| 罗非鱼热休克蛋白70在毕赤酵母中的表达与纯化 |
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| 引用本文： | 陈明,王秋华,王瑞,黄婷,梁万文,李超,雷爱莹,陈福艳,余晓丽,甘西.罗非鱼热休克蛋白70在毕赤酵母中的表达与纯化[J].大连水产学院学报,2011,26(1):58-62. |
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| 作者姓名： | 陈明 王秋华 王瑞 黄婷 梁万文 李超 雷爱莹 陈福艳 余晓丽 甘西 |
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| 作者单位： | 陈明,王瑞,黄婷,梁万文,李超,雷爱莹,陈福艳,余晓丽,甘西,CHEN Ming,WANG Rui,HUANG Ting,LIANG Wan-wen,LI Chao,LEI Ai-ying,CHEN Fu-yan,YU Xiao-li,GAN Xi(广西水产研究所,广西,南宁,530021);王秋华,WANG Qiu-hua(广西水产研究所,广西,南宁,530021;广西大学,动物科学技术学院,广西,南宁,530005) |
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| 基金项目： | 国家社会公益研究项目，国家科技支撑计划项目 |
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| 摘 要： | 以尼罗罗非鱼血液mRNA反转录获得编码罗非鱼Hsp70完整cDNA,重组构建罗非鱼真核表达载体pPIC9K/rtHsp70;在毕赤酵母Pichia pastoris KM71中表达重组罗非鱼的热休克蛋白70（rtHsp70）,对表达上清液进行SDS-PAGE及Western blot分析;采用亲和层析、HiTrap Desalting预装柱脱盐纯化目的蛋白。结果表明：克隆至pMD载体上的基因与目的基因完整编码区序列完全一致;诱导表达上清液经SDS-PAGE及Western blot分析,上清液中蛋白条带大小与目的蛋白相对分子质量大小一致;每升诱导上清液可以获得约2 mg纯化蛋白。本研究中获得的rtHsp70毕赤酵母工程菌及纯化的rtHsp70为后期rtHsp70-肽疫苗研究奠定了基础。
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| 关 键 词： | 重组罗非鱼热休克蛋白70（rtHsp70） 基因表达 毕赤酵母 |
Expression and purification of recombinant Tilapia heat shock protein 70 in yeast Pichia pastoris |
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| CHEN Ming,WANG Qiu-hua,WANG Rui,HUANG Ting,LIANG Wan-wen,LI Chao,LEI Ai-ying,CHEN Fu-yan,YU Xiao-li,GAN Xi.Expression and purification of recombinant Tilapia heat shock protein 70 in yeast Pichia pastoris[J].Journal of Dalian Fisheries University,2011,26(1):58-62. |
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| Authors: | CHEN Ming WANG Qiu-hua WANG Rui HUANG Ting LIANG Wan-wen LI Chao LEI Ai-ying CHEN Fu-yan YU Xiao-li GAN Xi |
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| Institution: | CHEN Ming1,WANG Qiu-hua1,2,WANG Rui1,HUANG Ting1,LIANG Wan-wen1,LI Chao1,LEI Ai-ying1,CHEN Fu-yan1,YU Xiao-li1,GAN Xi1(1.Guangxi Institute of Fisheries,Nanning 530021,China,2.College of Animal Science and Technology,Guangxi University,Nanning 530005,China) |
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| Abstract: | The recombinant cDNA of Tilapia Hsp70 was amplified from blood mRNA and inserted into vector pMD18-T.Sequencing revealed that the cDNA was subcloned into expression vector pPIC9K.The recombinant vector was transformed into the yeast Pichia pastoris KM71 via electroporation after sequencing.The transforming positive clones were screened by PCR and rtHsp70 in culture supernatant induced by methanol was identified by Western blot.Protein purification by the affinity chromatograph and gel filtration showed the ... |
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| Keywords: | rtHsp70(recombinant Tilapia heat shock protein 70) gene expression Pichia pastoris |
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