对虾白斑综合征病毒囊膜蛋白VP28和VP26的毕赤酵母组成型分泌表达
|
摘要
近年来,重组VP28和VP26蛋白作为蛋白亚单位疫苗,在增强对虾抗白斑综合征病毒(WSSV)感染的过程中具有重要作用。本研究根据Gen Bank中WSSV的基因序列设计引物,以WSSV粗提液为模板进行普通PCR扩增,得到VP28和VP26基因,再用引物悬挂法将Eco RⅠ和XbaⅠ酶切位点分别添加到VP28和VP26基因的5端和3端。目的基因经双酶切后插入到表达载体p GAPZαA,转化TOP10大肠杆菌,经博莱霉素(Zeocin)抗性筛选阳性重组酵母表达载体。AvrⅡ酶切线性化之后,电击转化X-33毕赤酵母感受态细胞,经Zeocin抗性筛选得到阳性重组酵母。SDS-PAGE电泳分析重组酵母表达上清液的目的蛋白,没有检测到VP28和VP26重组蛋白。随后,采用蛋白质银染法,结果显示,与空载p GAPZαA组相比,VP28和VP26表达上清液组有明显的条带,证明VP28和VP26在毕赤酵母中成功表达,蛋白分子量大小约为32 kDa。
WSSV has been a globally recognized highly harmful pathogen in shrimp farming industry that causes tremendous economic loss. The envelope proteins of WSSV, VP28 and VP26, play important roles in interacting with host cells, initiating virus infection and mediating virus intrusion. In this study, we used p GAPZαA as the expression vector and X-33 Pichia pastoris as the host cell to express VP28 and VP26 in a secretive manner. The coding sequences of VP28 and VP26(Gen Bank: AF332093.3) were amplified from WSSV using PCR, and the sequences of Eco R I(GAATTC) and Xba I(TCTAGA) were added to the 5? and 3? ends of the target genes. The purified PCR products were then cloned into the Eco R I/Xba I sites of the p GAPZαA vector. Sequencing analysis verified whether the target genes were correctly inserted into the reading frame. The construct was linearized by Bln I(Avr Ⅱ) and then was integrated into P. pastoris X-33 through electroporation while being screened by Zeocin. The expressed proteins were identified with SDS-PAGE. The VP28 and VP26 recombinant proteins could not be detected by coomassie brilliant blue R250 staining, however, the bands of the fusion proteins appeared after silver staining. The sizes of VP28 and VP26 fusion proteins were about 32 k Da. These results suggest that the P. pastoris system was effective in expressing WSSV envelope proteins VP28 and VP26, although the expression level was not sufficient. Nonetheless, our study still established a novel tool for the study of subunit vaccine, and provided basic information for the large scale vaccine production.
WSSV has been a globally recognized highly harmful pathogen in shrimp farming industry that causes tremendous economic loss. The envelope proteins of WSSV, VP28 and VP26, play important roles in interacting with host cells, initiating virus infection and mediating virus intrusion. In this study, we used p GAPZαA as the expression vector and X-33 Pichia pastoris as the host cell to express VP28 and VP26 in a secretive manner. The coding sequences of VP28 and VP26(Gen Bank: AF332093.3) were amplified from WSSV using PCR, and the sequences of Eco R I(GAATTC) and Xba I(TCTAGA) were added to the 5? and 3? ends of the target genes. The purified PCR products were then cloned into the Eco R I/Xba I sites of the p GAPZαA vector. Sequencing analysis verified whether the target genes were correctly inserted into the reading frame. The construct was linearized by Bln I(Avr Ⅱ) and then was integrated into P. pastoris X-33 through electroporation while being screened by Zeocin. The expressed proteins were identified with SDS-PAGE. The VP28 and VP26 recombinant proteins could not be detected by coomassie brilliant blue R250 staining, however, the bands of the fusion proteins appeared after silver staining. The sizes of VP28 and VP26 fusion proteins were about 32 k Da. These results suggest that the P. pastoris system was effective in expressing WSSV envelope proteins VP28 and VP26, although the expression level was not sufficient. Nonetheless, our study still established a novel tool for the study of subunit vaccine, and provided basic information for the large scale vaccine production.
