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Site-directed mutagenesis genetic engineering batroxobin and uses thereof

A site-directed mutagenesis and genetic engineering technology, applied in the field of batroxobin, can solve problems such as proteins with complex sugar chains in spatial structure

Active Publication Date: 2008-12-10
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, proteins with complex spatial structures and sugar chains are not suitable for expression in prokaryotic cells

Method used

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  • Site-directed mutagenesis genetic engineering batroxobin and uses thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] According to the nucleic acid sequence of batroxobin (X12747) published in the U.S. Genebank, select the genetic codes that yeast likes, artificially synthesize the structural gene sequence of the site-specific transformation of batroxobin, and mutate the code AGA of the 45th Arg into AAG encoding Lys, the yeast endogenous KEX2 protease recognition sequence Arg-Arg is mutated to Arg-Lys, so KEX2 protease will not degrade batroxobin secreted into the fermentation broth at this site, so that the target protein can be produced in large quantities accumulation. In order to recombine the synthetic target gene into the yeast secretory expression vector pPICZα, an Xho I restriction site was added to the 5' end of the gene, and a stop codon TAATGA and a SacII restriction site were added to the 3' end of the gene. In addition, the codon AAAAGA corresponding to the KEX2 protease recognition sequence Lys-Arg is added between the Xho I restriction site and the first amino acid Val ...

Embodiment 2

[0067] Linearize pPICZa-Bg with DNA restriction endonuclease SacI, prepare yeast host competent and transform according to the method in Multi-Copy Pichia Expression Kit Version B of Invitrogen Company, and spread the transformed cells on YPD+500ug / ml It grows on Zeocin medium, and a single colony can appear after 3-4 days at 30°C. Clones with large and full colonies were selected for expression screening.

Embodiment 3

[0069] The highly expressed engineered bacteria screened out in test tubes were inoculated into shake flasks for proliferation as seed liquid, and then transferred to a 10L fermenter for pilot-scale fermentation according to the conventional method of methanol yeast fermentation. Induction for 72 hours. Centrifuge the fermentation broth to collect the supernatant, or concentrate and desalt it by ultrafiltration, and use hydrophobic column chromatography, ion exchange column chromatography, affinity column chromatography, gel column chromatography and other biochemical separation techniques to obtain the activity with a purity of more than 97%. protein.

[0070] The method for measuring the activity of similar products was used for the in vitro coagulation test. The specific test method was as follows: take 0.2ml of citrate anticoagulated plasma, put it into a test tube with a diameter of 1cm, put it in a water bath at 37°C for 3 minutes, and add it to preheat at 37°C Mix 0.2m...

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Abstract

The invention relates to a site-directed mutagenesis genetic engineering batroxobin is capable of clotting animal plasma containing an anti-coagulant agent. The site-directed mutagenesis genetic engineering batroxobin is characterized in that: compared with the amino acid sequence of a natural batroxobin, the amino acid sequence of the site-directed mutagenesis genetic engineering batroxobin has a mutation from Arg to Lys at the 45th digit. The site-directed mutagenesis genetic engineering batroxobin having higher bioactivity can be produced in a large scale through a secreted expression mode.

Description

Technical field: [0001] The invention relates to a gene recombination method for preparing site-directed mutation batroxobin—Batroxobin. The key is the site-specific transformation, synthesis, cloning, expression of the batroxobin gene, fermentation expression and separation and purification of the product. The mutated batroxobin can be used not only as the main component of the hemostatic drug, but also directly as the hemostatic drug. Background technique: [0002] Since 1963, the Austrian scholar Von Klobusitzky isolated a serine proteolytic enzyme (thrombin-like enzyme) from the venom of the Brazilian spearhead viper (Bothropsatrox) for the first time, also known as batroxobin. So far, more than 30 kinds of snake venoms have been found to contain thrombin-like components, and more than 20 kinds have been isolated and purified successively. They can all act on Arg in the A chain of fibrinogen in mammalian plasma. 16 -Gly 17 The peptide bond between them releases fibrin...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N9/74C12N1/19C12N15/57A61K38/48A61P7/04
Inventor 薛百忠薛雁王宏英徐梅兰海英沈文彧苏珊李英高欣
Owner GRAND LIFE SCI (LIAONING) CO LTD
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