Construction of Lentiviral Expression Vector Containing Extracellular Domain of Human Hepatocyte Growth Factor Receptor and Its Expression in 293T Cell_Journal of Biomedical Engineering

Authors：

GUOJia , YINYanxin , JIANGMing , YULihua , JIANGYun , LIGuiqing ,  FANGJianmin

Center for Bio-Pharmacy, Suzhou Institute, TongJi University, Suzhou 215000, China;

Corresponding author：

FANGJianmin, Email: jianminfang@gmail.com

Keywords：

hepatocyte growth factor receptor; lentiviral expression vector; transfection; expression

DOI：

10.7507/1001-5515.20150072

Video：

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This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.

Citation： GUOJia, YINYanxin, JIANGMing, YULihua, JIANGYun, LIGuiqing, FANGJianmin. Construction of Lentiviral Expression Vector Containing Extracellular Domain of Human Hepatocyte Growth Factor Receptor and Its Expression in 293T Cell. Journal of Biomedical Engineering, 2015, 32(2): 400-404. doi: 10.7507/1001-5515.20150072 Copy

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2.	BIRCHMEIER C, BIRCHMEIER W, GHERARDI E, et al. Met, metastasis, motility and more[J]. Nat Rev Mol Cell Biol, 2003, 4(12):915-925.
3.	SPIGEL D R, ERVIN T J, RAMLAU R A, et al. Randomized phaseⅡtrial of Onartuzumab in combination with erlotinib in patients with advanced non-small-cell lung cancer[J]. J Clin Oncol, 2013, 31(32):4105-4114.
4.	GOYAL L, MUZUMDAR M D, ZHU A X. Targeting the HGF/c-MET pathway in hepatocellular carcinoma[J]. Clin Cancer Res, 2013, 19(9):2310-2318.
5.	CHRISTENSEN J G, BURROWS J, SALGIA R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention[J]. Cancer Lett, 2005, 225(1):1-26.
6.	SUN Bo, LIU Rui, XIAO Zhongdang, et al. c-MET protects breast cancer cells from apoptosis induced by sodium butyrate[J]. PLoS One, 2012, 7(1):e30143.
7.	BASILICO C, ARNESANO A, GALLUZZO M, et al. A high affinity hepatocyte growth factor-binding site in the immunoglobulin-like region of Met[J]. J Biol Chem, 2008, 283(30):21267-21277.
8.	SENNINO B, ISHIGURO-OONUMA T, WEI Ying, et al. Suppression of tumor invasion and metastasis by concurrent inhibition of c-Met and VEGF signaling in pancreatic neuroendocrine tumors[J]. Cancer Discov, 2012, 2(3):270-287.
9.	MERCHANT M, MA Xiaolei, MAUN H R, et al. Monovalent antibody design and mechanism of action of onartuzumab, a Met antagonist with anti-tumor activity as a therapeutic agent[J]. Proc Natl Acad Sci U S A, 2013, 110(32):E2987-E2996.
10.	MARTENS T, SCHMIDT N O, ECKERICH C, et al. A novel one-armed anti-c-Met antibody inhibits glioblastoma growth in vivo[J]. Clin Cancer Res, 2006, 12(20 Pt 1):6144-6152.
11.	YU Shan, YU Yiyi, ZHAO Naiqing, et al. C-Met as a prognostic marker in gastric cancer:a systematic review and meta-analysis[J]. PLoS One, 2013, 8(11):e79137.
12.	ZHU Kongkai, KONG Xiangqian, ZHAO Dan, et al. c-MET kinase inhibitors:a patent review (2011-2013)[J]. Expert Opin Ther Pat, 2014, 24(2):217-230.
13.	FONG J T, JACOBS R J, MORAVEC D N, et al. Alternative signaling pathways as potential therapeutic targets for overcoming EGFR and c-Met inhibitor resistance in non-small cell lung cancer[J]. PLoS One, 2013, 8(11):e78398.
14.	ELISEI R, SCHLUMBERGER M J, MVLLER S P, et al. Cabozantinib in progressive medullary thyroid cancer[J]. J Clin Oncol, 2013, 31(29):3639-3646.

1. LI Yunqing, LI A, GLAS M, et al. c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype[J]. Proc Natl Acad Sci U S A, 2011, 108(24):9951-9956.
2. BIRCHMEIER C, BIRCHMEIER W, GHERARDI E, et al. Met, metastasis, motility and more[J]. Nat Rev Mol Cell Biol, 2003, 4(12):915-925.
3. SPIGEL D R, ERVIN T J, RAMLAU R A, et al. Randomized phaseⅡtrial of Onartuzumab in combination with erlotinib in patients with advanced non-small-cell lung cancer[J]. J Clin Oncol, 2013, 31(32):4105-4114.
4. GOYAL L, MUZUMDAR M D, ZHU A X. Targeting the HGF/c-MET pathway in hepatocellular carcinoma[J]. Clin Cancer Res, 2013, 19(9):2310-2318.
5. CHRISTENSEN J G, BURROWS J, SALGIA R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention[J]. Cancer Lett, 2005, 225(1):1-26.
6. SUN Bo, LIU Rui, XIAO Zhongdang, et al. c-MET protects breast cancer cells from apoptosis induced by sodium butyrate[J]. PLoS One, 2012, 7(1):e30143.
7. BASILICO C, ARNESANO A, GALLUZZO M, et al. A high affinity hepatocyte growth factor-binding site in the immunoglobulin-like region of Met[J]. J Biol Chem, 2008, 283(30):21267-21277.
8. SENNINO B, ISHIGURO-OONUMA T, WEI Ying, et al. Suppression of tumor invasion and metastasis by concurrent inhibition of c-Met and VEGF signaling in pancreatic neuroendocrine tumors[J]. Cancer Discov, 2012, 2(3):270-287.
9. MERCHANT M, MA Xiaolei, MAUN H R, et al. Monovalent antibody design and mechanism of action of onartuzumab, a Met antagonist with anti-tumor activity as a therapeutic agent[J]. Proc Natl Acad Sci U S A, 2013, 110(32):E2987-E2996.
10. MARTENS T, SCHMIDT N O, ECKERICH C, et al. A novel one-armed anti-c-Met antibody inhibits glioblastoma growth in vivo[J]. Clin Cancer Res, 2006, 12(20 Pt 1):6144-6152.
11. YU Shan, YU Yiyi, ZHAO Naiqing, et al. C-Met as a prognostic marker in gastric cancer:a systematic review and meta-analysis[J]. PLoS One, 2013, 8(11):e79137.
12. ZHU Kongkai, KONG Xiangqian, ZHAO Dan, et al. c-MET kinase inhibitors:a patent review (2011-2013)[J]. Expert Opin Ther Pat, 2014, 24(2):217-230.
13. FONG J T, JACOBS R J, MORAVEC D N, et al. Alternative signaling pathways as potential therapeutic targets for overcoming EGFR and c-Met inhibitor resistance in non-small cell lung cancer[J]. PLoS One, 2013, 8(11):e78398.
14. ELISEI R, SCHLUMBERGER M J, MVLLER S P, et al. Cabozantinib in progressive medullary thyroid cancer[J]. J Clin Oncol, 2013, 31(29):3639-3646.

Journal of Biomedical Engineering

Construction of Lentiviral Expression Vector Containing Extracellular Domain of Human Hepatocyte Growth Factor Receptor and Its Expression in 293T Cell

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