摘要
目的 :探讨内含子对外源基因在真核细胞中表达的作用。 方法 :直接将含有 4个内含子和自身信号肽的 1.5 kb全长人生长激素基因直接克隆至真核表达载体 pc DNA3.0 ,然后利用脂质体转染家蚕 Bm N细胞 ,收取细胞上清 ,PAGE电泳和Western印迹分析鉴定表达产物 ;将全长基因序列、缺失第 1内含子基因序列和 c DNA序列的表达载体 ,分别转染细胞后检测各片段表达产物之间的差异。结果 :测序结果表明人生长激素基因组 DNA在细胞内正确转录、剪接。蛋白质电泳分析和免疫学检测证明转染细胞能够有效合成并分泌蛋白质。 EL ISA检测发现 3种基因片段中 ,全长的基因序列在表达效果上要高于后两者 (P<0 .0 5 ) ,其中缺失第 1内含子的序列表达效率较 c DNA序列明显降低 (P<0 .0 5 )。结论 :在家蚕细胞中能够正确表达带有自身内含子和信号肽的人源蛋白基因 ;
Objective: To study the effect of intron on the exogenous gene expression in the eukaryotic cells. Methods: A full length human growth hormone(hGH) genomic DNA fragment with 4 introns and the signal peptide sequence (1.5 kb) was cloned into the eukaryotic gene expressing vector pcDNA3.0, then the vector was transfected to the Bombyx mori N(BmN) cells with lipofectin. The cell total RNA was extracted and the hGH cDNA was amplified with RT PCR.The supernatant was collected at different time points. After cloning of 3 distinct fragments of hGH into expression vector,the difference among the 3 was measured. Results: The electrophoretic analysis and immunoblot of the recombinant protein showed that the transfected cell could synthesize and excrete the peptide effectively. ELISA assay found that of the 3 fragments,the expression efficacy of full length gene was higher than the others 2, and fragment lacking the first intron had the lowest expression. Conclusion: Our data indicate that in silkworm cells the human full length gene carrying the introns and signal peptide can be expressed,indicating that introns may have an important effect in the gene expression in body.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2003年第7期765-768,共4页
Academic Journal of Second Military Medical University
基金
国家"8 63"计划资助项目 ( 10 3 -12 -0 2 -0 6)
