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CONSTRUCTION OF cDNA LIBRARY BY YEAST TWO-HYBRID AND RECOMBINANT EUKARYOTIC EXPRESSION PLASMID OF PHASCOLOSOMA ESCULENTA
Author(s): 
Pages: 1506-1511
Year: Issue:  6
Journal: Oceanologia et Limnologia Sinica

Keyword:  Phascolosoma esculentaferritinyeast two-hybrid libraryeukaryotic expression;
Abstract: We constructed yeast two-hybrid library of Phascolosoma esculenta, containing 6.09×106 independent clones. The rate of recombination was 100% and the average size of inserts was larger than 1000bp. 200 clones were sequenced. The sequencing results show that there were 1 control plasmid and 67 unknown kinds of genes in total. Among all the 132 known genes, the genes related to mitochondrial protein accounted for 29.54%, hemerythrin 15.9%, ribosomal protein 10.6%, ferritin 8.3%, actin 6.1% and other genes 29.56%, including 5-amino levulinate synthase, bleomycin hydrolase-like, fibrinolytic protein, glutamine synthetase, heat shock protein 70, 90, nonspecific alkaline phosphatase-like, lipase, metalloprotein, thioredoxin, protein phosphatase, troponin C and ubiquitin. Based on the ferritin genes of P. esculenta, we constructed the recombinant eukaryotic expression plasmid pPink-HC-fer with primers designed by two enzyme sites EcoRⅠand KpnⅠ. The fragment was successfully constructed and judged by PCR and sequencing. The product was constructed successfully through identified by SDS-PAGE after transformed into yeast by electroporation. The molecular weight of the fusion protein is 23kDa.
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