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| Prokaryotic Expression of LINE-1 Encoded Protein L1-ORF1 and Preparation of Anti-L1-ORF1 Antibody |
| HU Ming-ming1,2, ZHU Yun-feng1, WANG Yue2, WANG Yu2, ZHANG Liang2, GAO Xu-dong1, DONG Jin-kai3, YU Ji-yun2 |
1. Affiliated Hospital in Academy of Military Medical Sciences, Beijing 100071, China;
2. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China;
3. Chinese PLA General Hospital, Beijing 100853 China |
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Abstract Aim:to detect L1-ORF1, an ubiquitous protein in tumor tissue, its polyclonal antibody with rabbits were prepared. Methods: L1-ORF1 gene was prepared by PCR amplification and cloned into pGEX-6P-1 with GST fusion. After expressing in E.coli, GST-L1-ORF1 fusion protein was purified by Glutathione Sepharose 4B and subjected to immunity with rabbits. The titer of anti-serum was measured by ELISA and the specificity was examined by Western blot with the cell lysates derived from HepG2 tumor cells. Result: The anti-L1-ORF1p antibody exhibits a high sensitivity and specificity, which was evaluated by L1-ORF1p transfected normal cells and tumor cells. Conclusion:Anti L1-ORF1p polyclonal antibody with high sensitivity and specificity were successfully acquired.
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Received: 07 June 2010
Published: 25 October 2010
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Corresponding Authors:
ZHU Yun-feng, YU Ji-yun
E-mail: zhuyf2004@163.com;yujyun@126.com
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[1] Smit A F. The origin of interspersed repeats in the human genome.Curr Opin Genet Dev, 1996, 6 (6): 743-748.
[2] Daskalos A, Nikolaidis G, Xinarianos G.Hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer. Int J Cancer,2009,124(1):81-87.
[3] Kirilyuk A, Tolstonog G V, Damert A. Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells. Nucleic Acids Res, 2008, 36(2):648-665.
[4] Bratthauer G L, Fanning T G. Active LINE-1 retrotransposons in human testicular cancer.Oncogene, 1992, 7(3):507-510.
[5] Martin S L. The L1-ORF1 protein encoded by LINE-1:Structure and function duringL1 retrotransposition.J Biomed Biotechnol, 2006, 2006(1):45621.
[6] Goodier J L, Ostertag E M, Engleka K A. A potential role for the nucleolus in L1 retrotransposition.Hum Mol Genet, 2004,13(10):1041-1048.
[7] 高旭东,胡明明,朱运峰.下调LINE-1编码蛋白L1-ORF1p对肺癌细胞A549生物学特征的影响.中国生物工程杂志,2010,30(6): 21-27. Gao X D, Hu M M, Zhu Y F. China Biotechnology, 2010,30(6):21-27.
[8] Crow M K. Long interspersed nuclear elements (LINE-1): Potential triggers of systemic autoimmune disease. Autoimmunity, 2010, 43(1): 7-16.
[9] Su Y, Davies S, Davis M. Expression of LINE-1 p40 protein in pediatric malignant germ cell tumors and its association with clinic opathological parameters: A report from the children’s oncology group. Cancer Lett, 2007,247(2):204-212.
[10] 杨宗义,田艳艳,李钰,等. 小鼠mP24基因的原核表达及多抗制备.现代生物医学进展,2009,19(13): 2428-2431. Yang Z Y, Tian Y Y, Li Y, et al. Progress in Modern Biomedicine, 2009,19(3):2428-2431.
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