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Current IssueEditorial BoardOnline First ArticlesArchive

Research Article

volume 53 issue 6 (june 2019) : 731-735,   Doi: 10.18805/ijar.B-963

Construction and expression of a prokaryotic expression vector for the goat sry gene

X
Xiao Wang
G
Guang-Xin E
S
Shu-Zhu Cheng
W
Wei-Wei Ni
Y
Yue-Hui Ma
M
Ming-Xing Chu
Y
Yong-Fu Huang
1<div style="text-align: justify;">College of Animal Science and Technology, Chongqing Key Laboratory of Forage &amp; Herbivore, Chongqing Engineering Research Centre for Herbivores Resource Protection and Utilization, Southwest University, Chongqing-400 716, China.</div>
Cite article:- Wang Xiao, E Guang-Xin, Cheng Shu-Zhu, Ni Wei-Wei, Ma Yue-Hui, Chu Ming-Xing, Huang Yong-Fu (2019). Construction and expression of a prokaryotic expression vector for the goat sry gene. Indian Journal of Animal Research. 53(6): 731-735. doi: 10.18805/ijar.B-963.

ABSTRACT

Goats are economically important animals in the world, and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization, and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl â-D-Thiogalactoside (IPTG), the cells were lysed, and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed, and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1:50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future.

REFERENCES

  1. Angelopoulou, R., Lavranos, G., Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model? Reproductive Biology & Endocrinology Rb & E. 10(1):13.
  2. Bradford, S. T., Wilhelm, D., Koopman, P. (2008). Comparative analysis of anti-mouse sry antibodies. Sexual Development, 1(5), 305-310.
  3. De, S. S., Kassahn, K. S., Mcintyre, L. C., Chong, C. E., Scott, H. S., Torpy, D. J. (2016). Case report of whole genome sequencing in the xy female: identification of a novel SRY mutation and revision of a misdiagnosis of androgen insensitivity syndrome. Bmc Endocrine Disorders, 16(1), 58.
  4. Daigle, M., Roumaud, P., Martin, L. J. (2015). Expressions of sox9, sox5, and sox13 transcription factors in mice testis during postnatal development. Molecular & Cellular Biochemistry, 407(1), 1-13.
  5. Daniel, E., Amy, M., Jason, B., Mat, C., Gail, D., Turner, M. E. (2007). Sry delivery to the adrenal medulla increases blood pressure and adrenal medullary tyrosine hydroxylase of normotensive wky rats. BMC Cardiovascular Disorders, 7(1), 6-6.
  6. Han Fengtong. (2008). bovine sry core promoter localization and gene funcational characteriation. Northeast Agricultural University. 
  7. He, Y., Zhang, Y., Li, X., Zhou, R., Li, L., Wang, Z. (2017). SRY and DelE Detection in Polled Intersex Tangshan Dairy Goat. Indian Journal of Animal Research. Print ISSN: 0367-6722 
  8. Kashimada, K., Koopman, P. (2010). Sry: the master switch in mammalian sex determination. Development, 137(23):3921-30.
  9. Kozhukhar’, V. G. (2012). SRY and SOX9: the main genetic factors of mammalian sex determination. Tsitologiia, 54(5), 390-404.
  10. Kono, N., Sun, L., Toh, H., Shimizu, T., Xue, H., Numata, O., Ato M., Ohnishi K., Itamura S. (2017). Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis. Biochem Biophys Res Commun, 487(2), 300-306.
  11. Larney, C., Bailey, T. L., Koopman, P. (2014). Switching on sex: transcriptional regulation of the testis-determining gene sry. Development, 141(11), 2195-205.
  12. Lau, Y. F., Li, Y. (2009). The human and mouse sex-determining sry genes repress the rspol/beta-catenin signaling. Journal of Genetics & Genomics, 36(4), 193-202.
  13. Ma Zhijun., Chen Xiang., Zhang Yong., Xu Houqiang., Xia Xianlin. (2007). Research progresses on the embryonic sex identification technique in mammal. Guizhou Agricultural Sciences. 35(2):116-120.
  14. Mohamed, E. S. (2013). Disorders of sexual differentiation: I. genetics and pathology. Arab Journal of Urology, 11(1), 19-26.
  15. Ortlieb, E., Cheek, E.H. (2012). Update on disorders of sex development. Current Opinion in Endocrinology Diabetes & Obesity. 19(1):28-32. 
  16. Polanco, J.C and Koopman, P. (2007). Sry and the hesitant beginnings of male development. Developmental Biology, 302(1):13-24.
  17. Sasaki, O., Kimura, H., Ishii, K., Satoh, M., Nagamine, Y., Yokouchi, K. (2011). Economic effects of using sexed semen in Japanese dairy herds. Animal Science Journal. 82(3):486-493.
  18. Sinclair, A.H., Berta, P., Palmer, M.S. (1990). A gene from the human sex-determining region encode a protein with homology to a conserved DNA -binding motif. Nature. 346: 240-244.
  19. Suntharalingham, J. P., Buonocore, F., Duncan, A. J., Achermann, J. C. (2015). Dax-1 (nr0b1) and steroidogenic factor-1 (sf-1, nr5a1) in human disease. Best Practice & Research Clinical Endocrinology & Metabolism, 29 (4), 607-619.
  20. Tanaka, S. S., Nishinakamura, R. (2014). Regulation of male sex determination: genital ridge formation and sry activation in mice. Cellular & Molecular Life Sciences, 71(24), 4781-4802.
  21. Terral, G., Champion, T., Debaene, F., Colas, O., Bourguet, M., Wagner-Rousset, E., et al. (2017). Epitope characterization of anti-    jam-a antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches. Mabs, 00-00.
  22. Wetering, M., Clevers, H. (1992). Sequence specific interaction of the HMG box proteins TCF 1 and SRY occurs within the minor groove of a Watson Crick double helix. Embo Journal. 11(8):3039-3044.
  23. Waters, P. D., Wallis, M. C., Graves, J. A. M. (2007). Mammalian sex—origin and evolution of the y chromosome and. Seminars in Cell & Developmental Biology, 18(3), 389-400.
  24. Wang, X., Xue, M., Zhao, M., He, F., Li, C., Li, X. (2018). Identification of a novel mutation (ala66thr) of SRY gene causes XY pure gonadal dysgenesis by affecting DNA binding activity and nuclear import. Gene, 651: 143-151.
  25. Xu Ying, Yang Ligu, Guo Aizhen. (2002). Research progress in the mammaiian sex determining Region of the Y. Biotechnology Bulletin. 22(1):328-330.
  26. Yan Lei., Zhang Xiaojie., Xu Houqiang., Lin Jiadong. (2009). Progress on sex-control of livestock . Animal Husbandry and Feed Science. 30(4):130-133.
  27. Zhang, M., Lu, K. H., Jr, G. E. S. (2003). Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls. Theriogenology, 60(9), 1657-1663. 
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    Research Article

