Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization
| Autor: | Shree Vidhya Srinivasan, Sayed Basha Nazar, K. S. Roy, Jyotirmoy Ghosh |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Gene isoform Antigenicity Molecular biology Veterinary medicine Buffalo Biology law.invention 03 medical and health sciences 0302 clinical medicine Antigen law medicine Coding region lcsh:Social sciences (General) lcsh:Science (General) Peptide sequence chemistry.chemical_classification Multidisciplinary medicine.diagnostic_test Recombinant PAG Pregnancy-associated glycoproteins 030104 developmental biology chemistry nervous system Immunoassay Recombinant DNA lcsh:H1-99 Glycoprotein 030217 neurology & neurosurgery lcsh:Q1-390 |
| Zdroj: | Heliyon, Vol 5, Iss 11, Pp e02863-(2019) |
| ISSN: | 2405-8440 |
| Popis: | Pregnancy-associated glycoproteins (PAGs) secreted from conceptus specific trophoblast cells are widely accepted biomarkers of ruminants. Limited information of PAGs in buffalo warrants further investigation for the development of sensitive homologous early pregnancy-specific diagnostic immunoassay. This experiment was thus designed to identify and clone the predominantly expressed early placentome-specific buffalo PAG (buPAG) isoform; to express this PAG isoform and verify its antigenicity by developing antisera and testing immuno-reactivity with recombinant proteins. Results indicated PAG 7 (buPAG 7) was the predominant isoform in buffalo early pregnant placentome. Attempt to express the full native glycosylated protein in the pcDNA3.3 vector and FreeStyle HEK 293F host was not successful. However, a partial 124 amino acid sequence selected from the non-glycosylated region of buPAG 7 could be expressed in E. coli BL21 (DE3) cells after codon optimization however; the yield was low. Antigenicity of the expressed protein was confirmed by successful immuno-reaction in rabbits indicating possibilities of using 124 aa partial PAG 7 protein as a putative antigen for monoclonal antibody production and development sensitive homologous immunoassay. In conclusion, our results confirmed the findings that buPAG 7 as the predominant early pregnancy-specific transcript. A selected partial 124 amino acid sequences of it could even be expressed in a heterologous host (E. coli). Based on our data presented here, we anticipate that the expressed recombinant protein can be useful as an antigen suitable for the development of PAG specific immunoassays in buffalo. |
| Databáze: | OpenAIRE |
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