人血清白蛋白-干扰素α2b融合蛋白在CHO细胞中的表达

Expression of Human Serum Albumin-interferon α2b Fusion Protein in Chinese Hamster Ovary Cells

干扰素α2b(interferonα2b,IFNα2b)是一种用于病毒性疾病和肿瘤性疾病治疗的多功能细胞因子,因其在体内的半衰期短限制了其在临床上的应用。将IFNα2b连接到人血清白蛋白(human serum albumin,HSA)的C端,构建融合蛋白HSA-IFNα2b。构建含融合蛋白的真核表达质粒p MH3/HSA-IFNa2b,经电转的方法转入中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞中。经G418抗性压力筛选和目的蛋白的表达量筛选,最终获得一株高表达的稳定细胞株(CHO/p MH3/HSA-IFNa2b)。表达的目的蛋白经Western blot验证显示,产物具有IFNα2b和HSA的双抗原性。经悬浮驯化稳定后,通过批次筛选得到一株稳定的高克隆表达株,产量约为65mg/L,进一步选取高表达克隆株在悬浮驯化中不同代数进行批次培养,不同代数之间蛋白质的表达量和生长情况没有明显的差异,获得一株稳定遗传表达的单克隆细胞株,3L摇瓶的流加培养结果显示,最佳发酵时间为15天,蛋白质表达量为121mg/L。经离心获得的发酵液,经两步纯化后获得蛋白质纯度高达96.8%的目的蛋白,总回收率为22.3%。参照《中国药典》2015版对IFNα2b的检测方法,结果显示,CHO表达的HSA-IFNα2b比活性为4.16×106IU/mg。首次将HSA-IFNα2b在哺乳动物细胞CHO中构建表达,表达获得高活性的HSA-IFNα2b融合蛋白。

Interferon α2b（ IFNα2b） is a multifunctional cytokine for the treatment of viral diseases and tumor diseases,and its clinical applications are limited by the short half-life. IFNα2b was fused to the Cterminus of human serum albumin（ HSA） and the p MH3 plasmid containing fusion protein（ HSA-IFNα2b）encoding gene was constructed. Fusion protein expression Chinese hamster ovary（ CHO） cell line was established by electroporation and high expression cell lines were screened by adding G418. The expressed fusion protein was analyzed by Western blot and results showed that the expressed protein was detected by both antibodies of HSA and IFNα2b. In suspension culture,fusion protein concentration achieved 65 mg / L at the end of batch fermentation. Different generations of fusion expression cell lines showed the similar cell growth and protein production and thus a stable expression cell line was obtained. In 3-L fed-batch culture,the optimized culture time was 15 days and final HSA-IFNα2b production was 121 mg / L. After two-step purification,the purity of fusion protein was 96. 8% and the total yield was 22. 3%. According to the ＂Chinese Pharmacopoeia 2015edition＂of the IFNα2b detection method,the results show that the anti-virus activity is 4. 1x106 IU / mg. In conclusion,fusion protein of HSA-IFNα2b was first expressed in mammalian cell CHO and the obtained fusion had high biological activity.