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Gene Cloning of Murine α-Fetoprotein Gene and Construction of Its Eukaryotic Expression Vector and Expression in cho cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2003年第4期23卷 392-395页

作者：易继林田耕 Department of General Surgery

To clone the murine α fetoprotein (AFP) gene, construct the eukaryotic expression vector of AFP and express in cho cells, total RNA were extracted from Hepa 1 6 cells, and then the murine α fetoprotein gene was amplified by RT PCR and cloned into the eukaryotic expression vector pcDNA3.1. The recombinant of vector was identified by restriction enzyme analysis and sequencing. After transient transfection of cho cells with the vector, Western blotting was used to detect the expression of AFP. It is concluded that the 1.8kb murine α fetoprotein gene was successfully cloned and its eukaryotic expression vector was successfully constructed.

关键词： gene cloning α fetoprotein gene eukaryotic expression vector cho cells

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Effects of X-rays with or without ELF magnetic fields exposure on induction of kinetochore positive and negative micronuclei in cho cells

Effects of X-rays with or without ELF magnetic fields exposu...

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第6届辐射研究与辐射工艺学术年会

作者： Ding Gui-Rong , Guo Guo-Zheng Department of Radiation Medicine, Fourth Military Medical University, Xi’an, 710032.

Induction of micronuclei by X-rays with or without extremely low frequency magnetic fields (ELFMF) exposure was investigated by immunofluorescence and cytokinesis-block method in cultured Chinese Ha

关键词： X-rays extremely low frequency magnetic fields (ELFMF) cho cells micronuclei

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Recombinant human bone morphogenetic protein-7 expressed from cho cells possessing the activity of bone-induced in vitro

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脊柱外科杂志 2006年第3期4卷 159-162,182页

作者： LI Xiaoyan WANG Hao YANG Yang TAN Min XUE Jingya NI Haidong GUO Yajun From International Joint Cancer Institute Second Military Medical University Shanghai 200433 P. R. China Shanghai Zhangjiang Biotechnology Ltd.

Objective To express the recombinant human bone morphogenetic protein-7 (rhBMP-7) in Chinese hamster ovary (cho) cells and to establish the in vitro biological activity assay of rhBMP-7. Methods Human BMP-7 cDNA was subcloned into pcDNA3.1 mammalian expression vector and transfected to cho cells by using the lipofectin transfection method. BMP-7 expression cell culture supernatants were harvested and purified for target protein. To analyze the bioactivity of the secreted rhBMP-7, a novel in vitro assay was established by measuring its alkaline phosphatase (ALP) stimulating of osteoblast cell line, W-20-17. Results BMP-7 stably expressing cell clone was selected, which secreted mature disulfide-linked homodimer form of hBMP-7 and had an apparent molecular weight of 36kDa. rhBMP-7 with >95% purity was obtained using 3 step chromatography method. Bioactivity assay showed that the purified protein specifically stimulated W-20-17 cell producing ALP, with a 4-fold increase of ALP activity at 100ng/ml or more, and the EC50 of 15.6ng/ml. Conclusion Purified rhBMP-7 from this cho expression system has significant biological activity in induction of osteoblast phenotype, which demonstrates potential bone regeneration activity.

关键词： bone morphogenetic proteins recombinant proteins alkaline phosphatase cho cells in vitro gel chromatography

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The Binding Ability Analysis of the Normal VLDL Receptor and Its Mutant

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2001年第3期21卷 177-180,194页

作者：屈伸冯宁刘志国周华邓耀祖冯宗忱 Department of Biochemistry Tongji Medical College Huazhong University of Science and Technology Wuhan

The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats. To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1-5. cho cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the cho cells transfected with pCD-VR. The results of binding experiments showed that the ability of the cho cells transfected with the full-length cDNA of VLDL-R binding DiI-labeled β-VLDL was higher than that of the cho cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on cho cells, and the receptor was almost inactivated when repeats1-5 were deleted.

关键词： VLDL receptor gene expression, β-VLDL cho cells

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DEHP and Its Metabolite MEHP Cause Genotoxicity and Mutagenesis in Mammalian Chinese Hamster Ovary cells

DEHP and Its Metabolite MEHP Cause Genotoxicity and Mutagene...

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第八届海峡两岸毒理学研讨会

作者： Yu-Jung Chang Chia-Yi Tseng Ming-Wei Chao Department of Bioscience Technology Chung Yuan Christian University Department of Biomedical Engineering Chung Yuan Christian University

DEHP,Di-（2-ethylhexyl）phthalate,the most common phthalate plasticizer used in the production of polyvinyl chloride（PVC）.Several in vivo studies have shown that DEHP absorbed in the body is metabolized to produce MEHP,mono-ethylhexyl phthalate and other *** metabolite can be absorbed through the villi and travel in the circulation system then causes series damages in endocrine and reproductive *** addition,investigations also demonstrated an association with elevated induction of rat hepatic cancer,testicular cancer,and developmental toxicity in reproductive system under MEHP ***,the mechanism regarding to carcinogenicity induced by DEHP or its metabolite MEHP remains unclear up to *** therefore are investigating the cell viability and mutagenicity of DEHP and MEHP in Chinese hamster ovary（cho）*** and mutagenicity induced by these chemicals in AS52 cell which inserted gpt *** show that the parental chemical,DEHP,as well as MEHP derivatives caused dose-dependent decreases in cell survival,but with very different potencies:MEHP has the high potency;and DEHP *** to these compounds induced dose-dependent production of ROS,as determined by Cm-H2DCF-DA *** was noted however that simultaneous treatment of ROS scavenger N-acetylcysteine（NAC）shows the protection against DEHP and MEHP induced DNA *** 6-TG selection assay,data indicate that exposure MEHP cause dose-dependent mutant frequency as compare to the *** summary,DEHP and MEHP are cytotoxic to the cells and require induction of ROS to exert their genotoxicity effects.

关键词： Cytotoxicity genotoxicity DEHP MEHP cho cells ROS

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Expression and characterization of biologically active recombinant human nerve growth factor

Expression and characterization of biologically active recom...

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医药生物技术国际研讨会

作者： Li Su-Qin,Pan Ming-Jie,Pan Ying,Li Yue-Xi~(**) (Institute of Military Medicine,Key Laboratory of Medical Biotechniques of Jiangsu Province, Nanjing Jinling Hospital,Nanjing,210002,China)

DNA fragment encoding proNGF was amplified by PCR from genomic DNA isolated from term placental tissue and was cloned into *** sequencing indicated that the cloned DNA fragment was identical as previously *** cells were then tranfected with the recombinant *** cultured in the selection medium containing G418 for 30 *** expressed product in the culture supernatant was *** culture supernatant of the cells harboring recombinant plasmid showed strong ability to promote the outgrowth of nerve fiber from the chicken dorsal root *** results of ELISA showed that the concentration of the recombinant human NGF in the culture supernatant was about 0.5 mg/*** specific activitiy was about 4×10 units per *** conclusion,the proNGF gene was successfully cloned and the protein was correctly expressed and processed in the cho *** expressed rhNGF has a high specific bioactivity.

关键词： recombinant human NGF PCR cho cells eukaryotic expression

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