Construction of a Fusion Gene Carrying E10A and K5 with 2A Peptide-Linked by Using Overlap Extension PCR

E10A is a kind of replication-defective adenovirus which carries the human endostatin gene to inhibit the growth of tumors. Kringle 5(K5) has almost the same function as angiostatin to also inhibit the growth of tumors since they are all the byproduct of the proteolytic cleavage of plasminogen. Tumor size increasing can be suppressed because both of the endostatin and K5 can restrain the angiogenesis process. Therefore, in order to improve the treatment effect on tumor, 2A peptide is used to construct a fusion gene carrying both E10A and K5. Using 2A peptide is an ideal strategy when a fusion gene is expressed because it can avoid many problems during the expression of more than one kind of protein. The overlap extension PCR is also used to connect 2A peptide with E10A and K5. The final construction of fusion gene E10A-2A-K5 can provide a possible new method of the anti-angiogenesis treatment with a better expression performance.


Authors:



References:
[1] Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries (J).CA Cancer J Clin, 2018,68(6):394-424.
[2] DI Maio M, Daniele B, Perrone F, et al. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function (J). Nat Rev Clin Oncol, 2009, 6 (9) :505-506.H. Poor, An Introduction to Signal Detection and Estimation. New York: Springer-Verlag, 1985, ch. 4.
[3] Robert S. Kerbel, Tumor angiogenesis: past, present and the near future, Carcinogenesis, Volume 21, Issue 3, March 2000, Pages 505–515.
[4] Folkman, J. (2003). Angiogenesis inhibitors: a new class of drugs. Cancer Biol. Ther. 2, S127-33.J. Wang, “Fundamentals of erbium-doped fiber amplifiers arrays (Periodical style—Submitted for publication),” World Academy of Science, Engineering and Technology J. Quantum Electron., submitted for publication.
[5] Dixelius, J., Cross, M., Matsumoto, T., Sasaki, T., Timpl, R. & Claesson-Welsh, L. (2002). Endostatin regulates endothelial cell adhesion and cytoskeletal organization. Cancer Res. 62, 1944-7.
[6] O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R. & Folkman, J. (1997). Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277-85.
[7] Perri, S. R., Martineau, D., Francois, M., Lejeune, L., Bisson, L., Durocher, Y. & Galipeau, J. (2007). Plasminogen Kringle 5 blocks tumor progression by antiangiogenic and proinflammatory pathways. Mol. Cancer Ther. 6, 441-9.
[8] Zhang Huan, Huang Si-chao, CAI Shao-hui. Development of 2A Peptide-based Strategies for Constructing Multicistronic Expression Vectors. China Biotechnology, 2013, 33(1): 104-108.
[9] Szymczak-Workman A L, Vignali K M, Vignali D A. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc, 2012, 2012 (2) :199-204.
[10] Szymczak-Workman A L, Vignali K M, Vignali D A. Generation of 2A-linked multicistronic cassettes by recombinant PCR. Cold Spring Harb Protoc, 2012, 2012 (2) :251-254.
[11] Shevchuk N A, Bryksin A V, Nusinovich Y A, et al.Construction of long DNA molecules using long PCR based fusion of several fragments simultaneously (J). Nucleic Acids Research, 2004, 32 (2) :19.
[12] Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice (J). PLoS One, 2011, 6 (4) :e18556.