Optimized HIL-17RA-HSA (human interleukin-17 receptor-human serum albumin) fusion gene encoding proteins

A technology of fusion gene and fusion protein, which is applied in the field of codon-optimized HIL-17RA-HSA fusion gene and its encoded protein, and can solve problems such as hindering the development prospect of cytokines and poor pharmacokinetic properties.

Inactive Publication Date: 2016-02-03
LANZHOU UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Poor pharmacokinetic properties severely hamper the development of these cytokines as drugs

Method used

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  • Optimized HIL-17RA-HSA (human interleukin-17 receptor-human serum albumin) fusion gene encoding proteins
  • Optimized HIL-17RA-HSA (human interleukin-17 receptor-human serum albumin) fusion gene encoding proteins
  • Optimized HIL-17RA-HSA (human interleukin-17 receptor-human serum albumin) fusion gene encoding proteins

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Experimental program
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Embodiment 1H

[0025] The codon optimization of embodiment 1HIL-17RA-HSA gene

[0026] In order to achieve a higher level of expression of the HIL-17RA-HSA gene in Escherichia coli and better function, according to the codon usage preference of HIL-17RA-HSA expressed in Escherichia coli, GenScript rare codon analysis software was used The cloned HIL-17RA-HSA gene was optimally designed to increase the codon adaptation index CAI value.

[0027] Rare codons in the HIL-17RA-HSA gene will reduce the transcription level and translation efficiency in E. coli. According to the codon preference of Escherichia coli, the HIL-17RA-HSA gene was optimized, and the new sequence obtained was designated as HIL-17RA-HSA (SEQ ID NO: 1). Sequence alignment results showed that the similarity with the wild-type HIL-17RA-HSA sequence was 77.3%, and the amino acid sequence remained unchanged. Among the 2799 amino acids, the codons of 1686 amino acids were optimized, and the optimization rate reached 60%.

[002...

Embodiment 2

[0029] Example 2 Recombination. Construction of pET30a / HIL-17RA-HSA expression vector

[0030] The whole gene synthesis HIL-17RA-HSA was connected into the pUC57-T vector ( image 3 ). The correct sequenced positive pUC57-T / HIL-17RA-HSA bacteria extracted plasmid (operate according to Sangong plasmid mini-extraction kit). Plasmid pET30a and pUC57-T / HIL-17RA-HSA were double digested with Xbal / HimdⅢ restriction endonuclease. The digested products were ligated, constructed into a prokaryotic expression vector pET30a / HIL-17RA-HSA, transformed into BL21(DE3) competent cells, and used Kan + The antibiotic plate was screened, and 10 single colonies were randomly selected for PCR identification with specific primers, and a bright band appeared at about 2800bp, which was consistent with the expected size ( Figure 4 ).

Embodiment 3

[0031] Example 3. Expression of recombinant plasmids in Escherichia coli

[0032] (1) Transform the recombinant expression plasmid pET30a / HIL-17RA-HSA and the empty plasmid pET30a into Escherichia coli BL21 (DE3) competent bacteria, plate and pick bacteria;

[0033] (2) Containing Kan + After shaking culture in the LB medium at 37°C overnight, take the empty vector bacterial solution and the recombinant bacteria into 3 mL and 30 mL LB medium containing kanapenicillin at a volume ratio of 1:100, respectively, and shake at 200 rpm at 37°C;

[0034] (3) When the measured OD600 value is 0.6-0.8, take out 3mL recombinant bacteria solution / tube, including the empty vector pET30a bacteria solution, and add 1mMIPTG, continue to culture at 15°C and 37°C for 4-16h, and induce for 4h, respectively. Take 1 tube each at 16 hours. In addition, take 1 mL of the bacterial liquid that has not been induced by IPTG as a control;

[0035] (4) Centrifuge the expressed bacterial solution at 6500...

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Abstract

The invention discloses a method for preparing codon-optimized human interleukin-17 receptor (HIL-17RA) and human serum albumin (HSA) fusion proteins and a product, and discloses construction and preparation of fusion proteins and application of the fusion proteins in the aspect of disease treatment. The method and the product have the advantages that expression of the modified (codon-optimized) fusion proteins in prokaryotic cells can be obviously improved; polypeptide fragments from the HIL-17RA and the human serum albumin (HSA) are fused with one another to obtain the fusion proteins, the problem of easy degradation of natural IL-17RA proteins can be solved by the optimized HIL-17RA-HSA fusion proteins, and the optimized HIL-17RA-HSA fusion proteins are high in biological activity and expression quantity; the method and the product have important actual significance and broad application prospects in the field of medicine and biological pharmacy.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and long-acting fusion protein medicine. Specifically, the present invention relates to a codon-optimized HIL-17RA-HSA fusion gene and its encoded protein. Background technique [0002] Interleukin 17 (Interleukin-17, IL-17) and its receptor (IL-17RAeceptor, IL-17RA) family is a unique immune molecule-receptor family, which plays an important role in the body's autoimmunity, inflammatory response, tumorigenesis and organ transplantation. It plays an important role in the reaction process (J.KKolls, A.Linden, Interleukin-17family members and inflammation. Immunity 21 (4) (2004) 467; T.A. Moseley, et al. Interleukin-17family and IL-17receptors. CytokineGrowthFactorRev14 (2) (2003) 155). IL-17A can stimulate a variety of tissue cells to release chemokines to attract neutrophils to the inflammatory response area (M.Laan, et al. Neutrophil recruitment by human IL-17 via C-X-Cchemokine rele...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C07K19/00C12N5/10A61K38/38A61P35/00A61P37/00A61P37/06A61P35/02
Inventor 张春江方媛文娟陈新君李晨辉
Owner LANZHOU UNIVERSITY
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