Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120

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	Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120
	Liu, FL ; Zhang, HB ; Shi, DJ ; Shang, ZD ; Lin, C ; Shao, N ; Peng, GH ; Zhang, XY; Zhang, HX ; Wu, JY ; Wang, J ; Xu, XD ; Jiang, YH ; Zhong, ZP ; Zhao, SJ ; Wu, M ; Zeng, CK
	1999-02-01
发表期刊	SCIENCE IN CHINA SERIES C-LIFE SCIENCES
ISSN	1006-9305
卷号	42 期号:1 页码:25-33
文章类型	Article
摘要	The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coli has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.; The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium Anabaena sp. PCC 7120 was reported. The 700-bp hTNF cDNA fragments have been recovered from plasmid pRL-rhTNF, then inserted downstream of the promoter PpsbA in the plasmid pRL439. The resultant intermediary plasmid pRL-TC has further been combined with the shuttle vector pDC-8 to get the shuttle, expression vector pDC-TNF. The expression of the rhTNF gene in Escherichia coil has been analyzed by SDS-PAGE and thin-layer scanning, and the results show that the expressed TNF protein with these two vectors is 16.9 percent (pRL-TC) and 15.0 percent (pDC-TNF) of the total proteins in the cells, respectively, while the expression level of TNF gene in plasmid pRL-rhTNF is only 11.8 percent. Combined with the participation of the conjugal and helper plasmids, pDC-TNF has been introduced into Anabaena sg PCC 7120 by triparental conjugative transfer, and the stable transgenic strains have been obtained. The existence of the introduced plasmid pDC-TNF in recombinant cyanobacterial cells has been demonstrated by the results of the agarose electrophoresis with the extracted plasmid samples and Southern blotting with alpha-(32)p labeled hTNF cDNA probes, while the expression of the hTNF gene in Anabaena sp. PCC 7120 has been confirmed by the results of Western blotting with extracted protein samples and human TNF-alpha monoclonal antibodies. The cytotoxicity assays using the mouse cancer cell line L929 proved the cytotoxicity of the TNF in the crude extracts from the transgenic cyanobacterium Anabaena sp. PCC 7120.
关键词	Human Tumor Necrosis Factor Alpha (htnf-Alpha) Anabaena Sp. Pcc 7120 Shuttle Expression Vector Triparental Conjugative Transfer Southern And Western Blottings Cytotoxicity
学科领域	Biology
收录类别	SCI
语种	英语
WOS记录号	WOS:000078524100004
引用统计	被引频次：13[WOS] [WOS记录] [WOS相关记录]
文献类型	期刊论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/3356
专题	实验海洋生物学重点实验室
作者单位	1.Chinese Acad Sci, Inst Bot, Lab Photosynth, Beijing 100093, Peoples R China 2.Guangzhou Gen Hosp, Guangzhou Mil Command, Guangzhou 510010, Peoples R China 3.Chinese Acad Med, Inst Oncol, State Key Lab Mol Oncol, Beijing 100021, Peoples R China 4.Chinese Acad Sci, Inst Oceanol, EMBL, Qingdao 266071, Peoples R China
推荐引用方式 GB/T 7714	Liu, FL,Zhang, HB,Shi, DJ,et al. Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120[J]. SCIENCE IN CHINA SERIES C-LIFE SCIENCES,1999,42(1):25-33.
APA	Liu, FL.,Zhang, HB.,Shi, DJ.,Shang, ZD.,Lin, C.,...&Zeng, CK.(1999).Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120.SCIENCE IN CHINA SERIES C-LIFE SCIENCES,42(1),25-33.
MLA	Liu, FL,et al."Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-alpha) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120".SCIENCE IN CHINA SERIES C-LIFE SCIENCES 42.1(1999):25-33.

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