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Pichia yeast expressing recombinant human blood coagulation factor VII, and preparation and use thereof

A human blood coagulation factor and blood coagulation technology, applied in the field of constructs for transforming yeast cells, can solve problems such as low yield, changing protein immunogenicity, shortening residence time, etc.

Inactive Publication Date: 2009-03-04
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] For example, Escherichia coli can express a variety of proteins as a host, but its existence (1) lacks post-translational modification and processing of eukaryotic proteins, such as cleavage, glycosylation, formation of disulfide bonds, etc.; (2) the expressed protein Insoluble inclusion bodies are often formed, and complex renaturation is required to restore conformation and activity; (3) Bacterial proteins are not conducive to purification and other disadvantages
[0010] The Saccharomyces cerevisiae system also has limitations: (1) the yield is usually low; (2) the expression plasmid is easy to lose; (3) lacks a strong and tightly regulated promoter; (4) excessive glycosylation modification of foreign proteins It will seriously change the immunogenicity of the protein, reduce the activity, and shorten the residence time; (5) The secretion efficiency is poor and the purification is difficult

Method used

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  • Pichia yeast expressing recombinant human blood coagulation factor VII, and preparation and use thereof
  • Pichia yeast expressing recombinant human blood coagulation factor VII, and preparation and use thereof
  • Pichia yeast expressing recombinant human blood coagulation factor VII, and preparation and use thereof

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preparation example Construction

[0088] The preparation method of the expression cassette of the present invention comprises the following steps:

[0089] (1) site-directed insertion of the human coagulation factor VII gene into a plasmid comprising the start signal element AOX and the stop signal element AOX (TT) to obtain a single-copy expression plasmid;

[0090] (2) Transforming the single-copy expression plasmid to obtain a complete human coagulation factor VII expression cassette.

[0091] In a preferred embodiment of the present invention, the plasmid used can be: pPIC9K, pPIC3.5K, pAO815 or pHIL-S1, etc., as long as it contains the start signal element AOX and the stop signal element AOX (TT); pPIC9K plasmid is preferred.

[0092] The human blood coagulation factor VII gene can be inserted between the 5'AOX1 and 3'AOX(TT) of the plasmid, preferably between the BamHI restriction site and the EcoR1 restriction site.

[0093] Conventional methods such as PCR can be used to amplify the FVII gene with the...

Embodiment 1

[0115] Example 1. Secretion and expression of recombinant human coagulation factor VII with different N-terminal primary structures in different strains of Pichia pastoris

[0116] 1. PCR amplification of FVII target gene

[0117] 1.1 Primer design

[0118] Primers were designed with reference to the full gene sequence of human blood coagulation factor VII (SEQ ID NO: 1), and synthesized by Shanghai Sangong.

[0119] Primer 1 5′-GC CTCGAG AAAAGAGCCAACGCGTTCCTGGAGGAG-3' (SEQ ID NO: 3)

[0120] wxya

[0121] Primer 25′-GC CTCGAG AAAAGA GAGGCTGAAGCT GCCAACGCGTTC-3' (SEQ ID NO: 4)

[0122] XhoI GluAlaGluAla

[0123] Primer 35′-GC GAATTC GCTGCTGGGCTAGGGAAATGG-3' (SEQ ID NO: 5)

[0124] EcoRI

[0125] 1.2 PCR reaction

[0126] Use P1, P3 primers to carry out PCR, and amplify to obtain pPIC9K / FVII target gene (excluding Glu-Ala repeat sequence); use P2, P3 primer to amplify to obtain pPIC9K / Glu-Ala-Glu-Ala-FVII target gene (containing...

Embodiment 2

[0174] Example 2. Construction and enzyme digestion identification of human coagulation factor VII high copy recombinant

[0175] The method of repeating and cascading the complete expression unit is used to construct the multi-copy recombinant of recombinant FVII. In this study, the special properties of two isotail enzymes BamHI:G↓GATCC and BglII:A↓GATCT at both ends of the complete expression element were used to construct high-copy recombinants.

[0176]After digesting the single-copy recombinants constructed in Example 1 with BamHI and BglII enzymes, the target fragments were recovered, and the insert fragments were self-ligated by using the properties of the homologous enzymes, and then treated with BamHI and BglII enzymes. connection system. If the expression unit is forward repeating series, the homologous enzyme at the junction of the two units will not become any restriction enzyme recognition site after joining, and the forward junction structure will be preserved;...

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Abstract

The invention provides a yeast cell which expresses a human coagulation factor seven; the yeast cell integrates a structure which expresses the human coagulation factor seven; the structure comprises two to fifteen human coagulation factor seven expression cassettes which are serially connected and lined; each expression cassette subsequently comprises the following elements from 5' to 3': (a) a start signal element AOX; (b) a human coagulation factor seven gene; (c) a stop signal element AOX (TT); and the yeast cell expresses the activated human coagulation factor seven under the induction of methanol. The invention also provides the structure and a preparation method thereof, a preparation method of the yeast cell and the human coagulation factor seven expressed by the yeast cell. The two to eighteen copied yeast cells structured by the invention can be used to improve yield and reduce cost, and are applicable in the mass production of human coagulation factor seven preparation.

Description

technical field [0001] The present invention relates to the fields of bioengineering and immunity, in particular to a yeast cell expressing recombinant human blood coagulation factor VII, a construct for transforming yeast cells, and their construction method and application. Background technique [0002] Coagulation factor VII (FVII) is a vitamin K-dependent zymogen with serine proteolytic activity, which is mainly synthesized in the liver. Most of FVII in plasma is a single-chain inactive zymogen with a concentration of 0.5 ug / ml (10 nmol / l). There are traces of activated FVIIa in plasma, at a concentration of 10-110 pmol / l. [0003] After vascular damage, tissue factor (TF) exposure, FVII or activated FVII (FVIIa) can form a complex with TF, and under the action of FXa, thrombin, etc., the arginine 152 and isoleucine in the FVII chain The 153 position is cleaved into double strands and activated. The residues from position 1 to arginine 152 of proto-alanine constitute ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/12C12N15/55C07K14/745C12N9/74A61K38/17A61K38/46A61P7/04
Inventor 朱威楼觉人蔡蓓蓓
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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