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Penetratin-hSOD1 with transmembrane function as well as preparation method and application of Penetratin-hSOD1

A membrane-penetrating and functional technology, applied in the fields of molecular biology and cell biology, can solve problems such as complex extraction and purification processes, and achieve the effects of improving antioxidant capacity, good medicinal prospects, and large yields

Pending Publication Date: 2022-07-29
盛世泰研(广东)健康科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main sources of SOD1 are as follows: one is to extract directly from animals and plants, which is rich in sources, but the extraction and purification process is relatively complicated

Method used

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  • Penetratin-hSOD1 with transmembrane function as well as preparation method and application of Penetratin-hSOD1
  • Penetratin-hSOD1 with transmembrane function as well as preparation method and application of Penetratin-hSOD1
  • Penetratin-hSOD1 with transmembrane function as well as preparation method and application of Penetratin-hSOD1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, preparation of Penetratin-hSOD1 with transmembrane function.

[0043] The cDNA corresponding to SOD1 of LO2 was used as a template for PCR amplification to obtain the Penetratin-hSOD1 gene. PCR amplification conditions:

[0044]

[0045] The result is as figure 1 As shown (M: DNA marker, 5K; 1: Penetratin-hSOD1 gene), the Penetratin-hSOD1 gene fragment is 507 bp.

[0046] The obtained Penetratin-hSOD1 gene fragment was connected to the expression vector pET15b plasmid with the recombinase Exnase, and the cloned competent cell DH5α was transformed, cultured on a plate containing ampicillin, and the single clone was picked and sent for sequencing. The sequencing result is as follows: figure 2 shown.

[0047] Transform the correctly sequenced plasmid into the expression competent BL21 Escherichia coli, and culture it on a plate containing ampicillin: pick a single clone in 5 ml of LB medium containing 100 μg / ml ampicillin, and cultivate at 37 °C and 200 rp...

Embodiment 2

[0051] Example 2, preparation of Penetratin-hSOD1 with transmembrane function, the part not described in detail in this example is consistent with Example 1.

[0052] The Penetratin-hSOD1 gene was obtained by PCR amplification with HK-2 cDNA as the template, which was ligated to the expression vector pET15b with recombinase, and transformed into cloned competent cell DH5α. The single clone was cultured in LB at 37°C and 200rpm until the OD600 was 0.5, IPTG was added to the final concentration of 0.5mM, and the induction was continued for 16h at 18°C ​​and 180rpm. The bacterial liquid was collected for sonication, protein purification was carried out with Ni-NTA column, and protein desalting was carried out with ultrafiltration tube to obtain Penetratin-hSOD1 protein with higher purity.

Embodiment 3

[0053] Example 3, preparation of Penetratin-hSOD1 with transmembrane function, the part not described in detail in this example is consistent with Example 1.

[0054] The Penetratin-hSOD1 gene was obtained by PCR amplification using the LO2 cDNA as a template, which was ligated into the expression vector pET15b with recombinase, and transformed into a cloned competent cell DH5α. The plasmid with the correct sequencing was selected to transform into the expression competent Transetta, and a single clone was picked. The cells were cultured in LB at 37°C and 200 rpm until the OD600 was 1.0, IPTG was added to a final concentration of 0.5 mM, and the incubation was continued for 16 h at 18°C ​​and 180 rpm. The bacterial liquid was collected for sonication, protein purification was carried out with Ni-NTA column, and protein desalting was carried out with ultrafiltration tube to obtain Penetratin-hSOD1 protein with higher purity.

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Abstract

The invention provides Penetratin-hSOD1 with a transmembrane function and a preparation method and application thereof, and belongs to the field of molecule and cell biology, and the preparation method specifically comprises the following steps: taking cell cDNA as a template, carrying out PCR amplification to obtain a Penetratin-hSOD1 fragment, carrying out homologous recombination on the fragment to a pET15b plasmid, transforming the plasmid into escherichia coli BL21, carrying out induced expression, crushing thalli, and carrying out freeze drying to obtain the Penetratin-hSOD1 with a transmembrane function. And carrying out Ni-NTA column purification on the supernatant total protein, so as to obtain the high-efficiency soluble Penetratin-hSOD1. The Penetratin-hSOD1 obtained by the preparation method disclosed by the invention can be applied to the preparation of antioxidant drugs, and the Penetratin-hSOD1 can efficiently penetrate through a cell membrane by adding the cell-penetrating peptide, so that the Penetratin-hSOD1 has stronger cell antioxidant capacity compared with SOD1 which cannot penetrate through the cell membrane. According to the invention, the cell-penetrating peptide Penetratin and the SOD1 protein are recombined for the first time, and compared with other similar products, the protein has stronger cell-penetrating capability and higher stability.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and in particular relates to a recombinant protein Penetratin-hSOD1 with membrane-penetrating function, and also relates to a preparation method of the recombinant protein Penetratin-hSOD1. The recombinant protein Penetratin-hSOD1 is used in the preparation of antioxidant drugs Applications. Background technique [0002] Superoxide dismutases (SODs) are an important class of antioxidant enzymes in organisms, which can remove excess reactive oxygen species in cells due to oxidative stress. According to the localization of SODs in cells, they can be divided into three types (SOD1, SOD2, SOD3), among which SOD1 is the most abundant in cells, distributed in the cytoplasm and mitochondria, accounting for 90% of the total SOD (Zelko, I.N., T.J.Mariani, and R.J.Folz, Superoxide dismutase multigene family: A comparison of the CuZn-SOD(SOD1), Mn-SOD(SOD2), and EC-SOD(SOD3) gene structur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70A61K38/44A61P39/06
CPCC12N9/0089C12N15/70C12Y115/01001A61K38/446A61P39/06C07K2319/10
Inventor 江仁望王晓璐陈健洲
Owner 盛世泰研(广东)健康科技有限公司
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