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Pig trypsinogen mutant and expression thereof in pichia pastoris

A technique for porcine trypsin and mutant, which is applied in the field of genetic engineering, can solve the problems of toxicity, high toxicity of Escherichia coli, environmental pollution, etc., and achieves the effects of simple fermentation process and purification process, avoiding complicated renaturation operation and broad application prospect.

Pending Publication Date: 2022-06-24
江苏万邦医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method cannot completely remove other proteins, and the extracted animal-derived viruses such as foot-and-mouth disease virus and mad cow disease virus cannot be completely removed, so there is an immunogenic hazard to the human body in medical applications.
The second is to produce animal-derived trypsin by genetic recombination, mostly pig-derived trypsin, and the host of genetic engineering is mostly Escherichia coli, but inclusion bodies often appear in the expression of Escherichia coli, and Escherichia coli is more toxic and low expression
[0005] Using the alcohol oxidase AOX1 promoter to express the target gene requires the use of methanol as a carbon source and inducer, but methanol is flammable and toxic, which may cause environmental pollution and fire hazards, resulting in residual methanol in the product
P GAP The promoter expresses porcine trypsin in Pichia pastoris X33, and this patent makes up for this deficiency

Method used

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  • Pig trypsinogen mutant and expression thereof in pichia pastoris
  • Pig trypsinogen mutant and expression thereof in pichia pastoris
  • Pig trypsinogen mutant and expression thereof in pichia pastoris

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Construction and enrichment of recombinant plasmid containing porcine trypsinogen

[0051] The recombinant plasmid was entrusted to Nanjing GenScript Company to synthesize, and CaCl was used after receiving the plasmid powder 2 The chemical transformation method was used to transform into Escherichia coli DH5α competent cells, and the operation steps were as follows:

[0052] Pick a small amount of Escherichia coli with an inoculating loop, inoculate it on the LB medium by streaking, and culture it in a constant temperature incubator at 37°C overnight. , 200r / min shaker culture, use a UV spectrophotometer to detect the OD of the bacterial suspension 600 value when OD 600 When the value is between 0.2-0.5, place on ice to stop growth. Take 1 mL of the above E. coli suspension with a pipette and place it in a centrifuge tube, centrifuge at 4°C and 4000 r / min for 5 min, discard the supernatant; add 100 μL of pre-cooled 0.1 mol / L CaCl to the centrifuge tube res...

Embodiment 2

[0054] Example 2 Construction of porcine trypsin yeast engineering bacteria

[0055] The Pichia pastoris expression plasmid was linearized with restriction enzyme AvrII, and electroporated into Pichia Pastoris X33 competent cells. The specific method is as follows:

[0056] The single colony Pichia X33 on the streak plate was inoculated into 10 mL of YPD liquid medium, and cultured at 30 °C and 250 r / min for 18 to 22 h;

[0057] Detect the OD of the bacterial suspension using a UV spectrophotometer 600 Put 50mL of YPD liquid medium in a 250mL conical flask, calculate the transfer amount according to the following formula, transfer Pichia X33 bacterial liquid, and shake at 30℃ and 250r / min for 18-22h;

[0058]

[0059] Where: OD 始 is the starting OD 600 value; V 1 is the transfer volume; V 2 is medium volume; t is culture time; OD 末 is the OD at the end of the culture 600 value. Example: Measured OD 始 = 16, put 50 mL of YPD liquid medium in a 250 mL conical flask, a...

Embodiment 3

[0067] Example 3 Expression of recombinant porcine trypsin yeast engineering bacteria

[0068] The above-mentioned recombinant engineered bacteria were inoculated in 10 mL YPD liquid medium according to 2% inoculum, and cultured at 30° C. at 250 r / min. After the cells grew, they were inoculated in 50 mL YPD liquid medium according to 1% inoculum, and 30 Cultivate at 250r / min and express for 48h. Recombinant porcine trypsinogen was secreted and expressed in liquid medium, and the expression of recombinant porcine trypsin was detected by SDS-PAGE. The results are shown in figure 1 (M is Marker, 1: supernatant protein of recombinant trypsin bacterial cell fermentation broth), a relatively obvious expression band appeared around 23kDa in the secreted fluid after expression.

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Abstract

The invention discloses a porcine trypsinogen mutant and an expression of the porcine trypsinogen mutant in pichia pastoris. The porcine trypsinogen mutant is cut by enterokinase to obtain a novel porcine trypsinogen mutant. According to the invention, PGAP is taken as the promoter, methanol induction is avoided, harm of methanol and pollution to the environment are reduced, and the method is safe and convenient. In addition, compared with wild type porcine trypsin, the porcine trypsin mutant has higher stability and activity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a porcine trypsinogen mutant and its expression in Pichia pastoris. Background technique [0002] Trypsin (Trypsin, EC 3.4.21.4) is a ubiquitous serine protease, especially in mammalian digestive system and insects. In mammals, trypsin is secreted from the pancreas and exists in the form of zymogen. The enterokinase secreted by the small intestine decomposes trypsinogen into an active digestive enzyme, which mainly acts on the peptide bond at the carboxyl terminus of arginine or lysine. It can also limit zymogen precursors such as chymotrypsinogen, carboxypeptidase, and phospholipase. [0003] Trypsin has a certain clinical effect on empyema, hemothorax, ulcer, surgical inflammation and surgical trauma in medicine, and also has a certain relieving effect on venomous snake bites. It is also commonly used in animal cell culture. deal with. It is clinically used for l...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/76C12N15/81C12N1/19C12R1/84
CPCC12N9/6427C12N15/815C12Y304/21004
Inventor 文良柱窦俊方宏清徐登圆辛中帅赵珊珊王媛朱家齐王玉刚
Owner 江苏万邦医药科技有限公司
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