Full-Text Search:
Home|Journal Papers|About CNKI|User Service|FAQ|Contact Us|中文
《Oceanologia et Limnologia Sinica》 2012-01
Add to Favorite Get Latest Update

PROKARYOTIC EXPRESSION, PURIFICATION AND IDENTIFICATION OF TWO HSP70s IN WUCHANG BREAM MEGALOBRAMA AMBLYCEPHALA

MING Jian-Hua1, 2, 3, XIE Jun2, XU Pao2, GE Xian-Ping2, LIU Wen-Bin3, YE Jin-Yun1 (1. College of Life Sciences, Huzhou Teachers College, Huzhou, 313000; 2. Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, 214081; 3. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, 214081)  
In this research, The fragments of Constitutive HSC70 and inducible HSP70 ORFs of Wuchang bream Megalobrama amblycephala were amplified by PCR, and then cloned into prokaryotic expression vector pET-22b(+). The two recombinant plasmids, confirmed by double-endonuclease digestion and DNA sequencing, were transformed into competent E. coli BL21 (DE3). The two recombinant strains were induced by 1mmol/L IPTG at different temperatures and times, establishing optimal conditions for inducible expression. The expression products were purified by Ni-NTA His Bind Resins affinity chromatography and DEAE-Sepharose FF anion-exchange chromatography, and detected by SDS-PAGE and Western-blotting. The results show that two recombinant expression plasmids of pET-22b(+)/Ma-HSC70 and pET-22b(+)/Ma-HSP70, each expressing a 72kDa protein that could be recognized by rabbit source anti-HSP70 polyclonal antibody, were successfully constructed, and the purity of two purified fusion proteins was more than 95%. A higher temperature (37℃) was conducive to the rapid expression of fusion protein, which was easy to form inclusion body; a lower temperature (25℃) was in favor of the soluble expression of fusion protein, but also reduced the expression rate of fusion protein. All things considered, the optimal conditions for expressions of two fusion proteins of Ma-HSC70 and Ma-HSP70 are induced for 7h. at 25℃ and 30℃, respectively. In this study, two higher purified fusion protein of Ma-HSC70 and Ma-HSP70 have been obtained by prokaryotic expression of two HSP70s of Wuchang bream, thus laying foundation for further research on molecular structure and function of two proteins and associated antibodies.
【Fund】: 农业部行业专项 200803013号;; 现代农业产业技术体系建设专项 nycytx-49-18号
【CateGory Index】: S917.4
Download(CAJ format) Download(PDF format)
CAJViewer7.0 supports all the CNKI file formats; AdobeReader only supports the PDF format.
【Citations】
Chinese Journal Full-text Database 4 Hits
1 SHI Hong-bo,DUAN Xiu-mei,LI Shu-lei,XU Shu-fen,CHE Yuan-yuan,LIU Xiao-lin,LIU Li-hua,JIANG Yan-fang,FANG Yan-qiu,TAN Yan~*(Central Laboratory,First Hospital,Jinlin University,Changchun 130021,China);Construction,expression,purfication and identification of prokaryotic expression plasmid of mycobacterium tuberculosis HSP70 protein[J];Journal of Jilin University(Medicine Edition);2006-06
2 LIU Zhen1,LUO Xiao-Hua1,2,LU Shuang-Qing1,XIE Di-Zhi1,2,FANG Zhi-Jia1,XIAO Ke-Yu2 (1.Department of Biotechnology&Environment Science,Changsha University,Changsha,410003;2.College of Animal Science and Technology,Hunan Agricultural University,Changsha,410128);CLONING AND PROKARYOTIC EXPRESSION OF GROWTH HORMONE GENE IN SINIPERCA CHUATSI[J];Oceanologia Et Limnologia Sinica;2010-03
3 SU Jie,CAI Hong,ZHU Yu-xian(National Laboratory of Protein Engineering and Plant Genetic Engineering,Peking University,Beijing 100871,China);Cloning,expression and identification of heat shock protein Hsp 70 of Mycobacterium tuberculosis in prokaryotic expression vector[J];Chinese Journal of Zoonoses;2006-08
