摘要
为了将可中和对虾白斑综合症病毒(WSSV)的单链抗体P1D3在酵母中实现表达,以原核表达载体M13噬菌粒为模板,设计带有SnaBⅠ和EcoRⅠ酶切位点的特异性引物,通过PCR方法扩增P1D3基因。经过酶切、连接反应将该基因连入大肠杆菌-酵母穿梭质粒pPIC9K上。重组质粒pPIC9K-scFvP1D3经BglⅡ线性化后,用电转化的方法转入毕赤酵母(Pichiapastoris)GS115中。通过PCR和DNA测序,挑选和鉴定阳性克隆。经甲醇诱导,P1D3在酵母中获得分泌表达。ELISA实验结果表明,酵母表达上清液中的单链抗体具有较高的WSSV结合活性,而且其活性要高于大肠杆菌所表达抗体的活性。表达条件优化后,单链抗体在酵母中最高表达量可达302mg/L,为开展对虾被动免疫研究提供了新的抗体来源。
White spot syndrome virus （WSSV） is a major pathogen in aquacuhure penaeid shrimp, which caused catastrophic economic losses in the worldwide. No adequate treatments against WSSV are available. In order to study infection mechanism of WSSV, a phage display scFv cDNA library against WSSV was constructed and a neutralizing antibody of scFv PID3 was selected in our lab previously, In this study, scFv P1D3 was expressed successfully in yeast, Firstly, the original expression vector of P1D3, M13 phagmid, was used as a template to design primers with restriction sites of SuaB Ⅰ and EcoR Ⅰ. Then the gene of P1D3 was amplified by PCR. After digested by SnaB Ⅰ and EcoR Ⅰ , the fragment of scFv PID3 with E-tag was inserted into yeast and E. coli shuttle plasmid pPIC9k. The recombinant plasmid pPIC9k-scFv P1D3-Etag was linearized with Bgt Ⅱ and then transformed into Pichia pastoris GS115 by electroporation. Positive clones were selected and verified by PCR and DNA sequencing. The scFv P1D3 was induced to express in yeast by methanol. The results of ELISA demonstrate that scFv P1D3 expressed in yeast still has high specificity to bind on WSSV and the binding activity is higher than that expressed in E. coli TG1. After several optimizing experiments, the results show that the expression amount of scFv P1D3 can reach to 302mg/L in yeast culture supernatant. This experiment has offered a new source of antibody for the researches on passive immunology for shrimp.
出处
《生物工程学报》
CAS
CSCD
北大核心
2006年第6期973-978,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目资助(No.30471339)。~~
关键词
毕赤酵母
对虾白斑综合症病毒(wssv)
单链抗体
分泌表达
Pichia pastoris, white spot syndrome virus （WSSV）, single chain fragment variable（scFv）, secreted expression
