人血清白蛋白-睫状神经营养因子突变体融合蛋白基因在毕赤酵母中的表达及表达产物的纯化和活性鉴定

为了延长人睫状神经营养因子突变体的体内半衰期 ,将人血清白蛋白 (HAS)的C 末端和人睫状神经营养因子突变体AX15 (R13K)的N 末端通过一个 11个氨基酸的连接肽融合在一起 ,构建了融合蛋白HAS-AX15 (R13K)。HAS-AX15 (R13K)融合蛋白基因在巴斯德毕赤酵母中进行表达后通过阳离子交换层析、反向层析和凝胶过滤对表达产物进行了分离纯化。体外TF 1细胞存活实验表明与HAS融合并未影响AX15 (R13K)的生物学活性。体内动物实验表明HAS-AX15 (R13K)融合蛋白的疗效比AX15 (R13K)更为持久 :每 3天注射一次 4 8mg/kg的HAS-AX15 (R13K)融合蛋白的治疗效果优于每天注射一次 1 6mg/kg的AX15 (R13K)的治疗效果。HAS-AX15(R13K)融合蛋白不但可以减少用药次数 ,提高病人的顺应性 ,而且还可以减少用药量和血药浓度的波动 ,从而降低副反应 ,在临床应用上具有一定的优势。

Purification and Activity Assay of HSA-AX15(R13K) Fusion Protein Expressed in Pichia pastoris

To increase the in vivo half-life of human CNTF mutein AX15(R13K), HS A -AX15(R13K) fusion protein was constructed by the fusion of the C-terminus of H SA to the N-terminus of AX15(R13K) via an 11 amino acids linker. HSA-AX15(R13K ) fusion protein was purified to homogeneity by cation exchange chromatography, re verse phase chromatography and gel filtration after expressed in pichia pastor is. TF-1 cell survival bioassay showed the biological activity of AX15(R13K) was n ot affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8mg/kg HSA-AX15(R13K) fusion protein could produce more potent anti-obesity ef fects on KM mice than daily injection of 1.6mg/kg AX15(R13K). The long-acting f orm of hCNTF variant has the potential to reduce discomfort by requiring fewer i njections and to minimize the side-effects by decreasing the dosage and fluctua tion of plasma concentration, and thus has superior clinical application.