鲤IGF2b基因内含子的克隆、基因组序列分析及慢病毒载体构建

Clone of IGF2b Introns,Analysis of Its Genomic Sequence and Construction of Lentiviral Vector(Lenti-IGF2-IRES-EGF) in Common Carp(Cyprinus carpio L.)

为了了解鲤IGF2b基因与鲤生长性状之间的关系,以建鲤为试验材料,克隆了IGF2b基因内含子,分析其基因组序列的特点,构建了IGF2基因的慢病毒载体,同时观察其在293T细胞中的表达活性。结果获得鲤IGF2b 5 173 bp长度的基因组DNA序列(HM755899),共有3个内含子,4个外显子;在3'非翻译区存在2个CpG岛,在5'端非翻译区存在(T)n重复序列;除此之外,成功构建了慢病毒载体质粒Lenti-IGF2b-IRES-EG-FP,转染293T细胞后,产生的重组慢病毒颗粒出现高表达绿色荧光,荧光定量PCR检测发现IGF2b基因在293T细胞中高表达。这些结果将为研究IGF2b基因在多态性、表达方面与鲤生长性状之间的关联奠定基础。

To explore the relationship between the IGF2b gene and growth traits, we cloned the introns of IGF2b in common carp （Cyprinus carpio ） muscle and analysis its characters. Further more, the lentivi- ral vector （Lenti-IGF2-IRES-EGF） was constructed and its activity was also researched. The results showed that： the length of 5 173 bp DNA sequence of IGF2b （HM755899） was get, including 3 introns and 4 exons. Two CpG islands in the 3＇untranslated region and （T） n repeat sequence in the 5 ＇untrans- lated region were found. Besides these, the lentiviral vector （Lenti-IGF2b-IRES-EGF） was constructedsuccessfully, which was cotransfected into 293T cells with the GFP expression. The higher expression of IGF2 was found in 293T cells by Real time PCR. These illustrated that these study may be lay on solid foundation for relationship between IGF2b genetic diversity, expression and common carp growth traits