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Updated: Apr 22, 2026
Separation and Fractionation of Cell Wall and Cell Membrane Proteins from Mycobacterium tuberculosis for Downstream Protein Analysis
Published on: September 26, 2025
Expression and diagnostic value of proteins in Mycobacterium tuberculosis
1Hainan Provincial Nongken General Hospital, Haikou, Hainan, China zhuzhongyuan197@163.com.
Abstract:
We constructed a prokaryotic expression vector expressing the Mycobacterium tuberculosis protein TB16.3, as well as 3 other proteins, including TB15.3, CFP-10, and Rv2626C, which were purified and analyzed for their effectiveness as detection antibodies. The TB16.3 genes of M. tuberculosis H37Rv genomic DNA were amplified by polymerase chain reaction, inserted into the expression vector pET-30a, and expressed in Escherichia coli. An enzyme-linked immunosorbent assay was used to detect the 4 M. tuberculosis antibodies. Engineered E. coli bacteria expressing TB16.3 and the 3 other proteins were constructed and found mainly to be soluble. For recombinant TB16.3 proteins, serum samples of 118 tuberculosis (TB) patients and 96 healthy controls were analyzed. Sensitivity, specificity, and adjusted concordance rate for the TB16.3 antibody were 72.9, 86.5, and 79.6%, respectively. The positive rate of Rv2626C antibody in TB patients (44.1%) was significantly lower than that in normal controls (75.0%, χ(2) = 20.8, P < 0.01). TB15.3 and TB16.3 were used for simultaneous detection and showed sensitivity, specificity, and repeatability rates of 69.4, 96.9, and 83.7%. The antibody positive rate and specificity for patients with lung disease was 9.6 and 90.4%, respectively. TB15.3 and TB16.3 were mixed and detected simultaneously. Combined with the results for TB15.3, the sensitivity, specificity, and concordance rates were 82.2, 95.9, and 88.9%, respectively. The concordance rate was the highest value observed. Target genes were cloned into a host strain and expressed successfully. The TB16.3 recombinant protein may be used as a new serological antigen for tuberculosis diagnosis.
Insights
Researchers developed a new diagnostic tool for tuberculosis (TB) using Mycobacterium tuberculosis proteins. The TB16.3 recombinant protein shows promise as a serological antigen for TB detection.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Tuberculosis (TB) remains a significant global health challenge, necessitating improved diagnostic methods.
- Current diagnostic approaches for TB have limitations, driving the search for novel biomarkers and antigens.
- Accurate and early detection of TB is crucial for effective treatment and disease control.
Purpose of the Study:
- To construct and express recombinant proteins from Mycobacterium tuberculosis, specifically TB16.3, TB15.3, CFP-10, and Rv2626C.
- To evaluate the diagnostic potential of these recombinant proteins as serological antigens for tuberculosis detection.
- To assess the sensitivity, specificity, and concordance rates of antibodies targeting these proteins in TB patients.
Main Methods:
- Prokaryotic expression vectors were engineered to express Mycobacterium tuberculosis proteins (TB16.3, TB15.3, CFP-10, Rv2626C) in Escherichia coli.
- Recombinant proteins were purified and analyzed for solubility and effectiveness as detection antibodies.
- Enzyme-linked immunosorbent assays (ELISA) were employed to detect antibodies in serum samples from TB patients and healthy controls.
Main Results:
- Recombinant TB16.3 protein demonstrated a sensitivity of 72.9% and specificity of 86.5% for TB detection.
- Simultaneous detection using TB15.3 and TB16.3 antibodies achieved a sensitivity of 69.4% and specificity of 96.9%.
- Combining TB15.3 and TB16.3 for simultaneous detection yielded the highest concordance rate (88.9%), with sensitivity of 82.2% and specificity of 95.9%.
Conclusions:
- The recombinant TB16.3 protein is a potential new serological antigen for tuberculosis diagnosis.
- The combined use of TB15.3 and TB16.3 antigens offers a highly specific and concordant method for TB detection.
- Further validation of these recombinant proteins could lead to improved serological diagnostic tools for tuberculosis.
