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Preparation and application of human galectin-9 deletant for enhancement of immune response

A galectin and immune-enhancing technology, applied in the field of protein preparation, can solve the problems of inconvenient application of two-way regulation, and achieve the effect of improving immunity

Inactive Publication Date: 2010-08-04
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The two-way regulation of Gal-9 molecule brings inconvenience to the application of this molecule

Method used

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  • Preparation and application of human galectin-9 deletant for enhancement of immune response
  • Preparation and application of human galectin-9 deletant for enhancement of immune response
  • Preparation and application of human galectin-9 deletant for enhancement of immune response

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 The acquisition of target gene and the construction of prokaryotic expression system

[0015] 1) Gene transfer

[0016] According to the gene sequence and the restriction site of the PET-32a vector, Sac I and Xho I double restriction sites were used to capture PCR Gal-9C and Gal-9, and the gene primers were designed as follows:

[0017] Gal-9CPs: 5'-ccggaattcttcatcaccacc-3' (SEQ ID NO: 1)

[0018] Gal-9C Pa: 5'-ccgctcgagctatgtctgcacatggg-3' (SEQ ID NO: 2)

[0019] Gal-9 Ps: 5'-ccggaattcgccttcagcggttcccagg-3' (SEQ ID NO: 3)

[0020] Gal-9 Pa: 5'-tccagctgacccatgtgcagacataggg-3' (SEQ ID NO: 4)

[0021] After the primers were synthesized, the cDNA generated by reverse transcription of total RNA extracted from human peripheral blood mononuclear cells was used as a template to capture the above two genes (SEQ ID NO: 5 and SEQ ID NO: 6). The PCR cycle conditions were set as 94°C for 5min; 94°C for 30s, 60°C for 30s, 72°C for 50s, 35 cycles; 72°C for 7min, 4°C hol...

Embodiment 2

[0024] Expression and purification of the protein of embodiment 2

[0025] 1) Culture of bacteria

[0026] Pick a single clone, inoculate it into a large amount of LB liquid medium after activation, place it in a shaker at 37°C for 3 or 4 hours, and induce the expression of the target protein until the OD value of the medium is about 0.6. The induction conditions were Gal-9: room temperature (25° C.), 0.05 mM IPTG, 1 L LB liquid medium; Gal-9C: room temperature (25° C.), 0.1 mM IPTG.

[0027] 2) Protein separation and purification

[0028]Induce for about 12 hours, collect the bacteria (7000rpm, 5mins), resuspend the bacteria expressing the two proteins in PBS, freeze and thaw two or three times in liquid nitrogen, and then add a small amount of lysozyme (4°C, 1 hour) try to lyse the bacteria, and finally ultrasonically disrupt (10 minutes each time, twice in total). Centrifuge (10000rpm, 10mins) to take the supernatant and filter it, then apply it to a nickel column for pu...

Embodiment 3

[0029] Example 3 Protein function verification

[0030] 1) Gal-9 and Gal-9C pro-inflammatory detection

[0031] Human DC cells and macrophages were respectively incubated with samples containing Gal-9 protein and samples containing Gal-9C protein at 37°C for 12-24 hours at different concentrations, and the cell culture supernatant was collected, and TNF-a was detected by ELISA Secretion. ELISA results such as figure 1 and figure 2 : Both Gal-9 and Gal-9C can promote the secretion of inflammatory factors.

[0032] 2) Detection of T cell apoptosis induced by Gal-9 and Gal-9C

[0033] After co-incubating human lymphocytes with Gal-9 and Gal-9C for 4 hours, the cells were stained with Annexin V-PE and CD4-FITC to detect CD4 + Apoptosis of T cells. The flow detection results are as follows: image 3 : Gal-9 significantly induces CD4 + T cell apoptosis.

[0034] The above two results show that Gal-9 can both enhance immunity and suppress immunity; while Gal-9C only retains...

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Abstract

The invention discloses a preparation method of human galectin-9 deletant for enhancement of immune response, which adopts an expression vector with a protein DNA sequence and a host cell transformed with the expression vector. The invention also provides application of the human galectin-9 deletant in improvement of cellular immunity. The protein has the function of promoting the activation of DC cells and macrophages, the demonstration of the function of the protein is the enhancement of the cellular immune response, and the immunity is suppressed in case of lack of the human galectin-9 holoprotein.

Description

Technical field: [0001] The invention relates to a protein preparation method and application. Specifically, the protein is a deletion body of human galectin-9, which is composed of 129 amino acid sequences at the C-terminal of human galectin-9. terminal gene, construct its prokaryotic expression system, separate and purify and prepare. The protein has the function of promoting the activation of DC cells and macrophages, and is characterized by enhancing cellular immune response, but lacks the immunosuppressive effect of human galectin-9 whole protein. The protein can be applied in the production of antiviral and antiinfective medicines. Background technique: [0002] Galectin exists in various organisms, not only in the nucleus, but also in the cytoplasm and even the extracellular matrix. Human galectin-9 (Gal-9), as a member of the family, not only induces eosinophil aggregation and activation, but also mediates cell differentiation, apoptosis, adhesion, intercellular a...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N1/19C12N1/21A61K38/17A61P37/04C12R1/19C12R1/645
Inventor 黎燕沈倍奋韩根成李育蓉冯健男
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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