4 XIE Shu-tao,WU Ren,XU Zhong-neng,LIN Xiao-tao,ZHANG Qi-zhong(Institution of Hydrobiology,Jinan University,Guangzhou 510632,China);Induced Protection of Sublethal Temperature Heat Shock in Litopenaeus vannamei[J];Acta Scientiarum Naturalium Universitatis Sunyatseni;2005-06
【Co-citations】
Chinese Journal Full-text Database 6 Hits
1 LI Yi-wen1,2,CAO Yu-cheng2,LI Zhuo-jia2,YANG Ying-ying2,WEN Guo-liang2 (1.Fishery College of Guangdong Ocean University,Zhanjiang 524000,China;2.South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510300,China);Review of the Relationship Between White Spot Syndrome(WSS) and Ambient Factors in Prawn-culture Waters[J];Advances in Marine Science;2008-04
2 HAO Yu1, 2, DENG Si-Ping1, 2, LIU Li1, 2, LIU Chu-Wu1, 2 (1. Fisheries College, Guangdong Ocean University, Zhanjiang, 524025; 2. Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong Higher Education Institutes, Guangdong Ocean University, Zhanjiang, 524025);LONING OF cDNA FOR RACHYCENTRON CANADUM GROWTH HORMONE AND ITS EXPRESSION IN PROKARYOCYTE[J];Oceanologia et Limnologia Sinica;2011-03
3 ZHANG Yong1, MA Xi-Lan2, 3, CHEN Yong-Zhi3, LI Shui-Sheng1, CHEN Hua-Pu1, LIU Xiao-Chun1, LIN Hao-Ran1 (1. Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, Sun Yat-Sen University, Guangzhou, 510275; 2. School of Life Sciences, South China Normal University, Guangzhou, 510631; 3. Department of Life Science, Institute of Biotechnology, Huizhou University, Huizhou, 516007);cDNAs CLONING AND mRNA EXPRESSION OF TWO PROLACTIN RECEPTORS IN ORANGE-SPOTTED GROUPER EPINEPHELUS COIOIDES[J];Oceanologia et Limnologia Sinica;2012-01
4 LI Shu-Lei,TAN Yan,LIU Li-Hua,DUAN Xiu-Mei,FANG Yan-Qiu,XU Shu-Fen,CHE Yuan-Yuan.Central Laboratory,the First Hospital of Jilin University,Changchun 130021,China;Mycobacterium tuberculosis heat shock protein 70 enhances immunization respond of the gene vaccine against hepatocellular carcinoma-associated antigens 661[J];Chinese Journal of Immunology;2008-06
5 Liu Kailin,He Lin,Miu Yinglin,Zhang Yunpeng,Wang Jinjun(Key Laboratory of Entomology and Pest Control Engineering,College of Plant Protection,Southwest University,Chongqing 400716);The Impact of High Temperature and Abamectin to Tetranychus cinnabarinus(Boiduval) and Expression of Heat Shock Protein[J];Chinese Agricultural Science Bulletin;2007-01
6 WU Ren,XIE Shu-tao,SUN Yong,WANYAN Xiao-qing,ZHANG Qi-zhong(Institution of hydrobiology,Jinan University,Guangzhou 510632,China);High level prokaryotic expression of heat shock protein 70 in Litopenaeus vannamei[J];Journal of Fishery Sciences of China;2006-02
【Secondary Citations】
Chinese Journal Full-text Database 10 Hits
1 DING Jie~(1,2),GU Ji-rui~2,WU Cui-fang~2,LI Yong-wen~1,ZHANG Tao~1,WU Jiang~2,XIE Xian-lian~2,XU Heng~(1,2)(1.Key laboratory of Bio-resources and Eco-environment(Ministry of Education);College of Life Science,Sichuan University,Chengdu 610064;2.Chengdu Tongwei Aquatic Science and Technology Co.Ltd,Chengdu 610081);The Construction and Detecting of Transgenic Spinibarbus sinensis With Growth Hormone Gene[J];Freshwater Fisheries;2006-03
2 Wang Wei, Wang Yaping, Sun Yonghua, Zhu Zuoyan (State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Science, Wuhan 430072);Growth Acceleration of Yellow River Carp Following Oral Administration of Recombinant Grass Carp Growth Hormone Expressed in Yeast[J];High Technology Letters;2000-12
