新型基因工程干扰素受体结合域的改造及其生物活性测定

目的 改造干扰素功能结合域，以提高干扰素的生物学活性。方法 在新型基因工程干扰素（IFNα1c/86D)AB环内通过点突变技术引入2个独立酶切位点EcoRV和EstEⅡ，用聚合酶链反应（PCR）技术在干扰素cDNA水平对母体干扰素LoopAB的31位甲硫氨酸换成天冬氨酸（M－D），32位天冬氨酸换成脯氨酸（D－P），将重组基因在大肠埃希菌中表达，用滤泡口炎病毒（VSV）－人羊膜传代细胞（WISH）系统检测抗病毒活性。用比色MTT实验检测抗增殖活性。结果 通过突变，31和32位氨基酸获得重组突变体3132IFNα1c/86D，经限制性内切酶图分析、DNA序列和抗病毒活性测定，初步表明3132IFNα1c/86D是母体干扰素IFNα1c/86D抗病毒活性的8倍，抗增殖作用与母体干扰素相比差异无显著性。结论 改造干扰素受体结合域，可提高干扰素的抗病毒活性。

Mutation of LoopAB in HuIFNα1c/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.