现代食品科技2017，Vol.33Issue(11)：55-62,8.DOI:10.13982/j.mfst.1673-9078.2017.11.009
cp4-epsps基因在毕赤酵母中的表达及鉴定
Expression and Identification of CP4-EPSPS Gene in Pichia pastoris
摘要
Abstract
Comtmcting the genetically modified (GM) Pichia pastoris stains is the key step forestablishing a new method to assess the safety of foreign protein.Based on the research model of the 5-enolpyruvyl s hikinmate-3-phosphate synthase derived from Agrobacterium CP4 strain (CP4-EPSPS),the cp4-epsps gene was chemically synthesized after optimizing thecodon usage bias.The codon adaption index (CAI) of optimized sequence was 0.85,and the GC content of optimized sequence was 52％.Cp4-epsps gene was amplified by PCR and cloned to the expression vector pPICZb of Pichia pastoris GS115 through homologous end-joining,and the recombinant vector pPICZb-EPSPS was constructed.The correct recombinant vector was transferred into GS 115 strain through electropomtion,and four positive strains were screened through the right site via PCR.Then,those positive strains were induced by 0.5％ methanol for four days at 28 ℃,250～300 r/min.Finally,the expression of cp4-epsps in GS115 was identified from CP4-EPSPS monoclonal antibody and C-MYC monoclonal antibody by SDS-PAGE and western blotting.The results showed that the target gene was expressed in GS115 strain and MW of protein was 50 ku,which coincided with the theoretical results.In conclusion,the construction of transgenic Pichia pastoris GS115 strains is beneficial for the safety assessment of transgenic CP4-EPSPS synthase in GM crops.关键词
转基因作物/外源蛋白/安全评价/cp4-epsps基因/毕赤酵母Key words
Cp4-epsps gene/Pichia pastoris/transgenic ingredients/safety assessment引用本文复制引用
赵慧,毛碧绮,梁治成,龚石娣..cp4-epsps基因在毕赤酵母中的表达及鉴定[J].现代食品科技,2017,33(11):55-62,8.基金项目
环保部公益性行业科研专项(201509044) （201509044）
感谢中国留学基金管理委员会资助(No.201608440425). （No.201608440425）
