Construction and expression method of recombinant g-csf(15-75) polypeptide vector

Abstract

The invention discloses a construction method of a recombinant G-CSF polypeptide (15-75) vector and a method for efficiently expressing the recombinant G-CSF polypeptide (15-75) in Pichia pastoris. The recombinant vector is formed by cloning a G-CSF polypeptide (15-75) gene into a pPIC9K expression vector, and transforms Pichia pastoris GS115, and methanol is utilized to induce the expression of the recombinant G-CSF polypeptide. The pPIC9K-G-CSF-GS115 recombinant Pichia pastoris constructed by the method can efficiently and stably express the recombinant G-CSF polypeptide (15-75). The expression level of the target protein is 2.8 g / L above, the purity of the purified recombinant G-CSF polypeptide (15-75) is up to 99.8%, and the biological specific activity is 1.5*10<8>IU / mg.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to the construction of an expression vector of G-CSF (15-75) polypeptide and its expression in Pichia pastoris. Background technique [0002] The main function of granulocyte stimulating factor (granulocyte colony stimulating factor, G-CSF) is to stimulate the proliferation and differentiation of hematopoietic cells of the neutrophil lineage, increase the number of neutrophils in peripheral blood, activate the function of neutrophils, and prevent tumors after chemotherapy. Patients injected with G-CSF can increase the level of circulating neutrophils, which may be related to shortening the time for some bone marrow cells to enter the S phase and increasing the number of progenitor cells that produce granulocytes. [0003] G-CSF was put on the market by the US FDA in 1991 for the treatment of neutropenia after radiotherapy. Now there are many companies in production and ...

AI Technical Summary

Problems solved by technology

[0005] Chinese patent CN103233053A discloses a production method of recombinant human granulocyte-stimulating factor, using recombinant plasmid pKG931 as the expression vector, and Escherichia coli HB101 as the recipient bacterium to improve production efficiency. Only 50% of human granulocyte-stimulating factor is expressed

[0006] Chinese patent CN103114115 discloses a method for preparing recombinant human granulocyte colony-stimulating growth factor by screening with microorganisms, and its pharmaceutical composition and preparation use engineering bacteria of recombinant human granulocyte colony-stimulating factor for fermentation and culture; Ultrasonic crushing of the obtained bacteria, collection of inclusion bodies, washing with inclusion body washing solution and centrifugation at low temperature; inclusion body dissolving solution was added to the inclusion bodies, and after 8 to 12 hours at 4°C, centrifugation to obtain inclusion body extracts; SephacrylS -200 gel column to separate the extract, collect the refolded recombinant human granulocyte colony-stimulating factor fraction; then use SephadexG25 column to separate, and finally obtain high-purity, high-activity G-CSF, but the recovery rate is only 47.5 %

[0008] At present, most of the G-CSF produced in China is the expression product of Escherichia coli. The use of E. coli requires renaturation during the production process, and the renaturation rate is low, and the specific activity is relatively low; the use of polyethylene glycol-modified G-CSF , its biological activity will decrease; the expression of Saccharomyces cerevisiae engineered strains belongs to non-integrated expression, the expression process is unstable, the product expression level is low and the gene is easily lost.

Images