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Genetically engineered bacterium for expressing insulin precursor, and preparation method and application of genetically engineered bacterium

A technology of insulin precursors and genetically engineered bacteria, applied in genetic engineering, insulin, botany equipment and methods, etc., can solve the problems of expensive culture medium, easy introduction of animal-derived viruses, and low yield

Pending Publication Date: 2020-09-15
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the low yield of insulin precursor at shake flask level or fermenter level of the bacterial strain expressing insulin precursor in the prior art, and the culture medium used in the production process is relatively expensive and easy to introduce into animals. Defects such as virus-derived viruses, providing a genetically engineered bacterium expressing insulin precursor, its preparation method and application, and a preparation method of insulin precursor

Method used

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  • Genetically engineered bacterium for expressing insulin precursor, and preparation method and application of genetically engineered bacterium
  • Genetically engineered bacterium for expressing insulin precursor, and preparation method and application of genetically engineered bacterium
  • Genetically engineered bacterium for expressing insulin precursor, and preparation method and application of genetically engineered bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1: Expression vector pPIC9K-IP (IP is insulin precursor, insulin precursor, and the plasmid map is as follows figure 1 shown) and the construction and screening of Pichia pastoris engineering bacteria

[0047] The invention adopts pPIC9K vector, the cloning sites are EcoR I and Not I, and the recombinant vector is pPIC9K-IP. The expression vector is transcribed from the AOX1 promoter.

[0048] The target gene fragment (the specific sequence of the gene sequence such as SEQ ID NO.1) was synthesized by the company (Shanghai Ruidi Biotechnology Co., Ltd.) and cloned on the pPIC9K vector (purchased from Invitrogen), and the cloning sites were EcoR I and NotI , the plasmid pPIC9K-IP was obtained.

[0049] 2. Extract the plasmid pPIC9K-IP obtained above, and use the following enzyme digestion system to perform plasmid linearization enzyme digestion reaction:

[0050]

[0051] Digest at 37°C for 4 hours, place the digested product at 70°C, and inactivate the rest...

Embodiment 2

[0063] Embodiment 2: Shake flask horizontal culture fermentation

[0064] Randomly pick the transformant in the MD plate of the third part in Example 1 and the YPD plate containing 5, 6, and 7 mg / mL G418 in the fourth part of Example 1 and transfer it to a test tube containing 2 mL of YPD liquid medium Cultivate YPD bacteria liquid at 28°C and 230rpm; transfer the obtained YPD bacteria liquid to contain 6mL BMGY (composed of: 10g / L yeast extract, 20g / L tryptone, 3.4g / LYNB, 100mM potassium phosphate buffer, 400μg / L biotin, 10g / L glycerol) in a 20mL shake flask for about 18 hours, and the shake flask was placed in a refrigerator at 4°C for free sedimentation for 6 hours; the supernatant in the shake flask was poured out carefully, and 20mL BMMY (composed of : 10g / L yeast extract, 20g / L tryptone, 3.4g / L YNB, 100mM potassium phosphate buffer, 400μg / L biotin, 0.5% methanol [% is the volume ratio of the whole culture medium]) continue to cultivate , adding 0.5% methanol in the med...

Embodiment 3

[0067] Example 3: 5L fermenter horizontal high-density fermentation

[0068] Inoculate the above-mentioned activated glycerol bacteria into a total of 6 tubes containing 2mL of YPD liquid medium, which is the primary seed solution;

[0069] Select 2 tubes of the first-grade seed liquid, a total of 4mL, and inoculate it in a 500mL shake flask containing 100mL of YPD liquid medium, and cultivate it at 28°C and 230rpm for 12 hours. seed liquid;

[0070]Configure BSM (40g / L glycerin, 26.7mL / L phosphoric acid (85%), 0.93g / L calcium sulfate dihydrate, 18.2g / L potassium sulfate, 14.9g / L magnesium sulfate heptahydrate, 4.13g / L potassium hydroxide , 4.0mL / L PTM1 (Pichia pastoris trace element 1 related components: 6g / L copper sulfate pentahydrate, 0.08g / L sodium iodide, 3g / L manganese sulfate monohydrate, 0.2g / L dihydrate and molybdic acid Sodium, 0.02g / L boric acid, 0.5g / L cobalt chloride, 20g / L zinc chloride, 65g / L ferrous sulfate heptahydrate, 0.2g / L biotin, 5mL / L sulfuric acid)),...

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Abstract

The invention provides a genetically engineered bacterium for expressing an insulin precursor. The genetically engineered bacterium is an engineered bacterium in which an expression vector of an insulin precursor gene is integrated in pichia pastoris, and the nucleotide sequence of the insulin precursor gene is shown as SEQ ID NO.1 in a sequence table. The invention also provides a preparation method of the insulin precursor. The strain yield of the genetically engineered bacterium fermented at a shake flask level is higher than the strain yield reported in the prior art. Through combination of the genetically engineered bacterium and the preparation method of the insulin precursor, the yield of the insulin precursor obtained by fermentation tank-level fermentation is remarkably increased.Besides, a culture medium used in the preparation method of the insulin precursor is relatively low in cost, and the risk of animal-derived virus pollution is avoided, so that cost of the preparationmethod is remarkably reduced.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a genetic engineering bacterium expressing insulin precursor, its preparation method and application, and the preparation method of insulin precursor. Background technique [0002] Diabetes is a chronic metabolic disease. Due to aging and rapid population growth, the number of people with diabetes worldwide may increase by 50.7% by 2030. At present, recombinant human insulin and its analogues are the most effective and main treatment for diabetes. The mainstream insulin drugs on the market include insulin aspart, insulin glargine, insulin detemir, insulin degludec, etc., among which the latter three are long-acting insulins, which can last for 24 hours in the human body and have no concentration peak. Existing technologies for insulin analogues mostly use Escherichia coli system and yeast system to prepare precursors, and then make human insulin through transpeptide or l...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/17C12N15/81C12P21/02C12R1/84
CPCC07K14/62C12N15/815
Inventor 谢丽萍胡又佳吴珺艺张伟韩姝龚桂花许人仁
Owner SHANGHAI INST OF PHARMA IND
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