A Novel Reconstruction and Expression of Endostatin with pDC316 Plasmid in Eukaryotic Cells

Student: Ziqi Wang
Table: MED1215
Experimentation location: Reseach Institution
Regulated Research (Form 1c): No
Project continuation (Form 7): No

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Abstract:

Endostatin is one of the strongest endothelial cell inhibitory factors that has been found so far, however, endostatin protein is extremely unstable and difficult to prepare and apply. The goal of this research is to investigate a novel biotechnology method to reconstruct endostatin. Subcloning endostatin gene into pDC316 plasmid and transfect into human embryonic kidney 293T cells is proposed in this research.

Endostatin gene is amplified by polymerase chain reaction (PCR). Purified endostatin gene fragments were ligated to eukaryotic expression vector pDC316. The reconstructed plasmids were transformed into competent cells E. coli DH5α. Single and clear-edged bacteria are tested by bacterial PCR and electrophoresis. Plasmid was extracted from the positive bacteria and was sequenced for further confirmation. The endostatin containing plasmid was transfected into 293T cells, the expression of the target gene in the infected cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) and gel electrophoresis.

The final product reconstructed by endostatin DNA with the pDC316 vector was confirmed by sequencing and endostatin mRNA was successfully expressed in transfected 293T cells. This research successfully reconstructed endostatin into pDC316 plasmid vector and transfected the identified plasmid into 293T cells. This recombinant endostatin has potential value angiogenesis gene therapy.

Keywords
Recombinant Endostatin, Angiogenesis, pDC316, 293T cells

Bibliography/Citations:

References

  1. Ribatti D. (2008). Judah Folkman, a pioneer in the study of angiogenesis. Angiogenesis11(1), 3–10. https://doi.org/10.1007/s10456-008-9092-6
  2. Wikipedia contributors. (2020, July 4). Angiogenesis. In Wikipedia, The Free Encyclopedia. Retrieved 14:01, September 4, 2020, from https://en.wikipedia.org/w/index.php?title=Angiogenesis&oldid=965958178
  3. Walia, A., Yang, J. F., Huang, Y. H., Rosenblatt, M. I., Chang, J. H., & Azar, D. T. (2015). Endostatin's emerging roles in angiogenesis, lymphangiogenesis, disease, and clinical applications. Biochimica et biophysica acta1850(12), 2422–2438. https://doi.org/10.1016/j.bbagen.2015.09.007
  4. Wikipedia contributors. (2020, April 15). Endostatin. In Wikipedia, The Free Encyclopedia. Retrieved 13:26, September 4, 2020, from https://en.wikipedia.org/w/index.php?title=Endostatin&oldid=951078398
  5. Javaherian, K., Lee, T. Y., Tjin Tham Sjin, R. M., Parris, G. E., & Hlatky, L. (2011). Two Endogenous Antiangiogenic Inhibitors, Endostatin and Angiostatin, Demonstrate Biphasic Curves in their Antitumor Profiles. Dose-response : a publication of International Hormesis Society9(3), 369–376. https://doi.org/10.2203/dose-response.10-020.Javaherian
  6. Rajabi, M., & Mousa, S. A. (2017). The Role of Angiogenesis in Cancer Treatment. Biomedicines5(2), 34. https://doi.org/10.3390/biomedicines5020034
  7. Yoo, S. Y., & Kwon, S. M. (2013). Angiogenesis and its therapeutic opportunities. Mediators of inflammation2013, 127170. https://doi.org/10.1155/2013/127170
  8. Gupta, K., & Zhang, J. (2005). Angiogenesis: a curse or cure?. Postgraduate medical journal81(954), 236–242. https://doi.org/10.1136/pgmj.2004.023309
  9. Nishida, N., Yano, H., Nishida, T., Kamura, T., & Kojiro, M. (2006). Angiogenesis in cancer. Vascular health and risk management2(3), 213–219. https://doi.org/10.2147/vhrm.2006.2.3.213
  10. Mohajeri, A., Sanaei, S., Kiafar, F., Fattahi, A., Khalili, M., & Zarghami, N. (2017). The Challenges of Recombinant Endostatin in Clinical Application: Focus on the Different Expression Systems and Molecular Bioengineering. Advanced pharmaceutical bulletin7(1), 21–34. https://doi.org/10.15171/apb.2017.004
  11. Mohajeri, A., Pilehvar-Soltanahmadi, Y., Pourhassan-Moghaddam, M., Abdolalizadeh, J., Karimi, P., & Zarghami, N. (2016). Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System. Advanced pharmaceutical bulletin6(2), 187–194. https://doi.org/10.15171/apb.2016.026
  12. Singh, A., Upadhyay, V., Upadhyay, A. K., Singh, S. M., & Panda, A. K. (2015). Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial cell factories14, 41. https://doi.org/10.1186/s12934-015-0222-8
  13. Gray D. (2001). Overview of protein expression by mammalian cells. Current protocols in protein scienceChapter 5(1), Unit5.9. https://doi.org/10.1002/0471140864.ps0509s10
  14. Vieira Gomes, A. M., Souza Carmo, T., Silva Carvalho, L., Mendonça Bahia, F., & Parachin, N. S. (2018). Comparison of Yeasts as Hosts for Recombinant Protein Production. Microorganisms6(2), 38. https://doi.org/10.3390/microorganisms6020038
  15. Li, L., Huang, J. L., Liu, Q. C., Wu, P. H., Liu, R. Y., Zeng, Y. X., & Huang, W. L. (2004). Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery. World journal of gastroenterology10(13), 1867–1871. https://doi.org/10.3748/wjg.v10.i13.1867
  16. Winter L.E., Barenkamp S.J. Construction and immunogenicity of recombinant adenovirus vaccines expressing the HMW1, HMW2, or Hia adhesion protein of nontypeable Haemophilus influenzaeClin Vaccine Immunol. 2010;17:1567–1575.
  17. Anderson, G. J., M Cipolla, C., & Kennedy, R. T. (2011). Western blotting using capillary electrophoresis. Analytical chemistry83(4), 1350–1355. https://doi.org/10.1021/ac102671n
  18. Lee, C. S., Bishop, E. S., Zhang, R., Yu, X., Farina, E. M., Yan, S., Zhao, C., Zheng, Z., Shu, Y., Wu, X., Lei, J., Li, Y., Zhang, W., Yang, C., Wu, K., Wu, Y., Ho, S., Athiviraham, A., Lee, M. J., Wolf, J. M., … He, T. C. (2017). Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine. Genes & diseases4(2), 43–63. https://doi.org/10.1016/j.gendis.2017.04.001
  19. Wold, W. S., & Toth, K. (2013). Adenovirus vectors for gene therapy, vaccination and cancer gene therapy. Current gene therapy13(6), 421–433. https://doi.org/10.2174/1566523213666131125095046
  20. Andrew L. Feldman, Steven K. Libutti, in The Cytokine Handbook (Fourth Edition), 2003
  21. Zhen-Yu Ding, Yu-Quan Wei, in Recent Advances in Cancer Research and Therapy, 2012

