Skip to main content
SpringerLink
Log in

Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

The bovine pregnancy-associated glycoproteins (bPAGs) have been widely used as robust markers for early diagnosis of pregnancy in the cattle. The current immune recognition methods for detecting bPAGs are limited and, to a certain extent, are associated with high costs and poor stability of the antibody. Aptamers that are more stable and easily synthesized than antibodies might serve as suitable candidates for the development of rapid detection methods. This paper describes selection and characterization of bPAG4 aptamers and theirs applicability to detect bPAG4 in the serum. In this work, the recombinant bovine pregnancy-associated glycoproteins 4 (bPAG4) with a relative molecular mass of about 48 kDa was successfully expressed in human embryonic kidney 293 (HEK 293) cells. Subsequently, the ssDNA aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) using magnetic beads (MB) coated with bPAG4 as target. After 9 rounds of selection, three aptamers with high affinity to bPAG4 (Kd = 11.7~40.2 nM) were identified. The selected aptamers were successfully used in enzyme-linked aptamer assay (ELAA) to detect bPAG4 at a detection limit of 0.09 ng/mL. Meanwhile, it has been successfully applied for the detection of bPAG4 in serum samples. This work demonstrated that the selected aptamers could be used as promising affinity probes in the development of inexpensive, simple, and sensitive analysis methods for detecting bPAGs.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. LeBlanc S. Assessing the association of the level of milk production with reproductive performance in dairy cattle. J Reprod Dev. 2010;56:1–7.

    Article  Google Scholar 

  2. Kossaibati MA, Esslemont RJ. The costs of production diseases in dairy herds in England. Vet J. 1997;154:41–51.

    Article  CAS  PubMed  Google Scholar 

  3. Youngquist RS. Pregnancy diagnosis. In: Youngquist RS, Threlfall WR, editors. Current therapy in large animal theriogenology. 2nd ed. Missouri: Saunder; 2007. p. 294–303.

    Chapter  Google Scholar 

  4. Romano JE, Thompson JA, Forrest DW, Westhusin ME, Tomaszweski MA, Kraemer DC. Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. Theriogenology. 2006;66:1034–41.

    Article  PubMed  Google Scholar 

  5. Karen A, Sousa NMD, Beckers JF, Bajcsy ÁC, Tibold J, Mádl I, et al. Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim Reprod Sci. 2015;159:31–7.

    Article  CAS  PubMed  Google Scholar 

  6. Nagappan M, Michael M, Robert S. Methods for early detection of pregnancy in cows. Monsanto Technology LLC, assignee. US Pat. No. 7,604,950 B2. 2009.

  7. Dufour S, Durocher J, Dubuc J, Dendukuri N, Hassan S, Buczinski S. Bayesian estimation of sensitivity and specificity of a milk pregnancy-associated glycoprotein-based ELISA and of transrectal ultrasonographic exam for diagnosis of pregnancy at 28 to 45 days following breeding in dairy cows. Prev Vet Med. 2017;140:122–33.

    Article  PubMed  Google Scholar 

  8. Commun L, Velek K, Barbry JB, Pun S, Rice A, Mestek A, et al. Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cows. J Vet Diagn Investig. 2016;28:207–13.

    Article  CAS  Google Scholar 

  9. Zoli AP, Guilbault LA, Delabaut P, Ortiz WB, Beckers JF. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis. Biol Reprod. 1992;46:83–92.

    Article  CAS  PubMed  Google Scholar 

  10. Yuan Q, Lu DQ, Zhang XB, Chen Z, Tan WH. Aptamer-conjugated optical nanomaterials for bioanalysis. Trac-Trend Anal Chem. 2012;39:72–86.

    Article  CAS  Google Scholar 

  11. Wang RH, Zhao JJ, Jiang TS, Kwon YM, Lu HG, Jiao PR, et al. Selection and characterization of DNA aptamers for use in detection of avian influenza virus H5N1. J Virol Methods. 2013;189:362–9.

