Preparation and identification of recombinant adenovirus containing the Toll-like receptor 2 extracellular domain gene of human / 解放军医学杂志

ABSTRACT

Objective To construct and identify the recombinant adenovirus vector containing the Toll-like receptor 2(TLR2) extracellular domain gene of human.Methods Human TLR2 extracellular domain cDNA was amplified by RT-PCR from peripheral blood mononuclear cells(PBMCs) and inserted into pMD18-T vector.After being confirmed by enzyme digestion and sequencing,the DNA fragment,digested with Kpn Ⅰ and Hind Ⅲ,was directionally cloned into adenovirus shuttle plasmid pAdTrack-CMV.After linearization by Pme Ⅰ digestion,the recombinant plasmid pAdTrack-CMV-TLR2 was transformed into competent AdEasier-1 germs and then homologically recombined with an adenoviral backbone plasmid pAdEasy-1 in bacteria BJ5183 to obtain the recombinant adenovirus plasmid.After confirmation,the recombinant adenovirus plasmid pAd-TLR2 was linearized with Pac Ⅰ digestion and transfected into 293 cells via liposome,and then package and adenovirus amplification were performed.The expression of green fluorescent protein(GFP) was observed,the virus titer was determined and the recombinant adenovirus was identified by PCR.ResultsThe gene fragment obtained by RT-PCR was of the same sequence as in GenBank.It was certified by restricted endonuclease digestion and PCR analysis that the recombinant adenovirus containing the TLR2 extracellular domain gene of human had been successfully constructed with a satisfactory high titer of 3?109pfu/ml.Conclusion The recombinant adenovirus containing TLR2 extracellular domain gene of human has been successfully constructed,which lays a foundation for further study on the structure and biological activity of TLR2.