 


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Research Plan:

A. Questions or Problem being addressed
Endostatin is one of the strongest endothelial cell inhibitory factors that has been found so far, however, endostatin protein is extremely unstable and difficult to prepare and apply. 
B. Goals/Expected Outcomes/Hypothesis
The goal of this research is to investigate a novel biotechnology method to reconstruct endostatin.
C. Methods/Procedures
Endostatin gene is amplified by polymerase chain reaction (PCR). Purified endostatin gene fragments were ligated to eukaryotic expression vector pDC316. The reconstructed plasmids were transformed into competent cells E. coli DH5α. Single and clear-edged bacteria are tested by bacterial PCR and electrophoresis. Plasmid was extracted from the positive bacteria and was sequenced for further confirmation. The endostatin containing plasmid was transfected into 293T cells, the expression of the target gene in the infected cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) and gel electrophoresis. Endostatin gene is amplified by polymerase chain reaction (PCR). Purified endostatin gene fragments were ligated to eukaryotic expression vector pDC316. The reconstructed plasmids were transformed into competent cells E. coli DH5α. Single and clear-edged bacteria are tested by bacterial PCR and electrophoresis. Plasmid was extracted from the positive bacteria and was sequenced for further confirmation. The endostatin containing plasmid was transfected into 293T cells, the expression of the target gene in the infected cells was detected by reverse transcription-polymerase chain reaction(RT-PCR) and gel electrophoresis. The final product reconstructed by endostatin DNA with the pDC316 vector was confirmed by sequencing and check whether it will be successfully be expressed in transfected 293T cells.

 

 

 

 

Questions and Answers

1. What was the major objective of your project and what was your plan to achieve it? 

Since endostatin protein is extremely unstable and difficult to prepare and apply, the goal of this project is to investigate a novel technology method to reconstruct endostatin. 

       a. Was that goal the result of any specific situation, experience, or problem you encountered?  

Not really for my personal experience, but after reading articles, I found it really interesting to study. 

       b. Were you trying to solve a problem, answer a question, or test a hypothesis?

Solve a problem. 

2. What were the major tasks you had to perform in order to complete your project?

Multiple biogenetic techniques need to be performed in order to reconstruct endostatin.

3. What is new or novel about your project?

In this project, the goal is to investigate a novel technology method to reconstruct endostatin. 

       a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?

There are some biogenetic techniques that I have never used before. 

       b. Is your project's objective, or the way you implemented it, different from anything you have seen?

No. From my perspective, this project follows the same approach as many other studies. Start with a hypothesis, set your own plan and do experiments to prove it.

       c. If you believe your work to be unique in some way, what research have you done to confirm that it is?

Through a lot of reading of related articles, I found that no one had tried the method used in this study. 

4. What was the most challenging part of completing your project?

Because I have not been experienced in a large laboratory before, sometimes unskilled or unregulated operation may occur

      a. What problems did you encounter, and how did you overcome them?

Sometimes I wasn't sure if I was doing the right thing, so asked my mentor to get the answer

      b. What did you learn from overcoming these problems?

I learned how to do operations properly, but more importantly, I learned how to communicate better with my mentors and observed what a researcher's day is like.

5. If you were going to do this project again, are there any things you would you do differently the next time?

I should have started my research earlier so that I could get more in-depth results

6. Did working on this project give you any ideas for other projects? 

Yes, my interest in doing biological research has increased a lot

7. How did COVID-19 affect the completion of your project?

Due to COVID-19, I could not complete the experiments in the school lab, but had to go back to China to complete them, and I had to communicate with my teacher through online channels at times.