    volume 53 issue 6 (june 2019) : 731-735,   Doi: 10.18805/ijar.B-963

    Construction and expression of a prokaryotic expression vector for the goat sry gene

    X
    Xiao Wang
    G
    Guang-Xin E
    S
    Shu-Zhu Cheng
    W
    Wei-Wei Ni
    Y
    Yue-Hui Ma
    M
    Ming-Xing Chu
    Y
    Yong-Fu Huang
    1<div style="text-align: justify;">College of Animal Science and Technology, Chongqing Key Laboratory of Forage &amp; Herbivore, Chongqing Engineering Research Centre for Herbivores Resource Protection and Utilization, Southwest University, Chongqing-400 716, China.</div>
    Cite article:- Wang Xiao, E Guang-Xin, Cheng Shu-Zhu, Ni Wei-Wei, Ma Yue-Hui, Chu Ming-Xing, Huang Yong-Fu (2019). Construction and expression of a prokaryotic expression vector for the goat sry gene. Indian Journal of Animal Research. 53(6): 731-735. doi: 10.18805/ijar.B-963.

    ABSTRACT

    Goats are economically important animals in the world, and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization, and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl â-D-Thiogalactoside (IPTG), the cells were lysed, and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed, and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1:50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future.

    REFERENCES

    1. Angelopoulou, R., Lavranos, G., Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model? Reproductive Biology & Endocrinology Rb & E. 10(1):13.
    2. Bradford, S. T., Wilhelm, D., Koopman, P. (2008). Comparative analysis of anti-mouse sry antibodies. Sexual Development, 1(5), 305-310.
    3. De, S. S., Kassahn, K. S., Mcintyre, L. C., Chong, C. E., Scott, H. S., Torpy, D. J. (2016). Case report of whole genome sequencing in the xy female: identification of a novel SRY mutation and revision of a misdiagnosis of androgen insensitivity syndrome. Bmc Endocrine Disorders, 16(1), 58.
    4. Daigle, M., Roumaud, P., Martin, L. J. (2015). Expressions of sox9, sox5, and sox13 transcription factors in mice testis during postnatal development. Molecular & Cellular Biochemistry, 407(1), 1-13.
    5. Daniel, E., Amy, M., Jason, B., Mat, C., Gail, D., Turner, M. E. (2007). Sry delivery to the adrenal medulla increases blood pressure and adrenal medullary tyrosine hydroxylase of normotensive wky rats. BMC Cardiovascular Disorders, 7(1), 6-6.
    6. Han Fengtong. (2008). bovine sry core promoter localization and gene funcational characteriation. Northeast Agricultural University. 
    7. He, Y., Zhang, Y., Li, X., Zhou, R., Li, L., Wang, Z. (2017). SRY and DelE Detection in Polled Intersex Tangshan Dairy Goat. Indian Journal of Animal Research. Print ISSN: 0367-6722 
    8. Kashimada, K., Koopman, P. (2010). Sry: the master switch in mammalian sex determination. Development, 137(23):3921-30.
    9. Kozhukhar’, V. G. (2012). SRY and SOX9: the main genetic factors of mammalian sex determination. Tsitologiia, 54(5), 390-404.
    10. Kono, N., Sun, L., Toh, H., Shimizu, T., Xue, H., Numata, O., Ato M., Ohnishi K., Itamura S. (2017). Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis. Biochem Biophys Res Commun, 487(2), 300-306.
