bioengineering, can solve problems that have not been reported, and achieve the effect of not easy to degrade, long half-life, and clear bands

Inactive Publication Date: 2020-03-24
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 of PPK2 protein as polyacrylamide gel electrophoresis 35kd standard substance
  • PPK2 protein, and application of PPK2 protein as polyacrylamide gel electrophoresis 35kd standard substance
  • PPK2 protein, and application of PPK2 protein as polyacrylamide gel electrophoresis 35kd standard substance

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_015420983.1), 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] The recombinant expression plasmid PPK2 / pET30a synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. was digested with restriction endonucleases NdeΙ and HindⅢ to carry out the identification of the whole plasmid digestion, and the results of DNA agarose gel electrophoresis experiments should have A 4216bp band and a 1964bp 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 correctly above was transformed into Escherichia coli BL21 (DE3) competent cells, and then spread evenly on the LB plate (containing 50 μg / ml kanamycin), and then inverted at 37 °C incubator overnight. Select a single clone from the transformed plate, inoculate it into 5ml of LB medium (containing 50μg / ml kanamycin), and cultivate it in a constant temperature shaker at 37°C and 220r / min until the OD600 value is 0.5- At 0.8, add IPTG to the test tube culture solution to make the final concentration 0.2mM / L, and then place them at 16°C, 25°C, and 37°C to induce expression. Centrifuge the induced culture medium at 12000rpm for 5min, remove the supernatant, add 50mM / L Tris-HCl (pH8.5) buffer to resuspend the pellet, and finally add SDS-PAGE loading buffer and heat the sample at 100°C for 10min , and then centrifuged to take the supernatant for SDS...

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Abstract

The invention discloses a PPK2 protein, and an application of the PPK2 protein as a polyacrylamide gel electrophoresis 35kd standard substance. A ppk2 gene is inserted into a prokaryotic expression vector pET30a by using NdeI and Hind III, the obtained recombinant expression vector is transferred into an Escherichia coli BL21 (DE3) strain through a physical method, IPTG is used to induce trial expression of the target protein PPK2 at 37 DEG C, 25 DEG C and 16 DEG C, and the obtained expression product is identified and analyzed through SDS-PAGE electrophoresis and Western blotting; and finally, amplification culture is carried out by utilizing 3 L of an expression bacterial liquid, and the expression product is separated and purified through a Ni-IDA affinity chromatographic column, an anion exchange chromatographic column and a gel filtration chromatographic column in sequence. Results show that a large intestine prokaryotic expression system can be stably and efficiently expressed under the induction of the IPTG with the temperature of 16 DEG C and the final concentration of 0.2 mM.L<-1>, and the PPK2 protein has the advantages of specific charged property, existence in a monomerform in a solution, stable properties, non-degradability and long half-life period, and has an application prospect as the polyacrylamide gel electrophoresis 35 kd standard substance.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a PPK2 protein and its application in polyacrylamide gel electrophoresis 35kd standard product. Background technique [0002] The main function of the protein standard is to indicate the molecular weight of the protein band on the polyacrylamide gel electrophoresis 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 blot or the degree of electrophoresis of proteins 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 other metazoans, s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/70C12N1/21G01N33/68C12R1/19
CPCC12N9/1229C12Y207/04001C12N15/70G01N33/68
Inventor 闫东科宫艳超吕平许凤霞李陇梅李冬
Owner TIANJIN VOCATIONAL INST
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