Construction of shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacterium, Anabaena sp. PCC 7120 |
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Authors: | LIU Feng-Long ZHANG Hong-BinSHI Ding-Ji SHANG Zhi-Di LIN Chen SHAO Ning PENG GuohongZHANG Xue-Yan ZHANG Hai-Xia WU Jin-Yin XU Xu-Dong WANG JieJIANG Yue-Hua ZHONG Ze-PuZHAO Shu-JinWU Min CENG Cheng-Kui |
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Affiliation: | (1) Laboratory of Photosynthesis, Institute of Botany, Chinese Academy of Sciences, 100093 Beijing, China;(2) The Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, 510010, China;(3) State Key Laboratory of Molecular Oncology, Institute of Oncology, Chinese Academy of Medicine, 100021 Beijing, China;(4) EMBL, Institute of Oceanology, Chinese Academy of Sciences, 266071 Qingdao, China |
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Abstract: | The construction of the shuttle, expression vector of human tumor necrosis factor alpha (hTNF-α) gene and its expression in a cyanobacteriumAnabaena 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 pDGTNF. The expression of the rhTNF gene inEscherichia 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 intoAnabaena sp 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 α-32P labeled hTNF cDNA probes, while the expression of the hTNF gene inAnabaena 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 c~anobacteriumAnabaena sp. PCC 7120. Project supported by the National Natural Science Foundation of China (Grant No. 39280016). |
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Keywords: | human tumor necrosis factor alpha (hTNF-α) Anabaena sp. PCC 7120 shuttle expression vector triparental conjugative transfer Southern and Western blottings cytotoxicity. |
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