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The Expression of Porcine Granulocyte-macrophage Colony Stimulating Factor Gene in Pichia Pastoris

Author: TangBinChuan
Tutor: WenXinTian；CaoSanJie
School: Sichuan Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Pig granulocyte - macrophage colony-stimulating factor Pichia Yeast expression
CLC: S858.28
Type: Master's thesis
Year: 2010
Downloads: 70
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Abstract


Granulocyte - macrophage colony stimulating factor (GM-CSF) is a multifunctional cytokine biological activity. GM-CSF on the body inflammation, cancer and other diseases have a better inhibitory or adjuvant therapy, but also has a significant immune enhancement, and has good application prospects. Having a biological activity of GM-CSF protein in vitro expression of great significance. The research was conducted porcine GM-CSF (pGM-CSF) gene expression in Pichia vitro studies. 1, Pichia expression vector pPIC9K-pGM study for the construction of all containing pGM-CSF coding region of the gene vector 19T-GM to design a pair of primers to amplify the signal pGM-CSF gene encoding mature peptide, the size of 384bp ; pGM-CSF gene was inserted into pMD19-T Simple Vector, successfully constructed pMD19-T-pGM cloning plasmid; pMD19-T-pGM by EcoR Ⅰ and Not Ⅰ double digestion, recall pGM-CSF gene was inserted into the same double digestion process vector pPIC9K to construct Pichia expression vector pPIC9K-pGM, right pPIC9K-pGM identified by PCR and restriction analysis showed that pPIC9K-pGM successfully constructed expression vectors. 2, GS115/pPIC9K-pGM recombinant screening will pPIC9K-pGM linearized by Sac Ⅰ and then recovered by ethanol precipitation, was transformed into Pichia pastoris host strain GS115 competent cells inoculated in YPD plates, and cultured at 30 ℃ 3d, medium grow milky white colonies, indicating pPIC9K-pGM recombinant transforming success; recombinants G418/YPD concentration gradient filter, which results in the G418/YPD 4mg/mL medium to obtain high copy recombinants; the high-copy recombinant phenotypic identification screening, successfully screened Mut-type high-copy recombinants; would Mut-type high-copy recombinants amplified by PCR amplification of the expected 384 bp fragment, indicating that the recombinants screened properly, and this high-copy recombinant bacteria named GS115 / pPIC9K-pGM. 3, GS115/pPIC9K-pGM recombinant expression in Pichia pastoris will GS115/pPIC9K-pGM activated in BMGY medium to an OD600 of 2.0 to 2.5 when the cells were harvested, BMMY medium was adjusted to OD600 = 1.0, at 30 ℃ , 300 r / m for air culture incubator expression, while methanol was added once every 24 h, to a final concentration of 1% (v / v). The expression products were indirect ELISA and SDS-PAGE, and the results show that pGM-CSF to obtain the expressed protein molecular weight of about 18 kD, and the expression best time 4 d. The western-bloting protein expression analysis, the results expressed proteins with pGM-CSF positive serum-specific binding, indicating that the product has reactogenicity. The pGM-CSF protein expressed after filter sterilization TF-1 cell proliferation assay, the protein can promote the TF-1 cell proliferation ability, and high activity (0.88 ~ 1.05) × 104IU/μg, showed the product to have a high biological learning activity.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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