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Optimization and high-level expression of insulin aspart precursor gene

A technology of insulin aspart and its precursor, which is applied in genetic engineering, plant genetic improvement, and microbial-based methods, etc., and can solve the problems of poor expression of Pichia pastoris and other problems

Active Publication Date: 2017-06-09
浙江海晟药业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Optimization and high-level expression of insulin aspart precursor gene
  • Optimization and high-level expression of insulin aspart precursor gene
  • Optimization and high-level expression of insulin aspart precursor gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Rational design of the optimized insulin aspart precursor gene and vector construction The first set of experiments

[0054] One experimental material

[0055] The primer design software was DNASTAR software, and the primers were synthesized by Shanghai Boshang Biotechnology 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; the plasmid The extraction kit and gel recovery kit were both products of Hangzhou Aisijin Biotechnology Co., Ltd.; gene sequencing was completed by Shanghai Boshang Biotechnology Co., Ltd.; other related reagents were purchased from the market.

[0056] Two method results

[0057] 1 The optimized insulin aspart precursor gene was obtained

[0058] The human insulin precursor gene sequence (Sequence NO.12) as the basis (the corresponding human insulin precursor amino acid sequence is Sequence NO.13), ...

Embodiment 2

[0073] Rational design of the target gene and vector construction of embodiment 2 The second group of experiments

[0074] One experimental material

[0075] The primer design software was DNASTAR software, and the primers were synthesized by Shanghai Boshang Biotechnology 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; the plasmid The extraction kit and gel recovery kit were both products of Hangzhou Aisijin Biotechnology Co., Ltd.; gene sequencing was completed by Shanghai Boshang Biotechnology Co., Ltd.; other related reagents were purchased from the market.

[0076] Two method results

[0077] 1 The optimized insulin aspart precursor gene was obtained

[0078] Based on the human insulin precursor gene sequence reported by Gurramkonda et al., the proline at position 28 of the B chain was changed to aspartic acid to obtain the origina...

Embodiment 3

[0093] Rational design of the target gene and the third group of experiments of the carrier construction of embodiment 3

[0094] One experimental material

[0095] The primer design software was DNASTAR software, and the primers were synthesized by Shanghai Boshang Biotechnology 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; the plasmid The extraction kit and gel recovery kit were both products of Hangzhou Aisijin Biotechnology Co., Ltd.; gene sequencing was completed by Shanghai Boshang Biotechnology Co., Ltd.; other related reagents were purchased from the market.

[0096] Two method results

[0097] 1 The optimized insulin aspart precursor gene was obtained

[0098] Based on the human insulin precursor gene sequence reported by Gurramkonda et al., the proline at position 28 of the B chain was changed to aspartic acid to obtain the ...

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Abstract

The invention discloses an optimized insulin aspart precursor gene sequence and its high-efficiency expression in yeast. The amino acid sequence encoded by the optimized insulin aspart precursor gene of the present invention is consistent with the original gene sequence, but the expression level in yeast is much higher than the original gene sequence. The expression results were detected by the shake flask fermentation test, and it was found that the protein expression of the insulin aspart precursor increased by 1.8 to 7.5 times after optimization; Increased by 10.8 times. The invention also discloses the modification method of the above gene sequence.

Description

technical field [0001] The present invention relates to gene modification and protein expression. By optimizing codons, the insulin aspart precursor gene sequence that can be used for high expression is obtained. Apply molecular biology techniques to synthesize the target gene and construct the expression vector, and evaluate the expression of the target protein. Background technique [0002] Insulin (Insulin, Ins) is a therapeutic drug mainly used in all type I diabetes and most type II diabetes patients. The ideal application mode of insulin is to simulate the situation of physiological insulin secretion to meet the needs of energy metabolism. For diabetes (diabetes mellitus, DM), insulin treatment can effectively reduce or alleviate the occurrence and development of symptoms of type I and type II diabetes. Therefore, insulin has become an ideal treatment method. Although ordinary short-acting human insulin is widely used, it also has some disadvantages, especially afte...

Claims

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Application Information

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IPC IPC(8): C12N15/17C12N15/81C12N1/19C12N15/10A61K38/28A61P3/10C12R1/84
Inventor 徐岩耿月兵聂磊王海彬叶小红白骅
Owner 浙江海晟药业有限公司
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