摘要
Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection.
Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection.
