引用本文:	张云云,周君,李晔,张春丹,蔡江佳,陈丽萍,李太武,苏秀榕.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.海洋与湖沼,2013,44(6):1506-1511.

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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
张云云¹, 周君¹, 李晔¹, 张春丹¹, 蔡江佳¹, 陈丽萍¹, 李太武², 苏秀榕¹
1.宁波大学海洋学院;2.宁波城市职业技术学院

摘要:

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。

关键词: 可口革囊星虫铁结合蛋白酵母双杂交文库真核表达

DOI：10.11693/hyhz201306014

分类号:

基金项目:国家自然科学基金资助项目, 40776075号, 41176123号; 海洋公益性行业科研专项经费资助项目, 201005016号; 宁波市科技局资助项目, 2008C50027 号。

附件

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

ZHANG Yun-Yun¹, ZHOU Jun¹, LI Ye¹, ZHANG Chun-Dan¹, CAI Jiang-Jia¹, CHEN Li-Ping¹, LI Tai-Wu², SU Xiu-Rong¹

1.School of Marine Science, Ningbo University;2.Ningbo City College of Vocational Technology

Abstract:

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.

Key words: Phascolosoma esculenta ferritin yeast two-hybrid library eukaryotic expression