    Article  CAS  PubMed  Google Scholar 

  12. Wang S, Dong YY, Liang XG. Development of a SPR aptasensor containing oriented aptamer for direct capture and detection of tetracycline in multiple honey samples. Biosens Bioelectron. 2018;109:1–7.

    Article  CAS  PubMed  Google Scholar 

  13. Liu C, Guo YJ, Luo F, Rao PF, Fu CL, Wang SY. Homogeneous electrochemical method for ochratoxin A determination based on target triggered aptamer hairpin Switch and exonuclease III-assisted recycling amplification. Food Anal Methods. 2017;10:1982–90.

    Article  Google Scholar 

  14. Wu SJ, Liu LH, Duan N, Li Q, Zhou Y, Wang ZP. An aptamer-based lateral flow test strip for rapid detection of zearalenone in corn samples. J Agric Food Chem. 2018;66:1949–54.

    Article  CAS  PubMed  Google Scholar 

  15. Liu CB, Lu CX, Tang ZG, Chen X, Sun FX. Aptamer-functionalized magnetic nanoparticles for simultaneous fluorometric determination of oxytetracycline and kanamycin. Microchim Acta. 2015;182:2567–75.

    Article  CAS  Google Scholar 

  16. Lu CX, Gao XX, Chen Y, Jiang T, Liu CB. An aptamer-based lateral flow test strip for the simultaneous detection of Salmonella typhimurium, Escherichia coli O157:H7 and Staphylococcus aureus. Anal Lett. 2020;53:646–59.

    Article  CAS  Google Scholar 

  17. Lu CX, Tang ZG, Liu CB, Kang LC, Sun FX. Magnetic-nanobead-based competitive enzyme-linked aptamer assay for the analysis of oxytetracycline in food. Anal Bioanal Chem. 2015;407:4155–63.

    Article  CAS  PubMed  Google Scholar 

  18. Green JA, Xie S, Quan X, Bao B, Gan X, Mathialagan N, et al. Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy. Biol Reprod. 2000;62:1624–1631.

  19. Wooding FB, Roberts RM, Green JA. Light and electron microscope immunocytochemical studies of the distribution of pregnancy associated glycoproteins (PAGs) throughout pregnancy in the cow: possible functional implications. Placenta. 2005;26:807–27.

    Article  CAS  PubMed  Google Scholar 

  20. Telugu BPV, Walker AM, Green JA. Characterization of the bovine pregnancy-associated glycoprotein gene family-analysis of gene sequences, regulatory regions within the promoter and expression of selected genes. BMC Genomics. 2009;10:185–201.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Xie S, Low BG, Nagel RJ, Beckers JF, Roberts RM. A novel glycoprotein of the aspartic proteinase gene family expressed in bovine placental trophectoderm. Biol Reprod. 1994;51:1145–53.

    Article  CAS  PubMed  Google Scholar 

  22. Hughes AL, Green JA, Garbayo JM, Roberts RM. Adaptive diversification within a large family of recently duplicated, placentally expressed genes. Proc Nati Acad Sci U S A. 2000;97:3319–23.

    Article  CAS  Google Scholar 

  23. Gajewski Z, Pertajitis M, Sousa MN, Beckers FJ, Pawlinski B, Janett F. Pregnancy-associated glycoproteins as a new diagnostic tool in cattle reproduction. Schweiz Arch Tierheilkd. 2009;151:577–82.

    Article  CAS  PubMed  Google Scholar 

  24. Liu CB, Shi GQ, Lu CX. Eukaryotic expression and purification of recombinant bovine pregnancy associated glycoprotein-9. Xinjiang Agr Sci. 2019;56:1552–9 (In Chinese).

    Google Scholar 

  25. Luo ZF, He L, Wang JJ, Fang XN, Zhang LY. Developing a combined strategy for monitoring the progress of aptamer selection. Analyst. 2017;142:3136–9.

