Wild type and mutant hFIX (human coagulation factor IX) Pichia pastoris expression vectors, and construction method and application thereof

Abstract

The invention discloses a construction method and application of expression vectors of a wild type hFIX (human coagulation factor IX) and three hFIX mutants in Pichia pastoris. The construction method comprises the following steps: according to a gene sequence provided by Genebank, cloning a hFIX full-length cDNA (complementary deoxyribonucleic acid) sequence from healthy human liver through a RT-PCR (reverse transcription-polymerase chain reaction) technology, constructing a yeast expression plasmid pPIC9K-hFIX which is used for Pichia pastoris expression and is fused with a yeast alpha-factor signal peptide and a full-length hFIX sequence, transferring the plasmid into Pichia pastoris SMD1168, and screening out a high-level expressing yeast strain; and then, constructing a yeast expression plasmid pPICAZ-hFIX, designing and constructing three yeast hFIX high-activity mutants on this basis, electrically transferring into Pichia pastoris SMD1168, and screening out high-level expressing yeast mutant strains. The coagulation activity of each of the obtained various hFIX yeast expression vectors is higher than that of a standard hFIX. The 50L pilot fermentation research on the wild type hFIX (pPIC9K-hFIX) shows that the maximum protein purification product secretion expression quantity can be up to 558 mg/L and the purity and coagulation activity are high.

Description

technical field [0001] The present invention belongs to the field of molecular biology. More specifically, it relates to a high-efficiency expression vector of a wild-type human coagulation factor IX (hFIX) and three hFIX highly active mutants in Pichia pastoris, and also involves a wild-type human coagulation factor IX (hFIX) and three hFIX A method for constructing a high-efficiency expression vector of a high-activity mutant in Pichia pastoris. The sequences of these hyperactive mutants are also involved. It also relates to the use of a wild-type human blood coagulation factor IX (hFIX) and three kinds of high-efficiency expression vectors of hFIX highly active mutants in Pichia pastoris, a wild-type hFIX product expressed by Pichia pastoris (SMD1168-pPIC9K-hFIX ) in the pilot-scale fermentation process. Background technique [0002] Research progress of coagulation factor Ⅸ: [0003] Coagulation factor IX is an important coagulation-promoting factor in the "cascade r...

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[0022] 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 sh

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