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Expression box, expression carrier, host bacteria and method for preparing non-labeled recombinant human IL (interleukin) 37 mature peptide

A technology of expression vector and expression cassette, which is applied in the field of preparing unlabeled recombinant human IL37 mature peptide, which can solve the problems of increasing purification steps and difficulty, limiting the clinical application of rIL37 products, and low purity

Inactive Publication Date: 2018-06-22
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some studies have used E. coli expression system to recombinantly express IL37b recombinant protein with His tag, but its purity is low
At the same time, rIL37 products with uncut tags cannot truly reflect the properties of IL37 itself, and these two factors greatly limit the clinical application of rIL37 products
However, the traditional method of removing the tagged protein needs to elute the fusion protein from the chromatographic column, and then remove the tag, which will lead to too many impurities in the sample, increasing the subsequent purification steps and difficulty

Method used

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  • Expression box, expression carrier, host bacteria and method for preparing non-labeled recombinant human IL (interleukin) 37 mature peptide
  • Expression box, expression carrier, host bacteria and method for preparing non-labeled recombinant human IL (interleukin) 37 mature peptide
  • Expression box, expression carrier, host bacteria and method for preparing non-labeled recombinant human IL (interleukin) 37 mature peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Construction of pGEX6P1-IL37(46-218) recombinant expression vector

[0046] 1. Preparation of IL37(46-218) clone fragment

[0047] The human IL37 gene (NM014439) was connected to the pDonR-223 vector by Nanjing GenScript Biotechnology Co., Ltd. and named pDonR-223-IL37. Then use 0.5 μl of the pDonR-223-hIL37 vector as a template, add 1 μl each of primers F46 (SEQ ID No: 2) and R218 (SEQ ID No: 3) at a concentration of 10 μM, 0.5 μl of PrimeSTAR HS DNA polymerase, 5×PrimeSTAR *Buffer(Mg 2+ plus) 5μl, 2.5mM dNTP Mixture 2.5μl, ddH 2 O 14.5 μl. The PCR reaction conditions are 98°C pre-denaturation for 5 minutes, 98°C / 10s, 58°C / 10s, 68°C / 45s (30 cycles), 72°C extension for 10 minutes, electrophoresis, and gel cutting to recover a DNA fragment of about 530bp. This fragment contains The IL37 (46-218) gene fragment at the BamHI and XhoI restriction sites was eluted from the DNA purification column with 30 μl sterile deionized water.

[0048] 2. Preparation of B...

Embodiment 2

[0052] Embodiment 2, construction of recombinant engineered bacteria and screening of high expression clones

[0053] 1. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) Rosetta.

[0054] First, prepare competent engineering bacteria (the following steps need to be carried out under sterile conditions): Take out the engineering bacteria BL21(DE3) Rosetta stored at -80°C, activate it on LB solid medium by streaking, and put it in a constant temperature incubator at 37°C Incubate overnight until a single colony grows; pick a single colony and inoculate it into a 250ml Erlenmeyer flask (containing 50ml LB medium), culture at 37°C, 220rpm for 6-8h, measure OD 600, stop culturing when it reaches 0.6-0.8; centrifuge at 2 500rpm for 5-10min at 4°C, collect the bacteria, and remove the supernatant; add 40ml of pre-cooled Inoue transformation buffer (10mM PIPES, 55mM MnCl 2 , 15mM CaCl 2 , 250mMKCl) resuspended bacteria. The whole process was operated on ice; ...

Embodiment 3

[0058] Example 3, expression, purification and purity estimation of recombinant protein

[0059] 1. Recombinant expression of protein

[0060] After thawing the engineered bacteria highly expressing the recombinant protein on ice, streak inoculate it on a 2×YT plate containing 100 μg / ml ampicillin, and culture it upside down in a constant temperature incubator at 37°C overnight until clearly visible single cells grow. colony. Then pick a single colony and inoculate it in 50ml of 2×YT medium containing 100μg / ml at 220rpm / 37℃ for overnight growth, and transfer it to a 500ml shaker flask with baffles according to the 1% inoculum size until the cell OD 600 When the value is about 0.8, add 1mM IPTG and continue shaking culture for 4h to induce the expression of recombinant protein. Then centrifuge at 8000rpm for 20min, discard the supernatant, collect the cells and freeze them at -20°C for later use.

[0061] 2. Shredding of engineered bacteria with induced expression

[0062] ...

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Abstract

The invention belongs to the technical field of biology, and discloses an expression box, an expression carrier, a host bacterium and a method for preparing non-labeled recombinant human IL 37 maturepeptide. The expression box comprises a promoter, a chaperonin-coding nucleotide sequence, a digestion recognition site coding nucleotide sequence, a human interleukin 37 mature peptide coding nucleotide sequence and a terminal transcripton. The method for preparing the non-labeled recombinant human IL 37 mature peptide (rIL 37) comprises the steps that a host bacterium containing an expression carrier of the expression box is cultured and subjected to induce recombinant protein expression, a thallus is collected and split, supernatant is collected and subjected to affinity chromatography, andthen protease capable of bonding with the affinity chromatography medium to online excise labeled protein, wherein the protease can recognize the digestion recognition site in the expression box. Thepreparation method can rapidly excise labeled protein, the preparation technology is simplified, and meanwhile, the purity of rIL 37 is improved greatly.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, the present invention relates to an expression cassette, an expression vector, a host bacterium and a method for preparing unlabeled recombinant human IL37 mature peptide. Background technique [0002] Interleukins are a type of cytokines that are produced by and act on various cells. Because it was originally produced by white blood cells and played a role among white blood cells, it got its name and is still in use. Initially, it refers to the cytokines produced by leukocytes and plays a regulatory role among leukocytes. Now it refers to a class of cytokines whose molecular structure and biological function have been basically clarified, and which have an important regulatory role and are uniformly named. Interleukin and blood cell growth factor belong to cytokines, and they coordinate and interact with each other to complete hematopoietic and immune regulation functions. Interleuk...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/62C07K14/54
CPCC07K14/54C07K2319/35C07K2319/50C12N15/62C12N15/63
Inventor 伍代朝马瑶
Owner NANHUA UNIV
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