嗜热四膜虫表达载体的优化
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摘要
近年来淡水纤毛虫嗜热四膜虫成为新的重组亚单位疫苗生产平台。为了进一步提高重组四膜虫的筛选效率,使其能更广泛地应用于重组亚单位疫苗制备,本研究利用基因工程手段,改造了原有的表达载体,把带有巴龙霉素抗性的neo基因换成嘌呤霉素抗性表达盒,从而提高重组四膜虫的筛选效率,达到优化表达载体的目的,同时将常用的氯化镉诱导的MTT1启动子替换成HSP70启动子,实现热诱导表达,为嗜热四膜虫表达平台在重组亚单位疫苗的进一步应用奠定基础。
The freshwater ciliate, Tetrahymena thermophila, has recently emerged as a novel manufacturingplatform for recombinant subunit vaccines. We aim to further improve the screening efficiency of recombinant Tetrahymena to make it more widely used in the preparation of recombinant subunit vaccine. In this study, weconstructed a new vector with the codon optimized puromycin resistance cassette replaced paromomycinresistance cassette to improve the screen efficiency and make the vector more optimum for expression. We alsosubstitute thermo-inducible promoter HSP70 for MTT1 promoter which was induced by CdCl_2. The expressionof foreign proteins was triggered simply by increasing the cultural temperature. Construction of this vectorwould widen the application of T. thermophila expression system to preparation of more subunit vaccines.
The freshwater ciliate, Tetrahymena thermophila, has recently emerged as a novel manufacturingplatform for recombinant subunit vaccines. We aim to further improve the screening efficiency of recombinant Tetrahymena to make it more widely used in the preparation of recombinant subunit vaccine. In this study, weconstructed a new vector with the codon optimized puromycin resistance cassette replaced paromomycinresistance cassette to improve the screen efficiency and make the vector more optimum for expression. We alsosubstitute thermo-inducible promoter HSP70 for MTT1 promoter which was induced by CdCl_2. The expressionof foreign proteins was triggered simply by increasing the cultural temperature. Construction of this vectorwould widen the application of T. thermophila expression system to preparation of more subunit vaccines.
引文
[1]Orias E,Hamilton E P,Orias J D.Tetrahymena as a laboratory organism:useful strains,cell culture,and cell line maintenance[J].Methods Cell Biol,2000,62:189-211.
[2]Karrer K M.Tetrahymena genetics:two nuclei are better than one[J].Methods Cell Biol,2000,62:127-186.
[3]Ruehle M D,Orias E,Pearson C G.Tetrahymena as a unicellular model eukaryote:genetic and genomic tools[J].Genetics,2016,203(2):649-665.
[4]Turkewitz A P,Orias E,Kapler G.Functional genomics:the coming of age for Tetrahymena thermophila[J].Trends Genet,2002,18(1):35-40.
[5]Cassidy-Hanley D M.Tetrahymena in the laboratory:strain resources,methods for culture,maintenance,and storage[J].Methods Cell Biol,2012,109:237-276.
[6]Cassidy-Hanley D,Smith H R,Bruns P J.A simple,efficient technique for freezing Tetrahymena thermophila[J].J Eukaryot Microbiol,1995,42(5):510-515.
[7]Gaertig J,Gao Y,Tishgarten T,et al.Surface display of a parasite antigen in the ciliate Tetrahymena thermophila[J].Nat Biotechnol,1999,17(5):462-465.
[8]俞婷,缪炜,万明亮,等.镉和铜对嗜热四膜虫金属硫蛋白基因的诱导表达[J].动物学报,2005(6):1115-1121.
[9]Boldrin F,Santovito G,Formigari A,et al.MTT2,a copperinducible metallothionein gene from Tetrahymena thermophila[J].Comp Biochem Physiol C Toxicol Pharmacol,2008,147(2):232-240.
[10]Formigari A,Boldrin F,Santovito G,et al.Functional characterization of the 5'-upstream region of MTT5 metallothionein gene from Tetrahymena thermophila[J].Protist,2010,161(1):71-77.
[11]Nanney D L,Simon E M.Laboratory and evolutionary history of Tetrahymena thermophila[J].Methods Cell Biol,2000,62:3-25.
[12]Frankel J,Nelsen E M.The effects of supraoptimal temperatures on population growth and cortical patterning in Tetrahymena pyriformis and Tetrahymena thermophila:a comparison[J].J Eukaryot Microbiol,2001,48(2):135-146.
[13]Mochizuki K.High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene[J].Gene,2008,425(1-2):79-83.
[14]Vazquez D.Inhibitors of protein biosynthesis[J].Mol Biol Biochem Biophys,1979,30:1-312.
[15]Iwamoto M,Mori C,Hiraoka Y,et al.Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena[J].Gene,2014,534(2):249-255.
[2]Karrer K M.Tetrahymena genetics:two nuclei are better than one[J].Methods Cell Biol,2000,62:127-186.
[3]Ruehle M D,Orias E,Pearson C G.Tetrahymena as a unicellular model eukaryote:genetic and genomic tools[J].Genetics,2016,203(2):649-665.
[4]Turkewitz A P,Orias E,Kapler G.Functional genomics:the coming of age for Tetrahymena thermophila[J].Trends Genet,2002,18(1):35-40.
[5]Cassidy-Hanley D M.Tetrahymena in the laboratory:strain resources,methods for culture,maintenance,and storage[J].Methods Cell Biol,2012,109:237-276.
[6]Cassidy-Hanley D,Smith H R,Bruns P J.A simple,efficient technique for freezing Tetrahymena thermophila[J].J Eukaryot Microbiol,1995,42(5):510-515.
[7]Gaertig J,Gao Y,Tishgarten T,et al.Surface display of a parasite antigen in the ciliate Tetrahymena thermophila[J].Nat Biotechnol,1999,17(5):462-465.
[8]俞婷,缪炜,万明亮,等.镉和铜对嗜热四膜虫金属硫蛋白基因的诱导表达[J].动物学报,2005(6):1115-1121.
[9]Boldrin F,Santovito G,Formigari A,et al.MTT2,a copperinducible metallothionein gene from Tetrahymena thermophila[J].Comp Biochem Physiol C Toxicol Pharmacol,2008,147(2):232-240.
[10]Formigari A,Boldrin F,Santovito G,et al.Functional characterization of the 5'-upstream region of MTT5 metallothionein gene from Tetrahymena thermophila[J].Protist,2010,161(1):71-77.
[11]Nanney D L,Simon E M.Laboratory and evolutionary history of Tetrahymena thermophila[J].Methods Cell Biol,2000,62:3-25.
[12]Frankel J,Nelsen E M.The effects of supraoptimal temperatures on population growth and cortical patterning in Tetrahymena pyriformis and Tetrahymena thermophila:a comparison[J].J Eukaryot Microbiol,2001,48(2):135-146.
[13]Mochizuki K.High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene[J].Gene,2008,425(1-2):79-83.
[14]Vazquez D.Inhibitors of protein biosynthesis[J].Mol Biol Biochem Biophys,1979,30:1-312.
[15]Iwamoto M,Mori C,Hiraoka Y,et al.Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena[J].Gene,2014,534(2):249-255.