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Published on: February 28, 2019
[Construction and evaluation of recombinant plasmid containing human triggering receptor expressed on myeloid cells-1
Yan Hu1, Li-Qi Shu, Qi-Xing Chen
1Department of Anesthesiology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China.
Objective:
To construct a eukaryotic expression plasmid containing human triggering receptor expressed on myeloid cells-1(TREM-1) gene.
Methods:
The entire gene coding region of human TREM-1 was amplified from total RNA of human peripheral blood by means of RT-PCR. The fragment of TREM-1 was cloned to vector pUCm-T. After digestion by restriction endonuclease BamH I and Pst I, the fragment was subcloned into the eukaryotic expressing vector pEGFP-C3. This recombinant vector was transfected into 293 cells using liposome. The expression level of TREM-1 was determined by fluorescence microscope and Western blot assay. The recombinant TREM-1 vector was transfected into THP-1 cells. After stimulation with 100 ng/ml LPS for 24 h, the mRNA levels of TNF-α and IL-1β were measured using RT-PCR.
Result:
The expression vector was constructed, and the result of the DNA sequencing showed that the constructed plasmid containing the TREM-1 gene. Fluorescence microscope and Western blot analysis showed that TREM-1 protein was expressed in 293 cells successfully. After transfection into THP-1 cells, recombinant TREM-1 could upregulate the mRNA levels of TNF-α and IL-1β.
Conclusion:
Eukaryotic expression plasmid pEGFP-TREM-1 is successfully constructed and showed biological activity.
