人γ干扰素突变体(rIFN-γ132-PGIL)的构建及其生物学活性的测定

CONSTRUCTION OF AN IFN-γ MUTANT (rlFN-γ 132-PGIL ) AND CHARACTERIZATION OF ITS ANTIVIRAL ACTIVITY

利用DNA重组技术,去掉人γ干扰素(IFNγ)基因3′端含编码多肽羧基(C)端11个氨基酸的核苷酸序列,与编码P,G,I,L的DNA序列相连接,构成IFNγ突变体(rIFN-γ132-PGIL)基因,将后者插入pBV220P_RP_L串连启动子下游,转化大肠杆菌DH5α,在CIts857基因的调节下,通过升温诱导,获得了高效表达,表达量约占菌体可溶性蛋白的30％以上,抗病毒活性平均可达4.9×10^-8U/L,比母体菌株高10倍。SDS—PAGE结果表明,rIFN-γ132—PGIL分子量为17kd。

An interferon gamma mutant (rIFN-γ132-PGIL) gene was constructed by removing the 3' termini coding 11 amino acids from the natural IFN-γ gene and replaced by the DNA sequence coding PGIL. This mutant gene was inserted downstream from the PL PR promoter transcription start site of the pBV220 and highly expressed in E.coli DH5α after temperature induction. The expressed mu.tant protein was estimated to constitute more than 30% of the total cellular soluble proteins. The average antiviral titer was about 4.9×109 U per liter culture, which was ten times higher than that of its parent. The result obtained from SDS-PAGE showed that he molecular weight of mutant protein was about 17kd. The hydrophobi-city of the mutant polypeptide was shown to be similar to that of its par-ent.The relationship the antiviral activity and its 3' terminal sequence was discussed.