融合基因GAD-IL-4的克隆及其在毕赤酵母中的表达

谷氨酸脱羧酶(GAD)是糖尿病的始动靶抗原,白细胞介素4(IL-4)与自身免疫性疾病密切相关。本研究通过重叠延伸PCR(Gene splicing by overlap extension,SOE PCR)技术扩增得到融合基因GAD-IL-4,构建重组酵母表达载体pPIC9K-GAD-IL-4。线性化pPIC9K-GAD-IL-4,通过电转化法转化毕赤酵母菌GS115。以0.5%的甲醇对酵母工程菌GS115/pPIC9K-GAD-IL-4进行诱导表达。经SDS-PAGE检测,在发酵上清中出现与预期大小相符的蛋白条带,表明融合基因GAD-IL-4在酵母细胞中实现正确表达,为下一步利用重组融合蛋白GAD-IL-4奠定了良好的实验基础。

Cloning and expression of GAD-IL-4 fusion gene in Pichia Pastoris GAD65 is an original target antigen of Diabetes. Interleukin4 is closely related to self-immune diseases.The fusion gene of GAD and IL-4 genes was constructed by SOE PCR technology.The fusion gene was inserted into the yeast expressionvectorpPIC9K.LinearizedplasmidpPIC9K-GAD-IL-4 withSalIwas transformedintoPichiaPastoris strainGS115 throughmethodofElectroporation. Methanol of 0.5% concentration was used to induce the expression of fusion protein in the recombinant pichia strain,and a band of target protein was found in the supernatant by SDS-PAGE,indicating that GAD-IL-4 fusion gene on the Pichia Pastoris was expressed correctly.GAD-IL-4 fusion protein will be further used in the research of prevention and treatment of diabetes.