[Mutation of LoopAB in HuIFN 1c/86D and enhancement of antiviral activity].
BACKGROUND
Based on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.


METHODS
Two unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.


RESULTS
The mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.


CONCLUSIONS
The authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.
Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.
Rong
R
Hu
Xuejun
X
Ma
Kaikun
K
Wei
Hong
H
Wang
Zhenchao
Z
Qian
Shuixing
S
Xue
Zhaojun
Z
Duan
Yunde
Y
Hou
D019943:Amino Acid Substitution; D000914:Antibodies, Viral; D000998:Antiviral Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D006801:Humans; D007370:Interferon Type I / Q000235:genetics* / Q000494:pharmacology; D016898:Interferon-alpha; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins
D000914:Antibodies, Viral; D000998:Antiviral Agents; D007370:Interferon Type I; D016898:Interferon-alpha; D010446:Peptide Fragments; D017471:Receptors, Interferon; D011994:Recombinant Proteins; C044342:interferon, alpha 1(111-166)