融合胸腺素α1的猪干扰素α在毕赤酵母中的表达及效价测定

Expression,characterization of the porcine interferon α-thymosin α1 fusion protein a in Pichia pastoris

猪干扰素α(IFN-α)是在特定条件下由细胞产生的一种具有广谱、高效抗病毒活性的可溶性糖蛋白,胸腺素α1(Tα1)是一种具有免疫调节的活性肽.本研究中将Tα1基因与去掉信号肽序列的猪IFN-αN端融合,构建酵母表达载体PHBM-Tα1-IFNα,同时构建单独表达IFNα的重组质粒PHBM-IFNα,重组质粒电转化至毕赤酵母(Pichia pastoris)菌株,甲醛诱导表达.利用SDS-PAGE法检测融合蛋白的表达,并用细胞病变抑制法测定干扰素的效价.实验结果表明,酵母表达上清中IFNα的效价为2.916×10~6U/m L;Tα1-IFNα的效价为2.860×10~7U/m L,该结果证实胸腺素α1可提高融合表达蛋白猪干扰素的活性,因此,通过酵母表达体系获得高活性胸腺素-猪干扰素融合蛋白,可为高效、低成本获得抗病毒药物提供一种新的模式.

Porcine interferon α（ IFNα） is a broad-spectrum,highly efficient antiviral glycoprotein which is produced by cells in the specific conditions. Thymosin alpha 1（ Tα1） is a kind of immune regulation peptide. Here,we constructed the fusion protein vector p HBM-Tα1-IFNα,putting Tα1 gene on the N terminal of porcine IFNα gene with the yeast expression vector p HBM respectively. The recombinant plasmids were electroporated into Pichia pastoris strain and induced to express by formaldehyde. The fusion proteins were detected by SDS-PAGE method. We tested the potency of fusion interferon by cytopathic inhibition method.Results showed that the titer in the supernatant of yeast expression of IFNα was 2. 916 × 10^6U/m L,and the Tα1-IFNα titer was 2. 860 × 10^7U/m L. These results confirmed that the Tα1 could increase the activity of fusion porcine interferon. Therefore,the higher activity of porcine interferon by fusing Tα1 in Pichia pastoris yeast expression system may provide a new model for developing high efficiency and low cost antiviral drugs.