Human interleukins-22 mutant as well as construction method and use thereof

An interleukin, mutant technology, applied in the direction of interleukin, cytokine/lymphokine/interferon, application, etc., can solve problems such as inability to accurately infer

Inactive Publication Date: 2008-07-23
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The hIL22 dimer is an asymmetric subunit. By comparing IL22 with hIFN-γ / hIFN-γRα and hL10 / hL10R1 complexes through homology modeling, it was found that the possible receptor binding site of IL22 is composed of helixA, loopAB, helixF composition (Ronaldo Alves Pinto Nagem, 1, 2 Didier Colau, Crystal Structure of Recombinant Human Interleukin-22 Structure, Vol.10, 1051-1062, August, 2002), however, it is not possible to precisely deduce which binding site is related to CRF2- 9 Binding, which site binds to CRF2-4

Method used

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  • Human interleukins-22 mutant as well as construction method and use thereof
  • Human interleukins-22 mutant as well as construction method and use thereof
  • Human interleukins-22 mutant as well as construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Fishing of wild-type hIL22 gene and construction of recombinant expression vector

[0084] 1.1 Extraction of total RNA

[0085] Dilute 1ml of fresh peripheral blood by 2 times with PBS containing 2mmol / L EDTA, spread it on an equal volume of lymphocyte separation medium (purchased from Shanghai Huajing Biological Company), centrifuge at 800r / min to make layers, and then use capillary Aspirate the buffy coat cells. After washing with Hanks solution for 3 times, the cells were suspended in 1640 culture medium (purchased from Tianrun Shanda Reagent Co., Ltd.) at 37°C, 5% CO 2 After culturing in the incubator for 24h, use ConA (2mg / ml, purchased from Xiasi Biological Company); and anti-CD 3 (4mg / ml purchased from Shanghai Wowu Biotechnology Co., Ltd.) co-stimulated cells and then at 37 ° C, 5% CO 2 After continuing to cultivate in the incubator for 24 hours, the total RNA was extracted with the total RNA extraction kit of Promega Company, the operation was car...

Embodiment 2

[0103] Example 2: Construction of a series of hIL22 mutants

[0104] Construction of mutant 6M that mutated all six amino acids of the receptor binding site (DNNTDV) into alanine (Ala)

[0105] First pair of primers:

[0106] Reverse R2: 5'GAGAATTCATATGGCACCCATCAGCTCCCCA3'

[0107] Forward FM: 5'AGCTGCAGCTGCAGCTGCAGCCAAGCTAGCCTCCTT3'

[0108] Second pair of primers:

[0109] Reverse RM: 5'GCAGCTGCAGCTGCAGCTCGTCTCATTGGGGAGAAA3'

[0110] Forward F2: 5′ACGGATCCTCAAATGCAGGCATTTCT3′

[0111] The PCR reaction system is as follows:

[0112] pBV220-hIL22 1ul

[0113] Forward primer R2 (or RM) 10umol / l 2ul

[0114] Reverse primer FM (or F2) 10umol / l 2ul

[0115] 10×buffer 5ul

[0116] 2.5mmol dNTP 4ul

[0117] Pyrobest High Fidelity Enzyme 0.5ul

[0118] Add water to 50ul

[0119] In the first round of PCR reaction, two fragments of R2-FM and RM-F2 were obtained, and then the second round of PCR was performed using these two fragments as templates

[0120] R2-FM 1ul

[01...

Embodiment 3

[0134] Construction of mutant 2NM that changes Asn at positions 68 and 69 to Ala

[0135] First pair of primers:

[0136] Reverse R2: 5'GAGAATTCATATGGCACCCATCAGCTCCCCA3'

[0137] Forward FM: 5′GTCTGTAGCTGCATCAGCCAAGCTAGCCTC3′

[0138] Second pair of primers:

[0139] Reverse RM: 5'GCTGATGCAGCTACAGACGTTCGTCTCATT3'

[0140] Forward F2: 5′ACGGATCCTCAAATGCAGGCATTTCT3′

[0141] The reaction system and parameters are the same as before. The amplification result is shown in FIG. 8 , and the amino acid sequence is shown in SEQ NO:3.

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Abstract

The invention discloses a receptor binding site of the interleukin-22, forming a basis for the utilization of the interleukin-22; the invention also discloses a mutant protein where the binding activity for the 67th and 71st Asp to change into Ala is improved, and the mutant protein can play a more significant role even than the interleukin-22. The receptor binding site of the interleukin-22 also provides a construction method for the interleukin-22 mutant, and the construction method has the advantages of high yield, low cost, and applicability to production and application in large scales.

Description

technical field [0001] The present invention relates to human interleukin-22, in particular to human interleukin-22 mutant and its construction method and application. Background technique [0002] Interleukin 22 (English name Interleukin 22, abbreviated as: IL22) is a cytokine discovered in 2000, which belongs to the interleukin 10 family member, and it is secreted by activated T cells and NK cells (Dumoutier L et al., J Immunol 2000; 164: 1814~9.), but its receptors are located on parenchymal cells of the skin, kidney, digestive system, and respiratory system, but have no effect on the immune system (WolkK et al., Immunity 2004; 21: 241 -54.), biological studies have shown that hIL22 can induce the expression of antimicrobial polypeptides in epithelial tissues such as the lung, intestinal tract, and skin, enhance the invasion of these cells against pathogenic microorganisms, inhibit the differentiation of epithelial cells, and enhance their proliferation and migration, so ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/54C12N15/09C12N15/24
Inventor 徐东刚邢微微蔡欣邹民吉徐涛刘深王园园吴伯灵杨旗
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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