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Codon-optimized Chi3L1 gene and application thereof

A codon optimization and gene technology, applied in the field of genetic engineering, can solve the problems of low biological activity and low expression of Chi3L1, and achieve the effects of strong immune activity, good foundation and clinical application prospects, and low cost.

Pending Publication Date: 2017-03-22
THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a codon-optimized Chi3L1 gene to realize heterologous expression of Chi3L1 protein, so as to solve the problems of low Chi3L1 expression and low biological activity in the prior art

Method used

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  • Codon-optimized Chi3L1 gene and application thereof
  • Codon-optimized Chi3L1 gene and application thereof
  • Codon-optimized Chi3L1 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of recombinant Chi3L1 protein expression plasmid.

[0029] Synthesize the Chi3L1 gene (as shown in ESQ ID No.: 1) first, and then subclone it into the pRSET-A plasmid by using NdeI and HindIII restriction sites to obtain the pRSET-A-Chi3L1 expression plasmid. The specific construction process as follows:

[0030] (1). Enzyme digestion: the synthesized DNA fragment of Chi3L1 was digested with restriction endonucleases NheI and HindIII, and the pRSET-A empty shuttle plasmid was digested with corresponding endonucleases.

[0031] Table 1 enzyme digestion reaction system

[0032]

[0033] Reaction conditions: 37°C water bath for 2h. The above digested products were separated by 1% agarose gel electrophoresis to recover the target fragment, and the purified target DNA fragment was obtained using Agarose Gel DNA Extraction Kit.

[0034] (2). Ligation: Ligate the expression fragment of Chi3L1 digested with the pRSET-A vector.

[0035] Table 3 Con...

Embodiment 2

[0038] Example 2: This example describes the expression and purification method of recombinant Chi3L1 protein

[0039] The expression plasmid pRSET-A has a continuous encoding of 6 histidines (His 6 ) base sequence, therefore, the expressed polypeptide has His at the amino terminal 6 Marker for easy purification and identification of expressed proteins. The expression plasmid was transformed into BL21 competent bacteria. After overnight culture, shake culture in 100ml SOB medium at 1:50 at 37°C. When the OD450 was about 0.5, add IPTG with a final concentration of 0.5mM and continue to culture for 4h; , 4h respectively sample 1ml, centrifuge at 6000r / min for 5min, add 30μl Laemmli buffer solution to the precipitate, heat at 95°C for 5min, take 8μl by SDS-PAGE, Coomassie brilliant blue staining analysis shows that with the prolongation of induction time, at the position of relative molecular mass 42kD The nearby protein bands were gradually enhanced, and the protein expression...

Embodiment 3

[0041] Example 3: Recombinant Chi3L1 protein promotes macrophage M2 differentiation

[0042]Macrophages were treated with 1000 μg / ml purified Chi3L1 protein for 4 hours, the cells were collected, RNA was extracted, and the expression levels of macrophage differentiation-related genes were detected by RT-PCR after reverse transcription. The results showed that CD163, CD206 and other macrophage M2 The expression of differentiation-related genes was significantly increased, suggesting that the Chi3L1 recombinant protein can effectively promote the M2 differentiation of macrophages ( figure 2 ).

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Abstract

The invention discloses a codon-optimized Chi3L1 gene. The nucleotide sequence of the gene is shown in SEQ ID NO.1. The invention further discloses a method for expressing and purifying Chi3L1 protein through the Chi3L1 gene. The method for obtaining Chi3L1 recombinant protein is provided and is low in cost and easy and convenient to implement, the Chi3L1 recombinant protein has very strong immunity activity and a function of promoting growth of tumors, can be taken as an active drug for immunity research and a drug target for development of anti-tumor drugs and has a good foundation and clinical application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a codon-optimized Chi3L1 (chitinase 3 sample 1) gene and its application. Background technique [0002] Chitin is an odorless and tasteless white amorphous substance that widely exists in nature, such as crustacean insects, crab shells, insect carapaces, and cell walls of bacteria and fungi. Chitin is a polymer substance with a molecular weight of up to 1×10 6 Above, insoluble in water, dilute acid, alkali, ethanol or other organic solvents. Chitinases and chitinase-like proteins belong to the glycosyl hydrolytic protein family18, they are widely distributed in prokaryotes and eukaryotes, both can bind chitin, the former has cleavage activity, while the latter does not. [0003] Chitinase 3-like 1 (Chi3L1), an important member of chitinase-like proteins, is an important regulator of innate immunity and acquired immunity, and is mainly secreted and expres...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N15/70C12N1/21C12N9/42A61K38/48A61P35/00
CPCC12N9/2434A61K38/00
Inventor 尤强潘金顺杨璟张晨胡石耿彪穆先敏徐澈赵婷
Owner THE SECOND AFFILIATED HOSPITAL OF NANJING MEDICAL UNIV
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