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Codon-optimized insulin degludec precursor gene and expression method thereof

A technology for insulin degludec and codon optimization, applied in the field of insulin degludec precursor gene and its expression, can solve the problems of poor expression effect of Pichia pastoris and other problems

Active Publication Date: 2019-02-01
JILIN HUISHENG BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insulin degludec precursor gene sequence is rarely reported. In our previous studies, we referred to Xu Yan et al. to optimize the insulin sequence and used it to optimize the insulin degludec precursor gene sequence, but the expression effect in Pichia pastoris still poor

Method used

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  • Codon-optimized insulin degludec precursor gene and expression method thereof
  • Codon-optimized insulin degludec precursor gene and expression method thereof
  • Codon-optimized insulin degludec precursor gene and expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Optimum Design of Insulin Degludec Precursor Gene

[0068] 1. Experimental materials

[0069] The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; the DNA polymerase, DNA ligase and restriction endonuclease used in the experiment were all products of TAKARA Company; the pPICZαA vector was purchased from Invitrogen Company. Figure 16 ; Plasmid extraction kit and gel recovery kit are products of Sangon Bioengineering (Shanghai) Co., Ltd.; gene sequencing was completed by Sangon Bioengineering (Shanghai) Co., Ltd.; other related reagents are commercially available.

[0070] 2. Method results

[0071] The human insulin precursor gene sequence (SEQ ID NO: 12) as the basis (the corresponding human insulin precursor amino acid sequence is SEQ ID NO: 13), obtain the original gene sequence (SEQ ID NO: 1) of the insulin degludec precursor, and replace the codons of individual amino acids with Pichia Yeast preferred codons, rationally optimized...

Embodiment 2

[0082] Example 2 Expression and identification of the optimized insulin degludec precursor gene in Pichia pastoris

[0083] 1. Experimental materials

[0084] Pichia strains were purchased from Invitrogen; the restriction enzyme SacI was a product of Invitrogen; the plasmid extraction kit and gel recovery kit were both products of Sangon Bioengineering (Shanghai) Co., Ltd.; gene sequencing was provided by Sangon Biotechnology Engineering (Shanghai) Co., Ltd. completed; other related reagents are commercially available.

[0085] 2. Method results

[0086] 1. Transformation of the optimized insulin degludec precursor gene into Pichia pastoris

[0087] Linearize the expression vector pPICZαA2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 containing the optimized insulin degludec precursor gene with restriction endonuclease SacI, the reaction system is 500 μL, including: 10× FD 50 μL, Sac I 25 μL, pPICZαA2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 50 μL, ddH 2 O 375 μL. The reaction conditions were enzyme digestion at 37°C for 1...

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Abstract

The invention relates to the technical field of genetic modification and protein expression, particularly to a codon-optimized insulin degludec precursor gene and an expression method thereof, whereinthe insulin degludec precursor gene is optimized based on the gene sequence represented by SEQ ID NO:1, and the optimizations comprise one or a plurality of the following cases that (a) the amino acid codon CAG at the site 4 is replaced with CAA; (b) the amino acid codon CAC at the site 5 is replaced with CAT; (c) the amino acid codon GTC at the site 12 is replaced with GTT; and (d) the amino acid codon CGT at the site 22 is replaced with AGA. According to the present invention, the amino acid sequence encoded by the optimized insulin degludec precursor gene sequence is consistent with the original gene sequence while the expression level of the gene sequence in yeast is much higher than the expression level of the original gene sequence.

Description

technical field [0001] The invention relates to the technical field of gene modification and protein expression, in particular to a codon-optimized insulin degludec precursor gene and an expression method thereof. Background technique [0002] Diabetes is a chronic disease caused by insufficient secretion or utilization of insulin in the body. Insulin is secreted by pancreatic islet β cells stimulated by endogenous or exogenous substances such as glucose, lactose, glucagon, etc. A protein hormone that allows glucose in the blood to enter cells and convert it into energy for body activities. Human insulin is composed of two peptide chains, α and β. Among them, the α chain has 11 kinds of 21 amino acids, and the β chain has 15 kinds of 30 amino acids. The lack or defect of insulin in diabetic patients keeps the glucose in the body in the circulating blood, and this high blood sugar may cause acute complications such as diabetic ketoacidosis, hyperglycemic hyperosmolar coma, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/17C12N15/81C07K14/62C12N1/19A61K48/00A61K38/28A61P3/10C12R1/84
CPCA61K38/28A61K48/005A61P3/10C07K14/62C12N15/815C12N2800/22
Inventor 曹海燕张世野纪晓影王宝泉王英明杨成龙王洪宇吴丹青徐霞
Owner JILIN HUISHENG BIOPHARMACEUTICAL CO LTD
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