根据细胞因子协同作用的特点,采用重组DNA技术构建了人干扰素(IFN)α2b-胸腺肽(THY)α1融合基因,克隆到pBacPAK8上,获得重组转移载体pBacPAK-IFN-THY.与线形化Bm-BacPAK6病毒基因组DNA共转染家蚕细胞,经过体内重组,筛选到重组病毒Bm-BacPAK-IFN-THY.将Bm-BacPAK-IFN-THY感染家蚕细胞进行表达.DNA印迹证明IFN-THY已插入Bm-BacPAK6中(4 kb左右的杂交带);SDS-聚丙烯酰胺凝胶电泳、蛋白质印迹证明IFN-THY在家蚕细胞中得到了表达(分子质量为23 ku左右),且具有IFN蛋白的免疫原性;微量细胞病变抑制法和玫瑰花结法显示96 h的表达产物IFN活性为3.72×104 U/ml,120 h表达产物IFN活性为3.10×105 U/ml,48~72 h表达产物IFN活性较低;48~120 h表达产物的玫瑰花结形成率均在10%以上.结果表明融合基因在家蚕细胞中得到了高效表达,表达的融合蛋白具有IFN-α2b和THY-α1的双重生物活性.
The baculovirus shuttle vector , pBacPAK-IFN-THY was constructed which contains the genes of IFN-α2b and THY-α1. Constructed vector was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The BmN cells were infected with the recombinant virus. The results showed that the protein was successfully prepared and its bioactivities were testified by WISH-VSV system and E-RFC. Results indicated that the expressed fusion protein have bioactivities of both IFN-α2b and THY-α1.
郭冬生,朱成钢,张耀洲,吴祥甫.人干扰素α 2b-胸腺肽α 1融合基因在家蚕细胞中的表达和活性研究[J].生物化学与生物物理进展,2003,30(5):803-807
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