Molecular Cloning, Primary Sequence Characterization and Phylogentic Analysis of Cathepsin B cysteine from Sheep (Ovis aries) Ovarian

DOI: 10.18805/IJAR.B-1290    | Article Id: B-1290 | Page : 420-425
Citation :- Molecular Cloning, Primary Sequence Characterization and Phylogentic Analysis of Cathepsin B cysteine from Sheep (Ovis aries) Ovarian.Indian Journal of Animal Research.2021.(55):420-425
Aiju Liu, Menghe Liu, Xin Qi, Xiaofei Ma, Heling Huang, Limeng Zhang, Shujun Tian tsj7890@126.com
Address : The Research Center of Cattle and Sheep Embryonic Technique of Hebei Province, Baoding 071000, P.R. China.
Submitted Date : 23-05-2020
Accepted Date : 30-07-2020


Background: CathepsinB (CTSB) is a lysosomal cysteine protease which has been implicated in a variety of physiological and pathological processes. However, the sequence and structure characteristics of sheep CTSB gene are still unclear, which severely restricts the further study about genetic variation, molecular evolution.
Methods: Therefore the coding sequence (CDS) of sheep CTSB gene from ovary was cloned by reverse transcription PCR (RT-PCR) amplification and regular sequencing technology. The characteristics of nucleotide and amino acid sequence of CTSB gene were analyzed through bioinformatics and the phylogenetic tree was constructed employing the Bayesian inference based on amino acid sequence of different species in this study.
Result: A complete coding sequence of 1008 bp of CTSB was obtained from ovary, which encoded 335 amino acids. Functional prediction of CTSB protein suggested that containing five conserved sequence motif sites in which the site of amino acids between 158 and 205 was the most conserved region. Secondary structure analysis showed that á-helices were the major structure at the N-terminus, while random coils were the major structure at the C-terminus. Additionally, the motif II in the random coils, in which two active sites of His278 and Asn298 were located therein, was more conserved. The high similarity of CTSB amino acids alignment between sheep and other species recommended that CTSB was highly conservative in the evolutionary process.


Cloned CTSB Protein structure Sheep


  1. Aboelenain, M., Balboula, A. Z., Kawahara, M., El-Monem Montaser, A., Zaabel, S. M., Kim, S. W., Nagano, M., Takahashi, M. (2017). Pyridoxine supplementation during oocyte maturation improves the development and quality of bovine preimplantation embryos. Theriogenology. 91: 127-133. 
  2. Anez, G., Grinev, A., Chancey, C., Ball, C., Akolkar, N., Land, K. J., Winkelman, V., Stramer, S. L., Kramer, L. D., Rios, M. (2013). Evolutionary dynamics of West Nile virus in the United States, 1999-2011: phylogeny, selection pressure and evolutionary time-scale analysis. PLoS Neglected Tropical Diseases. 7: e2245.
  3. Anja, P., Anahid, J., Janko, K. (2018). Cysteine cathepsins: Their biological and molecular significance in cancer stem cells. Seminars In Cancer Biology. 53: 168-177.
  4. Anzai, R., Asami, Y., Inoue, W., Ueno, H., Yamada, K., Okada, T. (2018). Evaluation of variability in high-resolution protein structures by global distance scoring. Heliyon. 4: e00510.
  5. Brix, K., Mcinnes, J., Al-Hashimi, A., Rehders, M., Tamhane, T., Haugen, M. (2015). Proteolysis mediated by cysteine cathepsins and legumain-recent advances and cell biological challenges. Protoplasma. 252: 755-774.
  6. Chauhan, J.H., Hadiya, K.K., Dhami, A. J., Sarvaiya, N.P. (2020). Cloning and Expression of Fasciola gigantica Cathepsin-B Recombinant Proteins. Indian Journal of Animal Research, online (B-3959).
  7. Chen, L., Li, X., Zhu, L., Li, Q. (2008). cDNA cloning and genetic polymorphism analysis of Cystatin B gene in pigs. Scientia Agricultura Sinica. 07: 2120-2127.
  8. Christensen, J. S., V Prasad J. (2015). Matrix-metalloproteinase-9 is cleaved and activated by Cathepsin K. BMC Research Notes. 8: 322.
  9. Hoffmann, J., Wrabl, J. O., Hilser, V. J. (2018). The role of negative selection in protein evolution revealed through the energetics of the native sate ensemble. Proteins. 86: 1313.
  10. Jain, A., Jain, T., Sachdeva, G. K., De, S., Datta, T.K. (2012). Effect of fsh on expression of cathepsin k and s during in vitro muturation of buffalo oocytes andtheir subsequent development competence. Indian Journal of Animal Research. 46(2): 137-142.
  11. Chauhan, J.H., Hadiya, K. K., Dhami, A. J., Sarvaiya, N. P. (2020). Cloning and Expression of Fasciola gigantica Cathepsin-B Recombinant Proteins. Indian Journal of Animal Research. online (B-3959).
  12. Kedzior, M., Pawlak, A., Seredynski, R., Bania, J., Platt-Samoraj, A., Czemplik, M., Klausa, E., Bugla-Ploskonska, G., Gutowicz, J. (2018). Revealing the inhibitory potential of Yersinia enterocolitica on cysteine proteases of the papain family. Microbiological Research. 207: 211-225.
  13. Lah, T. T., Kokalj-Kunovar, M., Turk, V. (2010). Stefins and lysosomal cathepsins B, L and D in human breast carcinoma. International Journal of Cancer. 50(1): 36-44.
  14. Menard, R., Carriere, J., Laflamme, P., Plouffe, C., Khouri, H.E., Vernet, T., Tessier, D.C., Thomas, D.Y., Storer, A.C. (1991). Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain. Biochemistry. 30: 8924-8.
  15. Schmitz, J., Li, T., Bartz, U., Gutschow, M. (2016). Cathepsin B inhibitors: combining dipeptide nitriles with an occluding loop recognition element by click chemistry. ACS Medicinal Chemistry Letters. 7: 211-6.
  16. Tam, S.W., Cote-Paulino, L.R., Peak, D.A., Sheahan, K., Murnane, M.J. (1994). Human cathepsin B-encoding cDNAs: sequence variations in the 3'-untranslated region. Gene. 139: 171-6.
  17. Turk, V., Stoka, V., Vasiljeva, O., Renko, M., Sun, T., Turk, B., Turk, D. (2012). Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochimica Et Biophysica Acta. 1824: 68-88.
  18. Zhou, J., Zhang, Y.Y., Li, Q.Y., Cai, Z.H. (2015). Evolutionary history of cathepsin L (L-like) family genes in vertebrates. International Journal of Biological Sciences. 11: 1016-25.
  19. Tian, Z.L., Wang, Y.Q., Shi, H.B., Wu, Z.B., Zhang, X.H., Wang, J.P., Li, Y.X., Yang, F., Liu Y.M., Chu, M.X. (2020). Molecular cloning, characterization and tissue specificity of the expression in the TAC1 genes from Henan Huai goat (Capra hircus). Indian Journal of Animal Research. 54: 679-684.

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