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Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon
Shree Vidhya Srinivasan1,2, Jyotirmoy Ghosh1, Sayed Basha Nazar1
1Molecular Biology Laboratory, Division of Physiology, ICAR-National Institute of Animal Nutrition and Physiology, Hosur Main Road, Adugodi-PO, Banglaore 560 030, Karnataka, India.
Heliyon
|December 18, 2019
Summary
Pregnancy-associated glycoproteins (PAGs) are key biomarkers for early pregnancy detection in buffalo. Researchers identified buPAG 7 as the predominant isoform, successfully expressing a partial protein for potential immunoassay development.
Area of Science:
- Reproductive biology
- Biotechnology
- Animal science
Background:
- Pregnancy-associated glycoproteins (PAGs) are crucial biomarkers for early pregnancy diagnosis in ruminants.
- Limited data exists on buffalo PAGs, hindering the development of specific diagnostic tools.
- Identifying and characterizing buffalo PAGs is essential for advancing reproductive technologies.
Purpose of the Study:
- To identify and clone the primary early placentome-specific buffalo PAG (buPAG) isoform.
- To express the identified buPAG isoform and confirm its antigenicity.
- To develop a homologous early pregnancy diagnostic immunoassay for buffalo.
Main Methods:
- Identification and cloning of the predominant early placentome-specific buPAG isoform.
- Expression of a partial buPAG 7 protein in *E. coli* after codon optimization.
- Development of antisera in rabbits and testing immuno-reactivity with the recombinant protein.
Main Results:
- PAG 7 (buPAG 7) was identified as the predominant early pregnancy-specific isoform in buffalo placentomes.
- Full-length glycosylated buPAG 7 expression was unsuccessful in a mammalian host.
- A partial 124 amino acid non-glycosylated sequence of buPAG 7 was expressed in *E. coli*, albeit with low yield.
- The expressed partial buPAG 7 protein demonstrated antigenicity, confirmed by immuno-reaction in rabbits.
Conclusions:
- buPAG 7 is the predominant early pregnancy-specific transcript in buffalo.
- A partial buPAG 7 sequence can be expressed in a heterologous host (*E. coli*).
- The recombinant partial buPAG 7 protein shows potential as an antigen for developing sensitive, homologous PAG-specific immunoassays in buffalo.
