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A kind of preparation method and application of recombinant protein swho1

A recombinant protein, HO1 technology, applied in the field of preparation of recombinant protein swHO1, to achieve the effect of improving serum total antioxidant capacity, increasing expression, and balancing oxidative stress

Active Publication Date: 2022-05-24
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whether the HO1 gene of rice field eel (Monopterus albus) can play a regulatory role in the oxidative stress induced by pathogenic bacteria and what effect it has on the expression of immune-related genes has not been reported.

Method used

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  • A kind of preparation method and application of recombinant protein swho1
  • A kind of preparation method and application of recombinant protein swho1
  • A kind of preparation method and application of recombinant protein swho1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Cloning of the eel HO1 gene

[0021] (1) Extract the total RNA of the eel liver according to the Trizol reagent instructions, use M-MLV reverse transcriptase to synthesize cDNA, and store the synthesized cDNA at -80°C for later use;

[0022] (2) According to the known HO1 gene sequence of fish, a pair of degenerate primers de-HO1-F (as shown in SEQ ID NO: 1, 5'-CACRGARCTBATGCTGAGCT-3') and de-HO1-R ( As shown in SEQ ID NO: 2, 5'-CTGSTGGCCCACGCKTACACC-3') is used to amplify the cDNA fragment of the eel HO1 gene obtained by the above-mentioned reverse transcription synthesis;

[0023] (3) After the amplification product is purified, it is connected to the pEASY-T1 vector, transformed into DH5a competent cells, and identified as a positive clone fragment;

[0024] (4) Gene-specific primers HO5GSP (as shown in SEQ ID NO: 3, 5'-ATCTACCAGGCCCTGGAGGAAGAGATGGACAGGAAC-3') and HO3GSP ​​(as shown in SEQ ID NO: 4, 5'-GGAGGTCAGGTCTTGGGTCGAATCGCTC-3') were designed accor...

Embodiment 2

[0026] Example 2 Construction of prokaryotic expression vector

[0027] According to the Trizol kit instructions, the total RNA from the liver tissue of the eel was extracted and the first strand of cDNA was synthesized by MMLV reverse transcriptase.

[0028] Using the full-length cDNA sequence of the eel HO1 gene prepared in Example 1 as a template, and re-ex-F and re-ex-R as primers, PCR amplification was performed. The PCR product and pET-28a(+) expression vector were double digested, recovered and purified, ligated and transformed into Escherichia coli DH5α, and the recombinant expression vector pET-HO1 was obtained.

[0029] Specifically, specific primers re-ex-F (as shown in SEQ ID NO: 6, 5'-CAAGGATCCATGGAAGCAGAGAAGAAAAC-3') and re-ex-R (as shown in SEQ ID NO: 7, 5'-CAA CTCGAG TAAAACGTAGATTCCCATA-3'), BamHI and XhoI restriction sites were added to the forward primer re-ex-F and reverse primer re-ex-R, respectively.

[0030] Using primers re-ex-F and re-ex-R to ampli...

Embodiment 3

[0032] Example 3 Induction expression and purification of recombinant protein pET-HO1

[0033] 1 ng of the recombinant expression vector pET-HO1 verified by sequencing in the above Example 1 was transformed into E.coli BL21 (DE3) bacteria, and cultured in LB medium containing 50 mg / L kanamycin to an OD value of about 0.6, then added IPTG (125μg·mL -1 ) were induced and cultured for 3 h. The bacterial cells were collected by centrifugation, sonicated, and then added with 5× loading buffer for 12% SDS-PAGE electrophoresis to detect the expression of the target protein.

[0034] The BL21(DE3) bacterial strain capable of expressing recombinant protein was inoculated into fresh LB medium containing 50 μg / mL kanamycin with 1% inoculation amount (mass volume ratio), and cultured at 200r / min at 37°C for 12-15h, Induced expression. Centrifuge the induced bacterial solution, collect the bacterial cells by centrifugation at 4°C, 10000 r·min-1 for 10 min, resuspend the pellet with PB...

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Abstract

The invention discloses a method for preparing the recombinant protein swHO1, extracting the total RNA of the liver tissue of rice field eel, and cloning the coding gene of rice field eel heme oxidase 1 into an expression vector through reverse transcription and PCR amplification to obtain the recombinant expression vector pET ‑HO1; transforming the recombinant expression vector pET‑HO1 into Escherichia coli BL21 (DE3) cells for expression and purification to obtain the recombinant protein swHO1. The application of a recombinant protein swHO1 in the regulation of oxidative stress induced by pathogenic bacteria and the expression regulation of immune-related genes in rice field eel. The invention constructs the prokaryotic expression vector of the eel heme oxidase 1 gene, induces the expression, and obtains the purified recombinant protein swHO1 by affinity chromatography. The rice field eel heme oxidase 1 prepared by the invention has the functions of improving the antioxidant capacity in the serum tissue treated with pathogenic bacteria and improving the expression of liver immune-related genes.

Description

technical field [0001] The invention belongs to the technical field of recombinant gene proteins, in particular to a preparation method and application of a recombinant protein swHO1. Background technique [0002] Heme oxidase is an important rate-limiting enzyme in the process of heme degradation in vivo, it can degrade heme into carbon monoxide (CO), biliverdin and Fe 2+ . These three products have antioxidant, anti-inflammatory, and autophagy-promoting and regulating cell apoptosis and proliferation effects in the body. It is currently known that mammalian heme oxidase has three main isozymes 1, 2 and 3. Whether the HO1 gene of yellow eel (Monopterus albus) can regulate the oxidative stress induced by pathogenic bacteria and what effect it has on the expression of immune-related genes has not been reported. SUMMARY OF THE INVENTION [0003] The purpose of the present invention is to provide a preparation method of recombinant protein swHO1, and the prepared recombina...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N15/66
CPCC12N9/0083C12N15/70C12N15/66C12Y114/99003
Inventor 李伟孙文秀臧彧伟
Owner YANGTZE UNIVERSITY
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