    Article  CAS  PubMed  Google Scholar 

  26. Wang Y, Wu JJ, Chen YJ, Xue F, Teng J, Cao JX, et al. Magnetic microparticle-based SELEX process for the identification of highly specific aptamers of heart marker–brain natriuretic peptide. Microchim Acta. 2015;182:331–9.

    Article  CAS  Google Scholar 

  27. Szenci O, Beckers JF, Sulon J, Bevers MM, Börzsönyi L, Fodor L, et al. Effect of induction of late embryonic mortality on plasma profiles of pregnancy associated glycoproteins in heifers. Vet J. 2003;165:307–13.

    Article  CAS  PubMed  Google Scholar 

  28. Patel OV, Takahashi T, Imai K, Hashizume K. Generation and purification of recombinant bovine pregnancy associated glycoprotein. Vet J. 2004;168:328–35.

    Article  CAS  PubMed  Google Scholar 

  29. Guruprasad K, Blundell TL, Xie S, Green J, Szafranska B, Nagel RJ, et al. Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases. Protein Eng. 1996;9:849–56.

    Article  CAS  PubMed  Google Scholar 

  30. Gao SX, Hu W, Zheng X, Cai S, Wu JH. Functionalized aptamer with an antiparallel G-quadruplex: structural remodeling, recognition mechanism, and diagnostic applications targeting CTGF. Biosens Bioelectron. 2019;142:111475.

    Article  CAS  PubMed  Google Scholar 

  31. Niazi JH, Lee SJ, Gu MB. Single-stranded DNA aptamers specific for antibiotics tetracyclines. Bioorg Med Chem. 2008;16:7245–53.

    Article  CAS  PubMed  Google Scholar 

  32. Handy SM, Yakes BJ, DeGrasse JA, Campbell K, Elliott CT, Kanyuck KM, et al. First report of the use of a saxitoxin-protein conjugate to develop a DNA aptamer to a small molecule toxin. Toxicon. 2013;61:30–7.

    Article  CAS  PubMed  Google Scholar 

  33. Duan N, Ding XY, He LX, Wu SJ, Wei YX, Wang ZP. Selection, identification and application of a DNA aptamer against Listeria monocytogenes. Food Control. 2013;33:239–43.

    Article  CAS  Google Scholar 

  34. Nie J, Deng Y, Deng QP, Zhang DW, Zhou YL, Zhang XX. A self-assemble aptamer fragment/target complex based high-throughput colorimetric aptasensor using enzyme linked aptamer assay. Talanta. 2013;106:309–14.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was financially supported by the National Natural Science Foundation of China (31860647).

Author information

Author notes

  1. Changbin Liu and Chunxia Lu contributed equally to this work.

Authors and Affiliations

  1. College of Animal Science and Technology, Shihezi University, Shihezi, 832003, China

    Changbin Liu & Guoqing Shi

  2. Institute of Animal Husbandry and Veterinary Science, Xinjiang Academy of Agriculture and Reclamation Science, Shihezi, 832000, China

    Changbin Liu & Guoqing Shi

  3. Life Science and Technology Institute, Yangtze Normal University, Chongqing, 408100, China

    Chunxia Lu

Authors
  1. Changbin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chunxia Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guoqing Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Guoqing Shi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Research involving human participants and/or animals

All experimental animal procedures were performed in compliance with the China Code of Practice Animals for the Care and Use of Animals for Scientific Purposes. The animal welfare was approved by Animal Experimental Ethical Review Form of the First Affiliated Hospital of Medical College, Shihezi University.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 706 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, C., Lu, C. & Shi, G. Selection, identification, and application of DNA aptamers against bovine pregnancy-associated glycoproteins 4. Anal Bioanal Chem 412, 4235–4243 (2020). https://doi.org/10.1007/s00216-020-02666-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-02666-w

Keywords

Search

Navigation