3 LU Shuang-Qing1, LIU Feng1, 2, 3, LIU Zhen1, ZHANG Jian-She1, XIE Xin-Min2 (1. Department of Biotechnology and Environment Science, Changsha University, Changsha, 410003; 2. College of Animal Science and Technology, Hunan Agricultural University, Changsha, 410128; 3. College of Aqua-life Science and Technology, Shanghai Ocean University, Shanghai, 200090);COMPARISON IN CLONING AND SEQUENCE OF GROWTH HORMONE GENE IN THREE SPECIES OF GENUS SINIPERCA[J];Oceanologia Et Limnologia Sinica;2008-04
4 Xie Shutao 1,2 He Jianguo1 Yang Xiaoming1 Lu Ling1 Jiang Jingbo1(1 School of Life Sciences, Zhongshan University, Guangzhou 510275; 2 Deptment of Veterinary Medicine, South China Agricultural University, Guangzhou 510642);Detection of White Spot Syndrome Virus(WSSV) in Penaeus Monodon Using Nested PCR[J];Journal of Ocean University of Qingdao;2001-02
5 LIU Shun-Mei,ZANG Xiao-Nan,ZHANG Xue-Cheng,LIU Bin,ZHANG Wei-Fen,SUN Ping-Nan,KUANG Cheng-Hong(College of Marine Life Science,Ocean University of China,Qingdao 266003,China);Safety Evaluation of Flounder(Paralichthys olivaceus) Fed with Transgenic Synechocystis[J];Periodical of Ocean University of China;2007-03
6 WU Cui-fang~1,GU Ji-rui~1,LIU Hui-ming~2,ZHANG Wen-lu~2, HONG Fang~2,WU Jiang~1,JIANG Tian-ming~1,XU Heng~2 (1.Chengdu Tongwei Aquatic Science and Technology Co.Ltd, Chengdu 610081,China; 2.College of Life Science, Sichuan University, Chengdu 610064, China);The Construction and Detecting of Transgenic Colorful Curcian carp with Growth Hormone Gene[J];Journal of Sichuan University (Natural Science Edition);2004-05
7 SUN Xiaowen, LIANG Liqun, YAN Xuechun, CAO Dingchen(Heilongjiang River Fisheries Research Institute, Chinese Academy of Fisheries Science, Harbin150070, China);The faster growth effect of transgenic common carp with growth hormone gene and its progenies[J];Journal of Fisheries of China;2002-05
8 LIU Bin1,ZANG Xiaonan1,LIU Shunmei1,ZHANG Xuecheng1,LEI Jilin2(1.College of Marine Life Science,Ocean University of China,Qingdao 266003,Shandong,China;2.Yellow Sea fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,Shandong,China);Molecular Cloning and Sequence Analysis of the Growth Hormone cDNAs from Seven Pleuronectiformes Fishes[J];Journal of Wuhan University(Natural Science Edition);2008-04
9 YANG Xue-ming1,2,ZHANG Li2,HUANG Guang-hua2,HE Jing-zhou1,ZHU Jia-jie1,JIANG He-sheng1 (1.College of Animal Science and Technology,Guangxi University,Guangxi Nanning 530004,China;2.Guangxi Fisheries Institute,Guangxi Nanning 530021,China);Gene cloning and sequence homology of growth hormone from Clarias lazera[J];Southwest China Journal of Agricultural Sciences;2008-02
10 ZHANG Jing-Nan~1,SONG Ping~(1,①),HU Jia-Rui~2,MO Sai-Jun~1,PENG Mao-Yu~1,ZHOU Wei~1,ZOU Ji-Xing~3,HU Yin-Chang~4(1.Key Laboratory of the Ministry of Education for Developmental Biology,College of Life Sciences,Wuhan University,Wuhan 430072,China;2.Zhongnan Hospital,Wuhan University,Wuhan 430071,China;3.South China Sea Institute of Oceanology,Chinese Academy of Science,Guangzhou 510301,China;4.Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510380,China)[WT5”F5];Molecular Cloning and Sequence Analysis of Full-Length Growth Hormone cDNAs from Six Important Economic Fishes[J];Acta Genetica Sinica;2005-01
©2006 Tsinghua Tongfang Knowledge Network Technology Co., Ltd.(Beijing)(TTKN) All rights reserved