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Preparation method of human active granzyme A recombinant protein

A technology of active particles and recombinant proteins, applied in the fields of molecular biology and genetic engineering, to achieve the effects of simple operation, good enzymatic activity and easy purification

Inactive Publication Date: 2014-02-05
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of recent studies have found that the presence of granzyme A was detected in the serum of some healthy donors, and when inflammation occurred, higher levels of granzyme A could be detected in serum and other body fluids, indicating that granzyme A, in addition to In addition to its cytotoxic function, it may also play a role in promoting inflammation and degrading the extracellular matrix outside the cell, thereby allowing cytotoxic cells to approach target cells in the tissue, but the specific mechanism remains to be further studied

Method used

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  • Preparation method of human active granzyme A recombinant protein
  • Preparation method of human active granzyme A recombinant protein
  • Preparation method of human active granzyme A recombinant protein

Examples

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Effect test

Embodiment 1

[0052] This embodiment is the construction and identification of the recombinant plasmid pET24a(+)-aGzmA in the method of the present invention, and its process is as follows figure 1 As shown, the specific process is as follows:

[0053] 1. Obtaining the DNA Fragment of Human Active Granzyme A

[0054] According to the full-length human granzyme A sequence (CR456968.1) in GenBank, the upstream and downstream primers of human active granzyme A were designed, and the corresponding restriction endonuclease cutting sites were introduced, and the upstream primer sequence (SEQ ID NO.1) For: 5'-GGAATTC CATATG ATTATTGGAGGAAATGAAG-3' (the underlined part is the Nde I restriction site, 85-103) downstream primer sequence (SEQ ID NO.2) is: 5'-CG CTCGAG AACTGCTCCCTTGATAGT-3' (the underlined part is the Xho I restriction site, 769-786). Using the plasmid pET24a-GzmA containing the full-length gene sequence of human granzyme A as a template, the specific primers SEQ ID NO.1 and SEQ ID ...

Embodiment 2

[0060] In this example, the recombinant plasmid pET24a(+)-aGzmA in Example 1 was transformed into Escherichia coli BL21 to obtain engineering bacteria pET24a(+)-aGzmA / BL21, and the induced expression of the target protein and the identification of the expression product were carried out. The specific process is as follows :

[0061] 1. Obtainment of human active granzyme A expressing strain pET24a(+)-aGzmA / BL21

[0062] (1) Take 4 μl of the recombinant plasmid pET24a(+)-aGzmA and add it to 200 μl of ice-cooled E. coli BL21 competent, place on ice for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and then quickly cool on ice for 3 minutes. Add 600 μl of LB medium without antibiotics, mix well and place it at 37°C for 60 minutes with shaking, then take the bacterial solution and spread it on the kan + LB plate, put the plate upright in the incubator at 37°C for 30 minutes, after it is completely absorbed, culture it upside down for 12-16 hours until a single co...

Embodiment 3

[0070] The recombinant protein in Example 2 was separated and purified using a nickel affinity chromatography column, and the activity of the purified protein was determined. The specific process is as follows:

[0071] 1. Separation and purification of human active granzyme A recombinant protein

[0072] (1) Determination of expression form of recombinant protein

[0073] After the expression cells were resuspended in PBS, they were placed in a sonicator for sonication. The ultrasonic conditions are working for 3s, intermittent for 3s, 400W, and the total ultrasonic time is 1h. Add ice cubes every 30min and wait for a while to lower the temperature of the bacterial liquid and prevent carbonization. After crushing, centrifuge at 12000rpm at 4°C for 20min, take a certain amount of supernatant and precipitate for SDS-PAGE analysis, the results are as follows: Image 6 As shown, the human active granzyme A recombinant protein mainly exists in the precipitate (fifth lane), indic...

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Abstract

The invention discloses a preparation method of a human active granzyme A recombinant protein. PCR amplification is carried out to obtain a human active granzyme A gene, the human active granzyme A gene is inserted to corresponding enzyme sites of a prokaryotic expression vector pET24a(+) to construct a recombinant expression plasmid pET24a(+)-aGzmA, and the pET24a(+)-aGzmA is transferred to Escherichia coli BL21 to obtain an engineering bacterium pET24a(+)-aGzmA / BL21. The engineering bacterium induced by IPTG can express the human active granzyme A recombinant protein, and the recombinant protein is expressed in an inclusion body form. The recombinant protein having a highest purity reaching above 95% can be obtained by separating through using a nickel affinity chromatography column, and an activity determination result of the recombinant protein by a BLT substrate solution shows that the recombinant protein has a good enzymatic hydrolysis activity. The method has the characteristics of low cost, simple operation, high protein expression level and the like, and the prepared recombinant protein has a biological activity and can be easily purified.

Description

technical field [0001] The invention relates to the fields of molecular biology and genetic engineering, in particular to a preparation method of human active granzyme A recombinant protein. Background technique [0002] Granzyme A (GzmA) is a serine protease present in cytotoxic T lymphocytes (Cytotoxic T lymphocytes, CTL) and natural killer cells (Natural killer, NK). Granzyme A mainly induces a kind of caspases-independent apoptosis, which is released through the exocytosis of granules, enters the cytoplasm of target cells under the synergistic action of Perforin, and accumulates in the nucleus. The released deoxyribonuclease NM232H1 forms a single-strand DNA strand cut (gap) on the chromosomal DNA, causing DNA breaks and eventually causing apoptosis of the target cells. A large number of recent studies have found that the presence of granzyme A was detected in the serum of some healthy donors, and when inflammation occurred, higher levels of granzyme A could be detected...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N15/70C12N9/64
Inventor 江文正胡雪菲
Owner EAST CHINA NORMAL UNIV
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