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人干扰素-γ cDNA新变体的克隆与表达

Cloning and expression of a novel variant of human interferon-gamma cDNA.

作者信息

Nishi T, Fujita T, Nishi-Takaoka C, Saito A, Matsumoto T, Sato M, Oka T, Itoh S, Yip Y K, Vilcek J

出版信息

J Biochem. 1985 Jan;97(1):153-9. doi: 10.1093/oxfordjournals.jbchem.a135039.

Abstract

A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.

摘要

用人外周血淋巴细胞制备了一个cDNA文库,这些淋巴细胞是用植物血凝素和佛波酯联合处理诱导产生的,从其中分离出了poly(A) mRNA。通过寡核苷酸杂交法鉴定出了含有人γ干扰素(HuIFN-γ)cDNA的重组质粒。核苷酸序列分析表明,质粒pIFNγ-G4中HuIFN-γ cDNA的核苷酸和氨基酸序列在第9位氨基酸处与已发表的数据不同(谷氨酰胺为CAA,而赖氨酸为AAA)。质粒pIFNγ-G4中的cDNA在猴COS细胞中由猿猴病毒40早期启动子控制表达,并且细胞分泌出了具有生物活性的HuIFN-γ。该cDNA也被插入到携带大肠杆菌色氨酸启动子的表达载体中,并在大肠杆菌中表达。结果表明,第9位氨基酸处从赖氨酸到谷氨酰胺的转变可能不影响HuIFN-γ的比活性。

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