【摘要】 依照血管钠肽氨基酸序列,根据毕赤酵母密码子偏爱性,人工合成了血管钠肽的编码基因,通过PCR技术,扩增出5’端含有组氨酸标签的血管钠肽基因片段,构建的分泌型表达质粒pPIC9K-VNP,pPICZαB-VNP分别经SalⅠ和SacⅠ线性化后,双质粒转化毕赤酵母GS115宿主菌后,G418/Zeocin双抗筛选出整合型His+Muts表达菌株,Tricine-SDS-PAGE和Western blot分析,证明His-VNP在毕赤酵母中可有效分泌表达,摇瓶产量可达70mg/L左右,双质粒整合较单质粒整合重组子目的多肽表达量提高了75%。生物学活性研究表明,表达产物在体外具有对巨噬细胞iNOS和TNFαmRNA的抑制作用。更多还原

【Abstract】 Synthesised the Vasonatrin peptide coding gene according to the Vasonatrin peptide amino sequence and Pichia pastoris codon preference,and then the 5′ end of Vasonatrin coding gene containing His tag marker was obtained by polymerase chain reaction(PCR) technology.The spliced gene was inserted into Pichia pastoris secretory expression vector pPIC9K and pPICZαB,linearized separately.First of all,transformed the linear pPIC9K-VNP into Pichia pastoris GS115 by electroporation,screened the excellent transformants on G418 plates.Similarly,transformed the linear pPICZαB-VNP into GS115/pPIC9K-VNP,and then selected the transformants on Zeocin plates.The expression efficiency of the GS115/pPIC9K-VNP/pPICZαB-VNP strains was analyzed by Tricine-SDS-PAGE and Western blot.The results showed that the yield is about 70mg/L in shake flask,which is increased by 75% compared with the single antibiotic marker screening method.And the research of biological activity indicated that the purified peptides can inhibit lipopolysaccharide(LPS) to induce iNOS and TNF-α in vitro macrophages.更多还原