大鼠神经肽Y前体融合蛋白在大肠杆菌中的超表达

OVEREXPRESSION OF RAT NEUROPEPTIDE Y PRECURSOR INE.COLIPROTEIN FUSION SYSTEM

应用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）从大鼠腋组织钓得神经肽Ｙ（ＮＰＹ）ｃＤＮＡ编码区序列。经ＤＮＡ序列测定证实其准确性后，将该ｃＤＮＡ定向亚克隆入一大肠杆菌的表达载体ｐＭＡＬ－Ｃ＿２的果糖结合蛋白（ＭＢＰ）基因中。ＤＮＡ测序表明ＮＰＹｃＤＮＡ与表达载体中ＭＢＰ开放阅读框架一致。将重组质粒转入大肠杆茵ＤＨ５α菌系中，该重组大肠杆菌在液体ＬＢ培养基中经１ｍｍｏｌ／Ｌ终浓度的ＩＰＴＧ诱导４ｈ，所表达的ＮＰＹ－ＭＢＰ融合蛋白产量高达大肠杆菌总蛋白量的６０％～７０％。超表达的ＮＰＹ经纯化后将为进一步进行结构与功能研究提供材料来源。

e report in this paper the overexpression of rat meuropeptide Y(NPY)precursor in an E.coli protein fusion and expression system. Rat neuropeptide Y cDNA coding sequence was ampli-fied by RT-PCR from rat brain tissues and into a PCR ⅡTA cloning vector. The clonedPCR product was sequenced to confirm the specific NPY coding region fragment and then sub-cloned into an E. coli protein fusion and expression vector pMAL-C_2 in the same open readingframe with a maltose binding protein(MBP)coding sequence in the vector.The MBP-NPY fu-sion protein expression is driven by a strong Ptac promoter.The recombinant plasmid pMAL-NPY was transformed into E .coli DH5α strain. Log phase recombinant E. coli Dh5α cells grownin LB Media were induced by IPTG at final concertration of Immol/L .for 4h. SDS-PAGE analy-sis showed that the yield of MBP-NPY fusion protein expression accounts up to 60%～ 70%oftotal E. coli proteins .The successful overexpression will provide sufficient material for hrtherNPY structural and functional studies.