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Preparation method of human LIGHT-Fc fusion protein

A fusion protein, pks-light-fc technology, applied in the expression of LIGHT-Fc fusion protein in Pichia pastoris, and the construction of recombinant plasmid LIGHT-Fc-pPIC9K, to achieve easy separation and purification, good stability and simple operation Effect

Inactive Publication Date: 2011-03-30
EAST CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the expression of human LIGHT protein in prokaryotic cells and mammalian cells has been reported, but no one has tried it in yeast

Method used

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  • Preparation method of human LIGHT-Fc fusion protein
  • Preparation method of human LIGHT-Fc fusion protein
  • Preparation method of human LIGHT-Fc fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The flow chart of the construction of the recombinant plasmid LIGHT-Fc-pPIC9K is as follows: figure 1 As shown, the specific steps are as follows: 1. Design the following primers according to the human LIGHT gene sequence published by Genebank:

[0038] Primer 1: 5′-CCG GAATTC CAGCTGCACTGGCGTCTAGG-3' (the underlined part is the EcoR I restriction site, 178-197)

[0039] Primer 2: 5′-CGC GGATCC CACCATGAAAGCCCCGAAGT-3' (the underlined part is the BamH I restriction site, 701-720)

[0040] The pET32a-LIGHT recombinant plasmid was used as a template, and Primer 1 and Primer 2 were used as upstream and downstream primers for PCR reaction.

[0041] Reaction program: 95°C for 2min, 94°C for 30s, 55°C for 30s, 72°C for 1min, after 30 cycles, continue to extend at 72°C for 10min.

[0042] After the PCR product was identified by 1% agarose gel electrophoresis, it was recovered and purified according to the instructions of the DNA gel recovery kit to obtain a PCR product wit...

Embodiment 2

[0045] Transform the recombinant plasmid constructed in Example 1 into Pichia pastoris competent and screen multi-copy transformants. The specific steps are as follows:

[0046] 1. Preparation of Competent Pichia pastoris GS115

[0047] (1) Take the frozen GS115 yeast strain, streak it on a YPD plate, and culture it statically at 30°C for 36-48 hours until it grows a monoclonal colony.

[0048] (2) Pick a single clone colony and inoculate it into 5mL of YPD medium, culture at 30°C with shaking at 250rpm for 12-16h.

[0049] (3) Transfer 0.2 mL of bacterial liquid to 100 mL of YPD medium, culture at 30° C., 250 rpm for 12-16 hours with shaking until OD600 = 1.3-1.5.

[0050] (4) Centrifuge at 1500 rpm for 5 minutes at 4°C, discard the supernatant, and add 100 mL of ice-cold sterile double-distilled water to resuspend the pellet.

[0051] (5) Centrifuge at 4°C and 1500 rpm for 5 minutes, discard the supernatant, and add 50 mL of ice-cold sterile double-distilled water to resus...

Embodiment 3

[0088] The yeast transformants that were screened by G418 and identified as positive in Example 2 expressed the LIGHT-Fc fusion protein under the induction of yeast, and the specific steps were as follows:

[0089] 1. Induced expression of LIGHT-Fc fusion protein in Pichia pastoris

[0090] (1) Pick a single clone, inoculate it into 5 ml of YPD medium, and culture it with shaking at 250 rpm at 30°C for 16-18 hours.

[0091] (2) Inoculate 200μl of the bacterial solution into 20mL of BMGY medium, culture at 30°C, shake at 250rpm until OD 600 =2-6.

[0092] (3) Centrifuge at 1500-3000 rpm for 5 minutes at room temperature, and remove the supernatant.

[0093] (4) Wash the bacterial cell pellet once with sterile water.

[0094] (5) Resuspend the bacterial pellet with BMMY medium (if it is Mut +For transformants, add an equal volume of BMMY medium, if it is Mut s Type transformants, then add 1 / 5 times the volume of BMMY medium), 30 ° C, 250 rpm shaking culture for 96 h, supple...

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Abstract

The invention discloses a method for preparing human LIGHT-Fc fusion protein by using a pichia expression system, comprising the following steps of: (1) constructing recombinant expression plasmid pPIC9K-LIGHT-Fc; (2) preparing and screening expression human LIGHT-Fc fusion protein yeast engineering bacteria; and (3) expressing and evaluating the human LIGHT-Fc fusion protein in pichia. The invention makes up the deficiency of the traditional human LIGHT preparation method and the expression mode of a prokaryotic expression system, otherwise, if being used for producing industrial human LIGHT protein, the invention has the advantages of simple operation, short growth cycle of raw organisms, large production scale (capable of carrying out high-density fermentation), low extraction cost, high activity of products, and the like and has important industrial application prospect and actual significance.

Description

technical field [0001] The invention belongs to the field of molecular biology and genetic engineering medicine, and specifically relates to the construction of recombinant plasmid LIGHT-Fc-pPIC9K and the expression method of LIGHT-Fc fusion protein in Pichia pastoris. Background technique [0002] LIGHT (homologous to lymphotoxins exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpesvirus entrymediator [HVEM], a receptor expressed by T lymphocytes) was first discovered by Mauri et al. It is HVEM-L (herpesvirus entrymediator-ligand), which belongs to the 14th member of the TNF superfamily (TNFSF14). It has been found that it has three receptors: HVEM, LTβR and TR6. LIGHT exerts different biological functions by interacting with these three receptors. Among them, HVEM is mainly expressed on T cells. After combining with LIGHT, it can co-stimulate T cell activation and regulate T cell immune response, mediating allograft rejection....

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/81C07K19/00
Inventor 江文正郝文丽闻洁君樊燕杜佳妮
Owner EAST CHINA NORMAL UNIVERSITY
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