引文
丁晶,彦波,傅玲琳.以枯草芽孢杆菌递呈VP28对南美白对虾免疫相关基因表达和细胞特异性吞噬的影响.水生生物学报,2013,37(4):705-711
陈文博,侯林,刘庆慧.对虾白斑综合征病毒亚单位疫苗研究进展.动物医学进展,2009,29(12):73-76
杨海,王芳宇.DNA疫苗的研究进展.中国畜牧兽医,2013,40(1):72-76
魏克强,许梓荣.家蚕蛹表达的重组VP28疫苗对克氏原螯虾的抗病毒保护效应.实验生物学报,2005,38(3):190-198
Chang YS,Liu WJ,Lee CC,et al.A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.PLo S One,2010,5(5):e10718
Delroisse JM,Dannau M,Gilsoul JJ,et al.Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.Protein Expres Purif,2005,42(2):286-294
Fu LL,Li WF,Du HH,et al.Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles.Lett Appl Microbiol,2008,46(5):581-586
Leu JH,Yang F,Zhang X,et al.Whispovirus//Lesser Known Large ds DNA Viruses.Springer Berlin Heidelberg,2009:197-227
Mu Y,Lan JF,Zhang XW,et al.A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease.Dev Comp Immunol,2012,36(2):442-449
Rajeshkumar S,Venkatesan C,Sarathi M,et al.Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus(WSSV).Fish Shellfish Immunol,2009,26(3):429-437
Satoh J,Nishizawa T,Yoshimizu M.Protection against white spot syndrome virus(WSSV)infection in kuruma shrimp orally vaccinated with WSSV r VP26 and r VP28.Dis Aquat Organ,2008,82(2):89-96
Sears IB,O'Connor J,Rossanese OW,et al.A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris.Yeast,1998,14(8):783-790
Tsai JM,Wang HC,Leu JH,et al.Identification of the nucleocapsid,tegument,and envelope proteins of the shrimp white spot syndrome virus virion.J Virol,2006,80(6):3021-3029
Wan Q,Xu L,Yang F.VP26 of white spot syndrome virus functions as a linker protein between the envelope and nucleocapsid of virions by binding with VP51.J Virol,2008,82(24):12598-12601
Waterham HR,Digan ME,Koutz PJ,et al.Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.Gene,1997,186:37-44
1)张平涛.鼠羧肽酶原B和蛇毒金属蛋白酶Alfimeprase两种蛋白在毕赤酵母中表达研究.厦门大学硕士研究生学论文,2008
陈文博,侯林,刘庆慧.对虾白斑综合征病毒亚单位疫苗研究进展.动物医学进展,2009,29(12):73-76
杨海,王芳宇.DNA疫苗的研究进展.中国畜牧兽医,2013,40(1):72-76
魏克强,许梓荣.家蚕蛹表达的重组VP28疫苗对克氏原螯虾的抗病毒保护效应.实验生物学报,2005,38(3):190-198
Chang YS,Liu WJ,Lee CC,et al.A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.PLo S One,2010,5(5):e10718
Delroisse JM,Dannau M,Gilsoul JJ,et al.Expression of a synthetic gene encoding a Tribolium castaneum carboxylesterase in Pichia pastoris.Protein Expres Purif,2005,42(2):286-294
Fu LL,Li WF,Du HH,et al.Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles.Lett Appl Microbiol,2008,46(5):581-586
Leu JH,Yang F,Zhang X,et al.Whispovirus//Lesser Known Large ds DNA Viruses.Springer Berlin Heidelberg,2009:197-227
Mu Y,Lan JF,Zhang XW,et al.A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease.Dev Comp Immunol,2012,36(2):442-449
Rajeshkumar S,Venkatesan C,Sarathi M,et al.Oral delivery of DNA construct using chitosan nanoparticles to protect the shrimp from white spot syndrome virus(WSSV).Fish Shellfish Immunol,2009,26(3):429-437
Satoh J,Nishizawa T,Yoshimizu M.Protection against white spot syndrome virus(WSSV)infection in kuruma shrimp orally vaccinated with WSSV r VP26 and r VP28.Dis Aquat Organ,2008,82(2):89-96
Sears IB,O'Connor J,Rossanese OW,et al.A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris.Yeast,1998,14(8):783-790
Tsai JM,Wang HC,Leu JH,et al.Identification of the nucleocapsid,tegument,and envelope proteins of the shrimp white spot syndrome virus virion.J Virol,2006,80(6):3021-3029
Wan Q,Xu L,Yang F.VP26 of white spot syndrome virus functions as a linker protein between the envelope and nucleocapsid of virions by binding with VP51.J Virol,2008,82(24):12598-12601
Waterham HR,Digan ME,Koutz PJ,et al.Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter.Gene,1997,186:37-44
1)张平涛.鼠羧肽酶原B和蛇毒金属蛋白酶Alfimeprase两种蛋白在毕赤酵母中表达研究.厦门大学硕士研究生学论文,2008