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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ＩＦＮａｌｃ／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、Ｍ１Ｔ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ＩＦＮａｌｃ／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。
Objective To construct the prokaryotic expression system of two novel IFN mutants and their parents, and to compare the expression of the two new IFN mutants and their anti-osteosarcoma activity. Methods The recombinant bacteriophage pCANTAB5E-IFNs was used as a template to amplify the DNA sequences of two IFNalc/86D mutants and their parents by PCR, and inserted into prokaryotic expression vector pBV3203. After being expressed and purified, the SDS-PAGE electrophoresis was used for identification, Western blot was used to detect its immunological activity, and M1T colorimetric assay was used to compare its activity against the proliferation of osteosarcoma cells (OS732). Results Restriction enzyme digestion and PCR showed that IFNs were successfully cloned in the expression vector. The anti-osteosarcoma activity of the two mutant IFNs was 4 and 16-fold higher than that of IFNalc/86D, respectively. Conclusion The prokaryotic expression system of two novel IFNs mutants was successfully constructed. In vitro experiments demonstrated that the IFNs protein product was more proliferative than OS732 cells.