    11. Larney, C., Bailey, T. L., Koopman, P. (2014). Switching on sex: transcriptional regulation of the testis-determining gene sry. Development, 141(11), 2195-205.
    12. Lau, Y. F., Li, Y. (2009). The human and mouse sex-determining sry genes repress the rspol/beta-catenin signaling. Journal of Genetics & Genomics, 36(4), 193-202.
    13. Ma Zhijun., Chen Xiang., Zhang Yong., Xu Houqiang., Xia Xianlin. (2007). Research progresses on the embryonic sex identification technique in mammal. Guizhou Agricultural Sciences. 35(2):116-120.
    14. Mohamed, E. S. (2013). Disorders of sexual differentiation: I. genetics and pathology. Arab Journal of Urology, 11(1), 19-26.
    15. Ortlieb, E., Cheek, E.H. (2012). Update on disorders of sex development. Current Opinion in Endocrinology Diabetes & Obesity. 19(1):28-32. 
    16. Polanco, J.C and Koopman, P. (2007). Sry and the hesitant beginnings of male development. Developmental Biology, 302(1):13-24.
    17. Sasaki, O., Kimura, H., Ishii, K., Satoh, M., Nagamine, Y., Yokouchi, K. (2011). Economic effects of using sexed semen in Japanese dairy herds. Animal Science Journal. 82(3):486-493.
    18. Sinclair, A.H., Berta, P., Palmer, M.S. (1990). A gene from the human sex-determining region encode a protein with homology to a conserved DNA -binding motif. Nature. 346: 240-244.
    19. Suntharalingham, J. P., Buonocore, F., Duncan, A. J., Achermann, J. C. (2015). Dax-1 (nr0b1) and steroidogenic factor-1 (sf-1, nr5a1) in human disease. Best Practice & Research Clinical Endocrinology & Metabolism, 29 (4), 607-619.
    20. Tanaka, S. S., Nishinakamura, R. (2014). Regulation of male sex determination: genital ridge formation and sry activation in mice. Cellular & Molecular Life Sciences, 71(24), 4781-4802.
    21. Terral, G., Champion, T., Debaene, F., Colas, O., Bourguet, M., Wagner-Rousset, E., et al. (2017). Epitope characterization of anti-    jam-a antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches. Mabs, 00-00.
    22. Wetering, M., Clevers, H. (1992). Sequence specific interaction of the HMG box proteins TCF 1 and SRY occurs within the minor groove of a Watson Crick double helix. Embo Journal. 11(8):3039-3044.
    23. Waters, P. D., Wallis, M. C., Graves, J. A. M. (2007). Mammalian sex—origin and evolution of the y chromosome and. Seminars in Cell & Developmental Biology, 18(3), 389-400.
    24. Wang, X., Xue, M., Zhao, M., He, F., Li, C., Li, X. (2018). Identification of a novel mutation (ala66thr) of SRY gene causes XY pure gonadal dysgenesis by affecting DNA binding activity and nuclear import. Gene, 651: 143-151.
    25. Xu Ying, Yang Ligu, Guo Aizhen. (2002). Research progress in the mammaiian sex determining Region of the Y. Biotechnology Bulletin. 22(1):328-330.
    26. Yan Lei., Zhang Xiaojie., Xu Houqiang., Lin Jiadong. (2009). Progress on sex-control of livestock . Animal Husbandry and Feed Science. 30(4):130-133.
    27. Zhang, M., Lu, K. H., Jr, G. E. S. (2003). Development of bovine embryos after in vitro fertilization of oocytes with flow cytometrically sorted, stained and unsorted sperm from different bulls. Theriogenology, 60(9), 1657-1663. 
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