摘要
利用重组PCR点突变技术将人IFNα 1c/ 86D的第 2 2、2 7位的Ser编码序列突变为Arg和Phe ,DNA测序正确 ,并在大肠杆菌BL2 1中表达 ,表达量为 19%。采用CM Sepharose、DEAE SepharoseFF纯化后WesternBlot检测为单一条带。细胞病变抑制法测定其比活为 3.5 1× 10 7U/mg ,较IFNα 1b( 1.5 5 10 7U/mg)略高。抗A5 4 9细胞的增殖活性与IFNα 1b相当。
In the recombinant human IFN α 1c/86D, we replace the residue Ser at the position 22 and 27 with the residue Arg and Phe respectively by PCR site specific mutagenesis. Analysis of DNA sequence demonstrated the IFN α Ic/86D/22R/27F was completely identical with expectation. Expression with high efficiency was obtained in E.coli BL21 and the expression level was 19% .The target protein was purified though cation exchange chromatograph(CM Sepharose) and anion exchange chromatograph(DEAE Sepharose). The result of Western blot showed as a single band. By the way of cell pathogenesis inhibition,the specifi cactivity of the purified protein was about 3.51 10 7 U/mg.The activity of IFN α 1c/86D/22R/27F Icseems higher than that of IFN α 1b.The activity of both IFN α 1c/86D/22R/27F and IFN α 1b is equivalente good on inhibiting growth rate ofcell A549.
出处
《微生物学免疫学进展》
2003年第4期28-32,共5页
Progress In Microbiology and Immunology
关键词
IFN—αlc
基因表达
点突变
生物活性
大肠杆菌
Interferon-α 1c
Gene expression
PCR Site-specific mutagenesis
Biological activity.
