摘要
人促红细胞生成素(EPO)是一种调控红系干细胞增殖、分化和成熟的糖蛋白激素。将合成的EPOcDNA插入杆状病毒转移载体pBlueBacⅢ,使其置于Ph基因强启动子控制之下,获得了转移载体pBlueBacEPO。将pBlueBacEPODNA与野生型BmNPVDNA共转染BmN细胞,经空斑纯化,获插入EPOcDNA的重组病毒rBmNPVEPO。经Sonthern杂交和PCR扩增鉴定证明人EPO基因已正确组建于BmNPV的预定位置。将重组病毒rBmNPVEPO穿刺接种5龄幼虫和蛹,收集感染第3～5d的幼虫血淋巴和3～65d蛹血淋巴。用ELISA检测幼虫血淋巴中EPO表达量高达62800u/mL,蛹血淋巴中表达量达74000u/mL。Westernblot结果显示幼虫血淋巴和蛹血淋巴均有一条明显的免疫杂交带,分子量均约为26kD。用TF1细胞对幼虫表达产物进行了生物活性测定,每毫升血淋巴中EPO活性约为63000u。
Erythropoietin (EPO) is a glycoprotein hormone produced primarily by the kidney,and is principal factor regulating red blood cell poduction. The synthesized EPO cDNA was inserted into the transfer vector pBlueBacⅢ to generate the recombinant transfer plasmid pBlueBacEPO. Cotransfection of BmN cells with pBlueBacEPO DNA and Wild BmNPV DNA generated the recombinant virus rBmNPVEPO carrying EPO gene driven by the strong promoter of AcNPV polyhedrin gene. The results of Southern blot and PCR reaction confirmed that EPO gene had been correctly inserted in the target position in the BmNPV genome. ELISA assay showed that the EPO gene was expressed with high level in the larvae and pupae of the silkworm. The larvae and pupae produced as high as 62 800 u and 74 000 u in 1mL hemolymph on the 4th day (larve) and the 5th day(pupae) after infection with the recombinant virus rBmNPVEPO,respectively. Western blot analysis showed the molecular weight of rhEPO produced in the lavae or pupae was about 26kD. Biologic assay showed the rhEPO had high activity (about 63 000 u in per milliliter of hemolymph) in vitro.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第1期46-50,共5页
Chinese Journal of Biotechnology
基金
国家高技术研究与发展计划项目部分内容
关键词
hEPO
杆状病毒-昆虫表达系统
家蚕
基因表达
Bombyx mori nuclear polyhedrosis virus,human erythropoietin gene,expression,Insect cells,silkworm larvae and pupae
