cDNA cloning and recombinant expression of a proliferation inducing ligand (APRIL) from Père David's deer
Cui Min1, Qi Wang, Yu-Qing Chen
1Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing 210046, China.
Abstract:
In the present study, the cDNA of Père David's deer APRIL (miAPRIL) was successfully amplified, and nucleotide sequence analysis showed that the open reading frame (ORF) of miAPRIL consists of 753 bases encoding 250-amino acids. Phylogenetic analysis exhibited high homology with the even-toed ungulates (artiodactyla). The extracellular soluble domain of the miAPRIL (misAPRIL) fragment was cloned into the expression vector pET43.1a and transformed into Escherichia coli BL21 (DE3) for recombinant expression. SDS-PAGE and western blotting analysis indicated that misAPRIL protein expression was successfully achieved. Indirect immunofluorescence staining demonstrated that misAPRIL has the ability to bind to the surface of Père David's deer lymphocytes. Flow cytometry analysis revealed that misAPRIL was able to enhance lymphocytes survival in a dose-dependent manner.
Insights
Researchers successfully amplified Père David's deer APRIL cDNA, expressing a soluble protein fragment (misAPRIL). This fragment enhances lymphocyte survival, offering potential insights into deer immunology.
Area of Science:
- Immunology
- Molecular Biology
- Genomics
Background:
- Père David's deer (Elaphurus davidianus) immunology requires further understanding.
- Apoptosis (programmed cell death) pathways are crucial in immune responses.
Purpose of the Study:
- To characterize the APRIL gene in Père David's deer.
- To express and analyze the function of the soluble APRIL protein fragment (misAPRIL).
Main Methods:
- cDNA amplification and nucleotide sequencing of Père David's deer APRIL.
- Recombinant expression of the soluble misAPRIL fragment in Escherichia coli.
- SDS-PAGE, Western blotting, indirect immunofluorescence, and flow cytometry for protein analysis and functional assays.
Main Results:
- The open reading frame (ORF) of miAPRIL was identified (753 bases, 250 amino acids).
- Phylogenetic analysis showed homology with other artiodactyls.
- Recombinant misAPRIL was successfully expressed and confirmed to bind Père David's deer lymphocytes.
- misAPRIL demonstrated a dose-dependent enhancement of lymphocyte survival.
Conclusions:
- The study successfully characterized and expressed a functional soluble APRIL protein from Père David's deer.
- The findings suggest misAPRIL plays a role in lymphocyte survival and immune regulation in this species.
- This research provides a foundation for further studies on Père David's deer immune mechanisms.
