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Recombined yeast strain secreting and expressing human Kallistatin and production process thereof

A yeast strain and secretory technology, applied in the field of molecular pharmacy, can solve problems such as uneven processing, incorrect processing, and difficulty in large-scale production, and achieve the effects of easy process production, high component level, and convenient purification.

Inactive Publication Date: 2010-06-09
刁勇 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inventors used Pichia pastoris to express human Kal protein, and found that the expression level of recombinant strains obtained by conventional technical methods was very low, and it was difficult to produce on a large scale
The purification process is also more complicated
The signal peptides used in the existing commercially available yeast secretory expression vectors are almost all α mating factor signal peptides. The post-translational processing of recombinant proteins requires the participation of two different enzymes, which often results in uneven and incorrect processing. The N-terminal sequence of the protein is deviated

Method used

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  • Recombined yeast strain secreting and expressing human Kallistatin and production process thereof
  • Recombined yeast strain secreting and expressing human Kallistatin and production process thereof
  • Recombined yeast strain secreting and expressing human Kallistatin and production process thereof

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Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0012] 1 Construction of engineered strains containing α mating factor signal peptide

[0013] 1.1 Construction of expression vector pPIC9-Kal01

[0014] Using pAAV-Kal containing the full-length Kal cDNA sequence as a template, the upstream primer 5'-aacctcgagaaaagagatggtgagagttgcagtaacagct-3' was used to introduce the Xho I restriction site, and the downstream primer 5'ccagaattcctatggtttcgtggggtcgacgacc-3' was used to introduce the EcoR I restriction site, and Pyrobest DNA polymerase, the DNA sequence corresponding to human Kal mature protein is obtained by PCR. The target fragment was recovered, digested and inserted between the Xhol / EcoRI sites of the pPIC9 vector. After transformation, the positive clones meeting the requirements were screened by colony PCR, enzyme digestion and other methods, and pPIC9-Kal01 was entrusted to Dalian Bao Biology Co., Ltd. for sequencing.

[0015] 1.2 Construction and identification of engineering strain Kal01

[0016] The pPIC9-Kal01 ex...

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Abstract

The invention relates to a method and a process for producing an antineoplastic drug human Kallistatin, relating to a key construction technology and a construction result of a yeast engineering strain secreting and expressing the human Kallistatin, an optimal fermentation condition for producing the human Kallistatin and a purification method of the human Kallistatin by the key construction technology and the construction result.

Description

technical field [0001] The invention relates to molecular pharmacy, biotechnology and disease prevention and treatment, in particular to the production process of human Kallistatin (Kal) produced by yeast strains, the construction of secreted expression vectors, fermentation conditions and purification methods. Background technique [0002] At present, malignant tumors have become the leading fatal diseases in developed countries and regions. Although surgery, radiotherapy and chemotherapy are still being developed as conventional treatments, due to their inherent limitations, their curative effect has basically reached the limit, so there is an urgent need to develop new treatments. [0003] Since Folkman first elucidated the relationship between tumor and neovascularization in 1971, and put forward a hypothesis about the molecular mechanism, anti-angiogenesis therapy targeting tumor neovascularization has become a hot spot in anti-tumor research. [0004] Kal is a new typ...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12P21/02C07K1/22C07K1/20C12R1/84
Inventor 黄晓平李招发刁勇许瑞安
Owner 刁勇