Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization
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Title |
Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization
Not Available |
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Creator |
ShreeVidhya S
Ghosh J Nazar SB Roy KS |
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Subject |
Veterinary medicine
Molecular biology Buffalo Pregnancy-associated glycoproteins Recombinant PAG |
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Description |
Not Available
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. ICAR- NIANP |
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Date |
2020-06-11T10:49:33Z
2020-06-11T10:49:33Z 2019-11-27 |
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Type |
Research Paper
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Identifier |
ShreeVidhya S, Ghosh J, Nazar SB, Roy KS. 2019. Buffalo early pregnancy biomarker coding sequence cloning and partial length expression in E. coli after codon optimization. Heliyon, 5:e02863
24058440 http://krishi.icar.gov.in/jspui/handle/123456789/37036 |
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Language |
English
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Relation |
Not Available;
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Publisher |
Elsevier BV
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