Construction Of Targeting Vector Of Human TLR4and MD2Gene

Objective To clone the Toll like receptor4(TLR4) and Myeloid differentia-tion pr-otein-2(MD2)gene from the peripheral blood mononuclear cells of human, then cost-ruct its eukaryotic ex-pression vector pcDNA.3.1/myc-His(-)B-TLR4、pcDNA.3.1/myc-His(-)B-MD2so as to provide basis for the estabolishment of cell model expressedTLR4and MD2.Method The total RNA of the human peripheral blood mononuclear cells was ext-racted. The completeencoded sequence of the human TLR4and MD2was amplifiedby RT-PCR.T-he ampilified fragment was inserted into colon vector pMD-19T to cons-truct the recombined plasmid.Then the recombined pMD-19T-TLR4and pMD-19T-MD2plasmid were digested with endonuclease,and the recovery fragment were inserted int-o eukaryotic expression vector pcDNA.3.1/myc-His(-)B to construct the recombined pl-asmid. The plasmid pcDNA.3.1/myc-His(-)B-TLR4and pcDNA.3.1/myc-His(-)B-MD2were introduced into HEK293cells through electroporation transfection.The mRNA express-ion of human TLR4and MD2were detected by reverse transcriptase-PCR(RT-PCR).Results TLR4and MD2cDNA were successful obtained from human peripheralblood mononuclear cells through RT-PCR.Then the recombinant plasmid were digestedwith endonuclease, then confirma-tion by sequencing and Nucleotide homology analysisto GeneBank, we successful obtained the recombinant plasmid pcDNA.3.1/myc-His(-)B-TLR4and pcDNA.3.1/myc-His(-)B-MD2. The recombinant plasmid pcDNA.3.1/myc-His(-)B-TLR4and pcDNA.3.1/myc-His(-)B-MD2were introduced into HEK293cells thro-ugh electroporation transfection respectively.The mRNA expression of human TLR4and MD2gene in the HEK293cells were confirmed by RT-PCR.Conclusion We successful construct eukaryotic expression vector of human TLR4and MD2gene from human peripheral blood mononuclear cells.