中华蜜蜂AccMRJP1基因克隆及在大肠杆菌中的表达  

Cloning of royal jelly AccMRJP1 gene from Apis cerana cerana and its expression in Escherichia coli.

作　　者：张瓅文[1] 丁美会[1] 张伟光[1] 金凤[1] 沈立荣[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院食品科学与营养系,浙江杭州310029

出　　处：《浙江大学学报（农业与生命科学版）》2010年第2期119-124,共6页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家高技术研究发展计划"863"专题资助项目(2007AA10Z324)

摘　　要：根据中华蜜蜂Acc MRJP1表达序列标签(EST)序列,从已完成EST测序的蜂脑cDNA文库中选出3个Acc MRJP1克隆,通过PCR检测和测序筛选获得1个cDNA全长为1 302 bp、可编码433个氨基酸残基的Acc MRJP1开放阅读框(ORF),该氨基酸序列与已报道的Acc MRJP1编码序列(AY279539)的同源性为99.8%.将该Acc MRJP1基因克隆到表达载体pGEX-4T-2,转化大肠杆菌BL21中进行融合表达.SDS-PAGE电泳分析显示一条分子量大小约76 ku、占细胞总蛋白17.7%的特异性条带;以谷胱甘肽转移酶(GST)多克隆抗体为一抗作Western blot分析,检测到Acc MRJP1与GST的融合表达蛋白印迹,并通过亲和柱分离获得了纯化的表达产物,证明Acc MRJP1基因已表达成功,这为开展MRJP1的生物工程利用提供了技术基础.Three clones of AccMRJP1 were screened out from the sequenced brain cDNA library of Chinese honeybee according to the expression sequence tag （EST） of AccMRJP1 . Through identifying with polymerase chain reaction （PCR） and sequencing, a AccMRJP1 clone containing an open reading frame （ORF） of 1 302 nucleotides encoding a protein of 433 amino acids was determined. The AccMRJP1 had 99.8% similarity with the previously reported AccMRJPI sequence （AY279539） in amino acid sequences. The AccMRJP1 was sub-cloned into the prokaryotic expression vector pGEX-4T-2 for fusion expression in Escherichia coli BL21. Analysis result of the SDS-PAGE showed that the expression product contained a specific band of protein about 76 ku in size and accumulated up to about 17.7 % of the total bacterial proteins. The fusion protein was cross reactive with glutathione S-transferase （GST） polyclonal antibody and was purified through affinity chromatography, which confirmed the successful expression of GST-AccMRJP1. This work provids a technical base for the utilization of MRJP1 with biological engineering.

关 键 词：中华蜜蜂 蜂王浆 AccMRJP1 基因克隆 原核表达 

分 类 号：Q781[生物学—分子生物学]