Preparation of PP7 bacteriophage-like particles carrying the polypeptide from 16 kD antigen and their value evaluation in the diagnosis of tuberculosis / 临床检验杂志

ABSTRACT

Objective To obtain the PP7 bacteriophage-like particles (BLPs) carrying the polypeptide from 16 kD antigen (16kD91-110) on their surface,and evaluate their diagnostic value in tuberculosis.Methods First,the PP7 capsid protein gene containing the encoding gene of polypeptide 16kD91-110 was amplified by PCR and inserted into the plasmid pETDuet-2PP7.Then,the obtained recombinant plasmid pETDuet-2PP7-16kD91-110 was transferred into Escherichia coli,and the recombinant protein was induced and identified by SDS-PAGE and western blot.Next,the purified 2PP7-16kD91-110 BLPs were used as stimulating antigen to inject into the blood of patients with tuberculosis,and their serum antibody levels against 16 kD antigen were detected by indirect ELISA.Results The results of SDS-PAGE and transmission electron microscope showed that the 2PP7-16kD91-110 BLPs were prepared,and that the 16kD91-110 polypeptide epitopes displayed on the surface of PP7 BLPs could bind with the antibodies against 16 kD antigen specifically.After using 2PP7-16kD91-110 BLPs as antigen to stimulate the blood cells from active tuberculosis and latent tuberculosis patients,respectively,the sensitivity of interferon-γrelease assay was 75.0％ and 82.9％,respectively,in the diagnosis of active tuberculosis and latent tuberculosis,which was similar to the results of Wantai TB-IGRA (interferon-γ release assay) kits from Beijing Wantai Biological Pharmacy Enterprise Co.,Ltd..Conclusion The PP7 BLPs with the 16kD91-110 polypeptide displayed on their surface are prepared successfully,which provides a kind of safe,stable and low cost stimulating antigen for the detection of interferon-γ release,and a new method for the diagnosis of tuberculosis.