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Human blood coagulation factor IX mutant pichia pastoris expression vector and construction method and application thereof

A plasmid and sequence technology, applied in the field of molecular biology, can solve the problems of relatively few reports on the fermentation process of fermenters

Inactive Publication Date: 2013-04-24
WUHAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] At present, the technical research on high-density expression of phytase is mainly concentrated on the shake flask level, and then on the basis of the shake flask, the scale-up culture is carried out in the fermenter, but there are relatively few detailed reports on the fermentation process at the fermenter level

Method used

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  • Human blood coagulation factor IX mutant pichia pastoris expression vector and construction method and application thereof
  • Human blood coagulation factor IX mutant pichia pastoris expression vector and construction method and application thereof
  • Human blood coagulation factor IX mutant pichia pastoris expression vector and construction method and application thereof

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Effect test

Embodiment 1

[0077] The construction method of the high expression vector of wild type and mutant hFⅨ in Pichia pastoris comprises the following steps:

[0078] Table 1

[0079] Primer name

Primer sequence

P01s

5'-GG GGATC CTAGCAAAGGTTATGCAGCGCGT-3′

P01a

5'-GG CTTAAG TTCAGGCACCTTACTTCATCCC-3'

P05a

5'-GG GCGGCCGC ATTAGTTAGTGAGAGGCCC-3'

P05s

5'-GG GAATTC TATAATTCAGGTAAATTGGAAGAGTT-3'

P06s

5'-GG CTCGAG AAAAGAGAGTATAATTCAGGTAAATTGG-3'

P07s

5'-CAAGGTGGTTTGCTCCTGTA-3'

P07a

5'-TAGCTGCATTGTAGTTGTGGTG-3'

5 AOXs

5′-GACTGGTTCCAATTGACAAGC-3′

3AOX

5′-GCAAATGGCATTCTGACATCC-3′

[0080] 1. Total RNA extraction:

[0081] (1) Extract total cellular RNA from the human fetal liver cell line L-02 (Kunming Cell Bank, Chinese Academy of Sciences Type Culture Collection Committee), add 1.3ml Trizol reagent, homogenize with a homogenizer for 10min, until all are homogenized into a p...

Embodiment 2

[0201] The application of hFⅨ Pichia pastoris high activity mutant expression vector in the production of human blood coagulation factor Ⅸ protein comprises the following steps:

[0202] 1. Seed activation and cultivation

[0203] (1) Recover strains: Take out about 1ml of hFIX wild-type Pichia expression strains stored in a sealed cryopreservation tube from the -80°C refrigerator, dissolve slowly at room temperature, take out 200ul in a clean bench and add 200mL of YPD liquid The culture medium was shaken at 28°C and 250rpm for 24h.

[0204] (2) Screening of bacterial strains: Sampling and detecting the OD600 value, and observing the growth of the bacteria, the strains can be screened if the OD value is in the range of 2 to 6, and the bacterial liquids with different dilution times are used for smearing bacteria, and are respectively applied on the YPD plate (plus or 2000ugml of G418 was not added), and the screening was carried out after 72 hours of observation in a 28°C in...

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Abstract

The invention discloses a human blood coagulation factor IX mutant pichia pastoris expression vector and a construction method and application thereof. The procedures of the construction method of the human blood coagulation factor IX mutant pichia pastoris expression vector includes that according to a hFIX gene sequence, the reverse transcription-polymerase chain reaction (RT-PCR) technology is used for cloning hFIX overall length complementary deoxyribonucleic acid (cDNA) from human hepatic cells, signal peptide mixed with yeast Alpha -factor and yeast expression plasmids pPIC9K-hFIX are constructed to express pichia pastoris, and then based on that the yeast expression plasmids pPICZ Alpha A-hFIX are constructed, four saccharomycetes hFIX high-activity mutants are constructed. Cruor activity of the achieved hFIX yeast expression vector is apparently higher than that of standard hFIX. One of the mutants is through 50-litre pilot scale test of fermentation, protein purification product secretory expression quantity is 702 milligrams / litre, so that a good foundation is laid for producing efficient hFIX products used for treating hemophilia B in a low cost and industrialized mode.

Description

technical field [0001] The present invention belongs to the field of molecular biology. It specifically relates to the expression vector of human blood coagulation factor Ⅸ (hFⅨ) mutant Pichia pastoris, and also involves the construction method of the expression vector of human blood coagulation factor Ⅸ (hFⅨ) mutant Pichia pastoris, and also relates to the human blood coagulation factor Ⅸ (hFⅨ) mutant Pichia pastoris Application of expression vector of red yeast in the production of hFⅨ protein. Background technique [0002] Research progress of coagulation factor Ⅸ: [0003] Coagulation factor IX is an important coagulation-promoting factor in the "cascade reaction" of blood coagulation. It is mainly synthesized by hepatic parenchymal cells and secreted into peripheral blood in the form of zymogen. When various internal and external factors activate the endogenous coagulation reaction, coagulation factor IX is activated, a series of coagulation chain reactions occur, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C07K14/745C12R1/84
Inventor 张同存周俊戴永刚王震宇吴尘宇成采莲唐文心
Owner WUHAN UNIV OF SCI & TECH
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