关键词：干扰素α；骨肉瘤；基因表达
【摘要】目的 构建两种新型IFN突变株及其母体的原核表达系统，表达后对比检测其抗骨肉瘤活性。方法 以重组噬菌体pCANTAB5E-IFNs为模板，PCR扩增出两种IFNα1c/86D突变株及其母体的DNA序列，插入原核表达载体pBV3203。表达、纯化后进行SDS-PAGE电泳鉴定、Westernblot检测其免疫活性、MTT比色法对比检测其抗骨肉瘤细胞(OS732)增殖的活性。结果酶切及PCR鉴定证明IFNs在表达载体上均已克隆成功。两种突变IFNs的抗骨肉瘤活性分别比IFNα1c/86D提高4和16倍。结论 两种新型IFNs突变体的原核高效表达系统构建成功，体外实验中证明其IFNs蛋白产物抗OS732细胞增殖的活性高于母体。
Cloning and Expression of Its Anti-osteosarcoma Bioactivity of Two New Types of IFNα
Hai , JIN Dadi, SHI Zhanjun, et al.
Department of Orthopaedics, Nanfang Hospital, Guangzhou 510515
【 Abstract 】 Objective In this study two prokaryotic expression systems of novel IFN mutants were constructed. After expression, the targetproteins were purified, and then their anti- osteosarcoma biological activity was compared with the parent substance. Methods Recombination phagemid- pCANTAB5E- IFNs were used in polymerase chain reaction (PCR) designed for specific amplification of two IFNα 1c/86D variants and their parent substance DNA sequences. Then the sequences were cloned into the bacterial expression vector- pBV3203, after induction, IFNs was secreted into the periplasm of JM103. Recombinant human IFNs(rhIFNs) were characterized by SDS/PAGE and Western blot as a monomeric 19× 10 3 protein expression and purification. The three IFNs' anti- proliferation of osteosarcoma cell (OS732) were compared by MTT(3- (4,5- dimethylthiazol- 2- yl)- 2,5- diphenyl tetrazolium bromide) assay. Results Restriction endonuclease digestion proved the success of IFNs DNA cloning pBV3203. Two IFNα 1c/86D variants were selected, of which the anti- proliferative activity on OS cell line were 4 and 16 fold more than IFNα 1c/86D. Conclusion The two new types of IFN mutants expression systems were constructed accurately, and the anti- osteosarcoma bioactivity of these two mutants was higher than their parent.
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