Recombinant human TSG-6 protein, preparation method thereof and application of recombinant human TSG-6 protein in treatment of acute inflammatory diseases

A technology of TSG-6 and protein, applied in the biological field, can solve the problems of severe degree and harmfulness to the body, and achieve good therapeutic effect

Inactive Publication Date: 2018-09-14
ANHUI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the inflammatory response lasts for a long time and is severe, it will be harmful to the body.

Method used

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  • Recombinant human TSG-6 protein, preparation method thereof and application of recombinant human TSG-6 protein in treatment of acute inflammatory diseases
  • Recombinant human TSG-6 protein, preparation method thereof and application of recombinant human TSG-6 protein in treatment of acute inflammatory diseases
  • Recombinant human TSG-6 protein, preparation method thereof and application of recombinant human TSG-6 protein in treatment of acute inflammatory diseases

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 TSG-6 gene cloning, expression

[0033] 1. Inoculate human embryonic lung fibroblasts (MRC-5 cells) in a 10cm cell culture dish, inoculate human cytomegalovirus (HCMV) with Moi=1, and extract total RNA on the third day after inoculating the virus;

[0034] 2. Use 500ng RNA as a template to synthesize cDNA by reverse transcription, and use self-designed specific primers to PCR amplify the full-length coding sequence of TSG-6. The specific primer sequences used are as follows:

[0035] Upstream primer: CATATGATCATCTTAATTTACTTATTTCTCTTGCTATGG

[0036] Downstream primer: CTCGAGTAAGTGGCTAAATCTTCCAGCT

[0037] 3. Connect the amplified TSG-6 coding gene to the prokaryotic expression vector pET30a to construct the recombinant expression vector pET30a-TSG-6. After sequencing and identification, transform Escherichia coli BL21(DE3) to obtain the recombinant expression strain pET30a-TSG-6- BL21;

[0038] 4. The overnight cultured recombinant bacteria were inoculate...

Embodiment 2

[0042] Purification of embodiment two recombinant proteins

[0043] 1. Centrifuge the enlarged cultured TSG-6 recombinant expression bacteria at 12000r / min for 10min, collect the bacteria, resuspend in PBS solution, and sonicate;

[0044] 2. Centrifuge the completely broken bacterial liquid at 12000r / min for 15min to collect the precipitate;

[0045] 3. Washing: Wash the inclusion body twice with Buffer A (50mM Tris, 100mM NaCl, 2mM urea, 0.1% EDTA, pH8.0), 3h each time; centrifuge at 12000r / min for 15min;

[0046] Denaturation: The precipitate after centrifugation was dissolved in Buffer B (50mM Tris, 100mMNaCl, 8M urea, 1% β-mercaptoethanol, pH8.0) at a ratio of 1g:20mL. After it was completely dissolved, centrifuge at 12000r / min for 15min. Discard the precipitate to obtain protein denaturation solution;

[0047]Renaturation: Dilute the resulting denaturing solution with Buffer C (50mM Tris, 100mM NaCl, 1% β-mercaptoethanol, pH8.0) to a volume ratio of 1:2, and then dilute...

Embodiment 3

[0051] Example 3 TSG-6 recombinant protein is used to treat acute pneumonia in mice caused by HCMV infection

[0052] 1. Proliferation of HCMV

[0053] Human embryonic lung fibroblast MRC-5 1×10 5 / mL inoculated in 10cm cell culture dishes, 10mL per dish. After the cells adhered to the wall, HCMV virus was inoculated according to Moi=0.02. After incubation at 37°C for 2 h, the MEM medium containing 10% fetal bovine serum was replaced. On the third day after the virus inoculation, the MEM medium containing 3% fetal bovine serum was replaced and the culture was continued for 5-7 days. The virus-containing supernatant was harvested when the cytopathic effect of the virus reached "++++". After centrifugation at 2000r / min for 20min, the virus-containing supernatant was collected, and the number of infectious virus particles was determined by plaque formation assay.

[0054] 2. Establishment of acute pneumonia model in mice infected with HCMV

[0055] BALB / c mice of 6 to 8 wee...

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Abstract

The invention discloses recombinant human TSG-6 protein, a preparation method thereof and application of the recombinant human TSG-6 protein in the treatment of acute inflammatory diseases. The aminoacid sequence of the recombinant human TSG-6 protein is as shown in SEQ ID No. 1, and the encoding gene nucleotide sequence of the recombinant human TSG-6 protein is as shown in SEQ ID No. 2. The recombinant human TSG-6 protein and the preparation method and application thereof have the advantages that a prokaryotic expression vector pET30a is utilized to build an Escherichia coli BL21(DE3) host strain capable of expressing the recombinant human TSG-6 protein, the strain is subjected to multiplication culture, isopropyl-beta-D-isopropylthiogalactoside induced expression and centrifuging are performed to obtain a thallus, the thallus is split and then inclusion body precipitate is collected centrifugally, and denaturation, renaturation and molecular-sieve purification are performed to obtain the recombinant human TSG-6 protein; the recombinant human TSG-6 protein has a good curative effect on the acute pneumonia, caused by HCMV virus infection, of mice and provides a new thought for thetreatment of the acute inflammatory diseases caused by virus infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant human TSG-6 protein, a preparation method and its application, in particular to the preparation of the recombinant human TSG-6 protein by prokaryotic genetically engineered bacteria and its application in treating inflammatory disease animal models. Background technique [0002] TSG-6 (tumor necrosis factor alpha stimulate gene 6), also known as tumor necrosis factor alpha stimulate gene 6, is a cytokine. Lee et al. discovered the TSG-6 gene for the first time when they screened the cDNA expression library of human diploid FS-4 fibroblasts stimulated by TNF-α (tumor necrosis factoralpha) in the 1990s. The TSG-6 gene exists in most mammalian genomes, and its amino acid sequence has high homology among different species. TSG-6 protein is mainly expressed in chondrocytes, synoviocytes, monocytes, smooth muscle cells, fibroblasts and stem cells with differentiation potential...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70A61K38/17A61P31/22
CPCA61K38/00A61P31/22C07K14/4718C12N15/70
Inventor 王明丽张文昌
Owner ANHUI MEDICAL UNIV
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