摘要
为进一步了解哲罗鲑IGF-I,实验采用逆转录聚合酶链式反应（RT-PCR）方法,从哲罗鲑肝脏的总RNA中扩增出胰岛素样生长因子-I（IGF-I）的c DNA开放阅读框（open reading frame,ORF）序列。利用原核表达载体p S构建重组表达质粒（IGF-I/p S）,并将其转化到宿主大肠杆菌Rosetta后经IPTG诱导获得重组哲罗鲑IGF-I蛋白。经SDS-PAGE电泳检测,在35~50 ku有条带与预期相符,且重组蛋白以包涵体的形式存在。包涵体经变性/复性实验后,获得纯化的IGF-I融合蛋白。ELISA鉴定结果显示,目的蛋白能特异性识别抗鱼类IGF-I抗体,表明获得了具有免疫活性的哲罗鲑IGF-I蛋白。细胞增殖实验（MTT法）结果显示,重组IGF-I蛋白对大麻哈鱼胚胎细胞（CHSE-214）、鲤上皮细胞（EPC）及虹鳟性腺细胞（RTG-2）均有显著促增殖作用,表明获得的重组IGF-I蛋白具有细胞水平的生物活性。本研究为深入了解IGF-I在哲罗鲑生长发育中的调控机制及绿色高效促生长制剂的研发奠定基础。
Insulin-like growth factor-I（IGF-I） plays a major role in the control of growth and development in fish.Total RNA was isolated from H. taimen liver tissue. The c DNA encoding insulin-like growth factor I（IGF-I）peptide was amplified by reverse transcription polymerase chain reaction（RT-PCR） strategy using isolated total RNA as template. Recombinant expression plasmid（IGF-I/p S） was constructed by prokaryotic expression vector p S and transformed into E. coli Rosetta. The expression of recombinant IGF-I protein of H. taimen was induced by IPTG. There was a clear target band with expected size between 35~50 k D in SDS-PAGE electrophoresis, and the recombinant protein existed in the form of inclusion body. Purified IGF-I protein was obtained after the inclusion body was denatured/renatured. ELISA results showed that target protein can identify commercial fish IGF-I antibody, indicating that H. salmon IGF-I protein with immune activity was obtained. Cell proliferation assay（MTT） results showed that recombinant IGF-I protein can significantly promote the propagation of CHSE-214,EPC and RTG-2, indicating that the recombinant IGF-I protein had biological activity. The study has laid a foundation for the research of IGF-I function in the growth and development of H. taimen.
出处
《水产学报》
CAS
CSCD
北大核心
2016年第11期1657-1663,共7页
Journal of Fisheries of China
基金
国家科技支撑计划(2012BAD25B10)
公益性行业(农业)科研专项(201003055-14)~~
关键词
哲罗鲑
胰岛素样生长因子-I
原核表达
生物活性
Hucho taimen
insulin-like growth factors-I
prokaryotic expression
bioactivity
