Prokaryotic expression, purification, and immunogenicity analysis of Mycobacterium tuberculosis specific excretive proteins / 中国医学科学院学报

<p><b>OBJECTIVE</b>To obtain the recombinant rv1837c and rv3803c of Mycobacterium tuberculosis using gene engineering technology and explore their prokaryotic expression, purification, and immunogenicity.</p><p><b>METHODS</b>The Mycobacterium tuberculosis rv1837c and rv3803c genes were amplified by polymerase chain reaction, and then cloned into the vector pTA2, followed by the subclone into the expression vector pET30a (+). The resulting plasmids, named pET30a (+): rv1837c and pET30a (+): rv3803c, encode recombinant protein containing a hexa-histidine tag on its N-terminus. pET30a (+): rv1837c and pET30a (+): rv3803c were introduced into E. coli BL21 (DE3) by transformation respectively, and the recombinant gene was induced with 0.4 mmol/L isopropyl-D-thiogalactopyranoside. The expressed products were identified by Western blot with hexa-histidine tag antibody and serum from tuberculotic patients. The histidine tagged protein was purified by nickel nitrilotriacetic acid His-Bind resin. Rabbits were immunized with purified recombinant Rv1837c and Rv3803c proteins. Then the purified recombinant Rv1837c and Rv3803c proteins were used to detect antibody in rabbit serum, which had been immunized by Western blot.</p><p><b>RESULTS</b>After transformation of the E. coli and induction with 0.4 mmol/L of isopropyl-D-thiogalactopyranoside, recombinant target proteins Rv1837c (relative molecular mass: 92000) and Rv3803c (relative molecular mass: 38 000) were expressed in pET30a (+): rv1837c and pET30a (+): rv3803c system. The expressed protein existed in cytoplasm in an unsoluble form and amounted to 30% and 50% of the total proteins of E. coli. The purity of the purified protein reached 90%. The immunogenicity of the recombinant proteins Rv1837c and Rv3803c was strong, as identified by Western blot.</p><p><b>CONCLUSION</b>The prokaryotic expression recombinant plasmids pET30a (+): rv1837c and pET30a (+): rv3803c was successfully constructed and the recombinant proteins Rv1837c and Rv3803c were obtained, which laid a basis for the optimized diagnosis of active tuberculosis.</p>

Establishment and evaluation of nitrate reductase combined with mycobacteriophage assay / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility.</p><p><b>METHOD</b>We used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method.</p><p><b>RESULTS</b>The sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide.</p><p><b>CONCLUSIONS</b>PhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.</p>

Relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the relationship between the resuscitation promoting role of resuscitation promoting factor and the initial bacteria amount of dormant Mycobacterium tuberculosis.</p><p><b>METHODS</b>Mycobacterium tuberculosis (dormant bacteria) was cultured for 100 days, then diluted into 1 mg/ml concentration with 7H9, and further diluted into 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/ml. Twelve new tubes added with 5 ml 7H9 and divided into two groups: the first group was added with the resuscitation-promoting factor protein, and the second group as control was added with 7H9. In each group the above diluted solutions were added. The tubes were located at 37 degrees C for culture. Optical density (OD) was detected on day 15, 25, 30, and 35. From each tube 1 microl culture solution was plated on 7H11 medium for colony counting.</p><p><b>RESULTS</b>OD detection showed that bacteria proliferation in each group had positive linear correlation (P < 0.05, P < 0.01), indicating that the resuscitation-promoting factor played a similiar role in solutions with different dilution concentrations. 7H11 results and the OD results show that these two detection methods in each group had linear correlation (P < 0.05, P < 0.01), indicating that these two methods showed consistent test results.</p><p><b>CONCLUSION</b>The resuscitation-promoting factor has no effect on the resuscitation of dormant Mycobacterium tuberculosis and its initial bacteria amount.</p>