克隆的大鼠α干扰素基因在大肠杆菌中的结构与表达

Structure and expression in Escherichia coli of a cloned rat interferon-alpha gene.

作者信息

Dijkema R, Pouwels P, de Reus A, Schellekens H

出版信息

Nucleic Acids Res. 1984 Jan 25;12(2):1227-42. doi: 10.1093/nar/12.2.1227.

DOI:10.1093/nar/12.2.1227

PMID:6320120

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC318569/

Abstract

DNA synthesized by in vitro transcription on rat interferon (IFN) mRNA has been cloned and amplified as recombinant DNA. The nucleotide sequence of these rat IFN cDNA clones revealed i. the partial presence of the coding region of the gene and ii all cDNA clones were derived from the same subtype of rat IFN-alpha mRNA. Purified inserted fragments were used as a hybridisation probe against chromosomal "Southern blots" to show that at least twelve rat IFN-alpha-related sequences are present in the genome. A lambda-linked rat gene library was screened with the cDNA probes, resulting in an equivalent number of rat IFN-alpha-related hybrid phages. By use of a 3'-noncoding region as a probe, the chromosomal counterpart of the cDNA clones could be detected and the nucleotide sequence of its coding region has been determined. Expression of the coding region in E. coli yielded biologically active IFN, when tested for in vitro or in vivo antiviral activity.

摘要

通过对大鼠干扰素（IFN）mRNA进行体外转录合成的DNA已被克隆并作为重组DNA进行扩增。这些大鼠IFN cDNA克隆的核苷酸序列显示：i. 该基因编码区部分存在；ii. 所有cDNA克隆均源自大鼠IFN-α mRNA的同一亚型。纯化的插入片段用作针对染色体“Southern印迹”的杂交探针，以表明基因组中至少存在12个与大鼠IFN-α相关的序列。用cDNA探针筛选λ噬菌体连接的大鼠基因文库，得到了数量相当的与大鼠IFN-α相关的杂交噬菌体。以3'非编码区为探针，可检测到cDNA克隆的染色体对应物，并确定了其编码区的核苷酸序列。当对其体外或体内抗病毒活性进行测试时，编码区在大肠杆菌中的表达产生了具有生物活性的干扰素。