Method for improving expression of recombinant human macrophage inhibitor-1(MIC-1) in pichia yeast

A macrophage, inhibitory factor technology, applied in the field of molecular biology, can solve the problems of difficulty in mass production, low yield, and high cost of CHO cell expression system, and achieve production yield improvement, production cost reduction, and simple and easy process. Effect

Inactive Publication Date: 2008-07-23
张伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art is to express wild-type recombinant MIC-1 protein in CHO cells and Pichia pastoris eukaryotic cell expression system, but the cost of CHO cell expression system is high and the yield is low; Only 3~8mg / L, it is difficult to meet the demand of mass production

Method used

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  • Method for improving expression of recombinant human macrophage inhibitor-1(MIC-1) in pichia yeast
  • Method for improving expression of recombinant human macrophage inhibitor-1(MIC-1) in pichia yeast
  • Method for improving expression of recombinant human macrophage inhibitor-1(MIC-1) in pichia yeast

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1) Obtain and optimize the MIC-1 gene fragment

[0026] According to the sequence characteristics of human macrophage inhibitory factor-1 gene and the sequence characteristics of the multiple cloning site of Pichia pastoris expression vector pPIC9K plasmid, three upstream primers and downstream primers were designed. First select the upstream primer 1 and the downstream primer, and amplify the MIC-1 wild-type gene fragment from human placenta tissue by RT-PCR method; then use the upstream primer 2, upstream primer 3 and downstream primer to perform 2 rounds of PCR amplification , and finally obtain the mutant MIC-1 gene fragment with optimized codons. 50μl PCR reaction system:

[0027] 10×Buffer

dNTP (2.5mM)

Upstream primer (50pM)

Downstream primer (50pM)

template

Pfu DNA polymerase (5U / μl)

ddH2O

5μl

1μl

1μl

1μl

1μl

1μl

40μl

total capacity

50μl

[0028] Reaction pro...

Embodiment 2

[0045]1) Induced expression of Pichia pastoris MIC-1 strain:

[0046] Each transformant was cultured in 5ml BMGY medium at 28-30°C with shaking at 200-250rpm / min; the next day, take an appropriate amount of bacterial liquid to re-inoculate in 100ml BMGY medium, and culture at 28-30°C for 6-8 hours. to OD600=2~6; discard the supernatant after centrifugation under sterile conditions, resuspend the cells with 100ml BMMY medium, and start induction by shaking culture at 200~250rpm / min at 28~30°C; during the induction process, supplement methanol every 24 hours to The final concentration was maintained at 3%, and the supernatant medium of each transformant was collected by centrifugation after culturing for 72 hours, and induced without adding methanol was used as a blank control.

[0047] 2) Electrophoresis detection and yield determination of the target protein:

[0048] Collect the induced expression medium, and use reducing and non-reducing Tricine SDS-PAGE protein electrophor...

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Abstract

The invention relates to a method for improving expression of recombination human macrophage inhibitory factor in pichia yeast, which is characterized in that gene sequence of the recombination human macrophage inhibitory factor is modified by molecular biological method, facilitating the expression thereof in the pichia yeast; external secretion expression vector containing the recombination human macrophage inhibitory factor is constructed, GS115 pichia yeast is transformed through PEG1000 method, multi-copy clone is screened through G418 resistance concentration increasing method, and high expression pichia yeast engineering bacteria GS115 / pPIC9K-MIC-1 is obtained; the macrophage inhibitory factor pichia yeast engineering bacteria is expressed through methanol induction, and the expression product is directly secreted outside the cell; the obtained supernatant is subject to centrifugation, and then the recombinant protein expression product-macrophage inhibitory factor is contained in the supernatant. The method for improving expression of recombination human macrophage inhibitory factor in pichia yeast has the advantages of simple purification process, low cost, and large-scale expression, and lays the foundation for function research and antibody preparation through antigen in the future.

Description

Technical field: [0001] The invention relates to a Pichia pastoris engineering bacterium (Pichia pastoris) for increasing the expression level of recombinant human macrophage inhibitory factor-1 (MIC-1) and a construction method thereof, belonging to the field of molecular biology. technical background: [0002] MIC-1 is a new gene found in the cDNA library of U937 cells when Bootcov et al. studied macrophage-related active factors in 1997. The protein encoded by it belongs to the TGF-β superfamily member. With the discovery in other tissues and organs, MIC-1 was named PTGF-β, PDF, GDF-15, PLAB, NAG-1 and so on. Studies in the past 10 years have shown that MIC-1 plays different roles in the growth and development of the fetus, the inflammatory response involving macrophages, the occurrence and development of tumors, and acute injury. Current studies have found that MIC-1 may be used as a new tumor marker and early warning indicator of miscarriage, and its application value ...

Claims

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Application Information

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IPC IPC(8): C12N15/67C12N15/81C12N15/12C07K14/475
Inventor 程东婉张伟田海梅王小兵李茉李艳芬
Owner 张伟
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