Ribokinase screened from T7 phage displayed Mycobacterium tuberculosis genomic DNA library had good potential for the serodiagnosis of tuberculosis

Abstract

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) is the leading cause of death among infectious diseases in the worldwide. Lack of more sensitive and effective diagnostic reagents has increased the awareness of rapid diagnosis for tuberculosis. In this study, T7 phage displayed genomic DNA library of M. tuberculosis was constructed to screen the antigens that specially bind with TB-positive serum from the whole genome of M. tuberculosis and to improve the sensitivity and specificity of tuberculosis serological diagnosis. After three rounds of biopanning, results of DNA sequencing and BLAST analysis showed that 19 positive phages displayed four different proteins and the occurrence frequency of the phage which displayed ribokinase was the highest. The results of indirect ELISA and dot immunoblotting indicated that representative phages could specifically bind to tuberculosis-positive serum. The prokaryotic expression vector containing the DNA sequence of ribokinase gene was then constructed and the recombinant protein was expressed and purified to evaluate the serodiagnosis value of ribokinase. The reactivity of the recombinant ribokinase with different clinical serum was detected and the sensitivities and specificities in tuberculosis serodiagnosis were 90% and 86%, respectively by screening serum from tuberculosis patients (n = 90) and uninfected individuals (n = 90) based on ELISA. Therefore, this study demonstrated that ribokinase had good potential for the serodiagnosis of tuberculosis.

T7 Phage as an Emerging Nanobiomaterial with Genetically Tunable Target Specificity

“…Besides, Luo et al constructed the T7 phages' genomic DNA library of mycobacterium tuberculosis, and screened the specific antigen binding to the positive serum of mycobacterium tuberculosis from the whole genome of mycobacterium tuberculosis to achieve higher specificity and sensitivity of serological diagnosis of tuberculosis. [122] The combined use of multiple biomarkers has the potent capacity to promote the early diagnosis of diseases efficiently and accurately. [28] Traditional diagnostic methods based on a single biomarker are proved imperfect diagnostic tools in clinical application due to their inefficiency, high cost, and sophisticated use.…”

“…constructed the T7 phages’ genomic DNA library of mycobacterium tuberculosis, and screened the specific antigen binding to the positive serum of mycobacterium tuberculosis from the whole genome of mycobacterium tuberculosis to achieve higher specificity and sensitivity of serological diagnosis of tuberculosis. [ 122 ]…”

Mycoplasma genitalium Protein of Adhesion Promotes the Early Proliferation of Human Urothelial Cells by Interacting with RPL35

“…The SV40 immortalized human urothelium cells (SV-HUC-1) (ATCC, CRL-9520) containing the SV40 genome were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The preparation of the T7 phage-displayed cDNA library of SV-HUC-1 cells was performed as described by Luo et al [43]. Approximately 150 µg of the total RNA of SV-HUC-1 cells was isolated using the nucleic acid isolation kit (Invitrogen, AM1975, Carlsbad, CA, USA).…”

Phage display technology for target determination of small-molecule therapeutics: an update

“…Thus, with this approach, the physicochemical properties of the protein sample or its interaction properties are irrelevant. Furthermore, this cell-free style approach enables drug and therapeutic target discovery even for infectious pathogens, such as tuberculosis [13,14] and malaria [15,16] that are difficult to handle in normal laboratories.…”