密码子优化的(GGGGS)_3对重组人血清白蛋白在毕赤酵母中表达的影响  

Effect of codon optimizated(GGGGS)_3 on the expression of the recombinant human serum albumin in Pichia pastoris

作　　者：王参军[1] 陈克平[1] 芦慧霞[1] 邹文艺[2] 宋礼华[2] 

机构地区：[1]东南大学附属中大医院检验科,南京210009 [2]安徽安科生物工程(集团)股份有限公司,合肥230088

出　　处：《生物学杂志》2015年第6期17-19,29,共4页Journal of Biology

基　　金：青年科学基金项目(81200877)

摘　　要：为探讨密码子优化的(GGGGS)_3对重组人血清白蛋白在毕赤酵母中表达的影响,分别用PCR的方法扩增出hsa和hsa-(ggggs)_3,经BamHⅠ/EcoRⅠ双酶切后与BamHⅠ/EcoRⅠ双酶切的pPIC9K连接,构建成pPIC9KHSA与pPIC9K-HSA-(GGGGS)_3重组分泌表达载体,转化大肠杆菌感受态细胞DH-5α后,表达载体分别用SalⅠ线性化后电击转化至毕赤酵母SMD1168感受态细胞中,将阳性转化子进行PCR鉴定并用甲醇诱导表达。SDS-PAGE分析表明HSA-(GGGGS)_3比HSA的表达量高约170.57%,Western Blotting表明HSA与HSA-(GGGGS)_3均具有与人血清白蛋白相同的免疫原性。密码子优化的(GGGGS)_3可以提高重组人血清白蛋白在毕赤酵母中的表达。To investigate the effect of codon optimizated （ GGGGS ） 3 on the expression of the recombinant human serum albumin in Pichia pastoris, the sequence of hsa and the hsa-（ggggs）3 were amplified with PCR, digested by BamHⅠand EcoRⅠ, ligated with pPIC9K digested by BamHⅠand EcoRⅠ, and then transformed into competent cell of Escherichia coli named DH-5α. After being lin-earized by Sal I, the recombinated vectors were transformed into competent cells of Pichia pastoris SMD1168 by electroporation, re-spectively. The positive recombinant Pichia pastoris clones were screened and identified by PCR. The target proteins secreted by the re-combinants Pichia pastoris were induced by methanol. Through phenotype selection and inducing assay, the expression engineering strains were obtained. The analysis of SDS-PAGE indicated that the expression of HSA-（GGGGS）3 was about 170. 57% higher than that of HSA. Western Blotting analyses showed both the expression productions had the immunogenicity of HSA. The expression level of the recombinant human serum albumin may be increased by codon optimization of （ GGGGS） 3 in Pichia pastoris.

关 键 词：连接肽 人血清白蛋白 毕赤酵母 

分 类 号：Q786[生物学—分子生物学]