可口革囊星虫(Phascolosoma esculenta)酵母双杂交文库和铁结合蛋白真核表达载体的构建

CONSTRUCTION OF cDNA LIBRARY BY YEAST TWO-HYBRID AND RECOMBINANT EUKARYOTIC EXPRESSION PLASMID OF PHASCOLOSOMA ESCULENTA

构建了可口革囊星虫(Phascolosoma esculenta)的酵母双杂文库,总克隆数为6.09×106CFU,重组率100%,平均插入片段长度>1kb。随机挑取200个克隆子测序,结果 1个空载,67个无法对应的基因类型,已知的132个基因中线粒体蛋白占29.54%,蚯蚓血红蛋白15.9%,核糖体蛋白10.6%,铁结合蛋白8.3%,肌动蛋白6.1%,其它基因29.56%。这些基因分别为:5-氨基乙酰丙酸合成酶、类博莱霉素水解酶、纤溶蛋白、谷氨酰胺合成酶、热休克蛋白、非特性类碱性磷酸酶、脂肪酶、金属蛋白、线粒体、硫氧还蛋白、蛋白磷酸酶、肌钙蛋白C、泛素。根据已获得的可口革囊星虫铁结合蛋白基因,以EcoRⅠ和KpnⅠ为双酶切位点设计引物,构建了可口革囊星虫铁结合蛋白真核表达载体pPink-HC-fer,经PCR及测序检验,序列完全正确,然后电转化至酵母细胞中诱导表达,经SDS-PAGE检验其融合蛋白分子量大小约为23kDa。

We constructed yeast two-hybrid library of Phascolosoma esculenta, containing 6.09×106independent 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. e s c u -lenta, 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.