Construction and identification of nucleotide-binding oligomerization domain-like receptor protein 6 recombinant adenoviral overexpression vector

Objective To construct and identify an overexpressing recombinant adenovirus vector carrying the mouse nucleotide-binding oligomerization domain-like receptor protein 6 (NLRP6) gene (NM_133 946.2).

Methods The coding sequence of the mouse NLRP6 gene was synthesized, digested with enzymes, and inserted into the adenovirus vector (GL2003). The transformed cells were then cultured in strain DH-5α, transformants were selected, and after identification by polymerase chain reaction (PCR), samples were sent for sequencing and comparison. The expression of NLRP6 protein in the recombinant plasmid was validated using the Western blot. The recombinant adenovirus vector Ad-NLRP6 was obtained using the Admax packaging system, then transduced into HEK293 cells, amplified, purified, and the viral titer was determined afterwards.

Results The successful integration of NLRP6 into the expression plasmid was confirmed through double enzyme digestion and DNA sequencing. Concurrent detection of the FLAG-tagged protein indicated a significant increase in the expression level of NLRP6. Recombinant adenovirus carrying NLRP6 were packaged and produced, followed by infecting HEK293 cells with the virus. The cellular expression of green fluorescent protein in Ad-NLRP6 indicated successful infection. The virus was collected, amplified, purified, and the viral titer was determined to be 4×10¹⁰ PFU/mL.

Conclusion The overexpression recombinant adenovirus vector carrying the mouse NLRP6 gene has been successfully constructed, providing an effective tool for further exploring the molecular function of NLRP6.