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Eukaryon Expression Of The Recombinant PGH In Pichia Pastoris And Its Bioactivity Study

Posted on:2007-09-14Degree:MasterType:Thesis
Country:ChinaCandidate:R B FuFull Text:PDF
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Firstly in my research, Porcine growth hormone (pGH) gene was correctLy inserted into pMD-T pLasmid in order to construct the expression pLasmid pPIC3.5k-pGH. The recombinant plasmid pMD-T was transformed into E.coli JM109competent cells. The positive clone was selected out by colony PCR and plasmid PCR. We used BamH I and EcoRI digestion to identify the recombinant pLasmid. Then, we obtain pGH gene fragment by BamH I and EcoRI digestion, use gel to harvest, and directly insert into pPIC3.5k plasmid to produce a recombinant plasmid, which was named pPIC3.5k-pGH. After linearly cleavage, the pPIC3.5k-pGH plasmid was used to transform GS115 yeast by lightning stroke,then I used MD and MM mediums to screen the recombinants, cracked the cells and extracted the genome DNA,doing further identification by PCR, applied G418 antibiotic to copy number screening,employed methanol to induce protein expression,the protein product was analyzed by SDS-PAGE and western—blot,thus I have got a pGH high expression recombinant yeast,which was denominated as pPIC3.5k-pGH-GS115. SDS-PAGE and Western—blot assayes have proved the recombinant yeasts have been induced to express the pGH protein, whose molecular weight was close to 25kDa,which is in accordance with expected 24.4kDa pGH fusion protein. After pPIC3.5k-pGH-GS115 yeasts was induced by methenol,I sampled the fementation broth in 48h, 72h, 96h and 120h respectively in order to find out the best condition and time of expression. I havested the correctly expressed fusion protein from thr polyacrylamide gel.,then...
Keywords/Search Tags:pGH, pPIC3.5k plasmid, eukaryotic expression, Mus musculus albus, growth promotion activity
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