Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis.

BACKGROUND: Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage. METHODS: MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique. RESULTS: RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS. CONCLUSIONS: MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

[Effects of isocitrate lyase from Mycobacterium tuberculosis on the survival of Mycobacterium smegmatis in macrophage and mechanism thereof].

OBJECTIVE: To investigate the effects of isocitrate lyase (ICL) from Mycobacterium tuberculosis (MTB-icl) on the survival of Mycobacterium smegmatis (MS) in macrophage and illuminate the possible mechanisms. METHODS: MTB-icl gene was amplified by PCR and cloned into Ecoli-Mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmid pUV15-icl expressing ICL-GFP. The recombinant shuttle plasmid pUV15-icl and blank plasmid pUV15 were induced into MS of the line 1-2c so as to obtain rMS-pUV15-icl and rMS-pUV15. Shuttle plasmid rMS-pUV15-IG expressing ICL-green fluorescent protein (GFP) was constructed. rMS-pUV15-IG and MS 1-2c were used to infect the murine macrophages of the line RAW264.7, fluorescence microscopy was used to observe the expression of ICL-GFP. The expression of ICL in the MS swallowed by the macrophages was verified by RT-PCR and Western blotting. Another macrophages RAW264.7 were cultured and infected with rMS-pUV15-icl and rMS-pUV15 respectively. 0, 24, and 48 hours later macrophages were collected and the number of MS colonies was calculated. The interferon (IFN)-gamma and nitrogen oxide (NO) concentrations in the culture supernatants of macrophages infected by rMS-pUV15-icl and rMS-pUV15 were measured by ELISA and Griess assay respectively. The apoptotic rate of the macrophages was assayed by in situ TUNEL technique. RESULTS: Western blotting showed that the MTB ICL protein expression of the rMS-pUV15-icl was significantly higher than that of rMS-pUVI5. Fluorescence microscopy showed green fluorescence in the RAW264.7 cells infected with rMS-pUV15-IG, but not ion the RAW264.7 cells infected with MS 1-2c. 0 h after the infection of the macrophages there was not significant difference in the MS amount in the macrophages between the rMS-pUV15-isl and rMS-pUV15 groups, and 24 h and 48 h later the MS amounts of the rMS-pUV15-icl group were (32.78 +/- 2.90) x 10(3) and (23.33 + 2.34) x 10(3) respectively, both significantly higher than those of the rMS-pUV15 group [(14.67 +/- 2.45) x 10(3) and (2.28 +/- 0.25) x 10(3) respectively, both P < 0.01]. There were not significant differences in the concentrations of IFN-gamma and NO in the culture supernatants between the 2 groups (both P >0.05). 48 h after the infection the apoptotic rate of the rMS-pUV15-icl group was significantly lower than that of the rMS-pUV15 group (P <0.01). Conclusion MTB-ICL promotes the intracellular survival of MS. Suppressing the apoptosis of the host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

[Construction of a fusion gene expression vector containing mouse Ipr1 and EGFP and its expression in murine macrophage].

AIM: To construct an eukaryotic expression vector containing the fusion gene of mouse Ipr1 and EGFP and to study its expression in murine macrophage RAW264.7. METHODS: The coding sequence of Ipr1 gene was amplified from the total RNA of C57BL/6J mouse thymus by RT-PCR. The gene was cloned into pEGFP-C1 and the recombinant plasmid was identified by PCR, restrict endonuclease digestion and sequencing. Then the pEGFP-Ipr1 was transiently transfected into RAW264.7. The expression of Ipr1 gene and fusion protein was detected by RT-PCR and laser scanning confocal microscopy. RESULTS: The whole coding sequence of Ipr1 was successfully amplified. The recombinant plasmid was identified by PCR, restrict endonuclease digestion and sequencing. The fusion protein was successfully expressed in the targeted cells and its localization was in nucleus. CONCLUSION: The eukaryotic expression vector pEGFP-Ipr1 has been successfully constructed. The fusion protein can be expressed in murine macrophage RAW264.7 and located in nucleus.

[Therapeutic effects of granulysin/interleukin-12 recombinant M. smegmatis in a murine model of M. tuberculosis infection].

OBJECTIVE: To study the effects and mechanisms of recombinant Mycobacterium smegmatis (M. smegmatis) encoding human granulysin and murine interleukin-12 (IL-12) on murine M. tuberculosis infection. METHODS: Balb/c mice infected with M. tuberculosis were treated with normal saline, M. smegmatis, pZM03, recombinant M. smegmatis respectively. The numbers of viable bacteria in the lung and the spleen were counted. The levels of IL-12, IgG(2a) in serum and interferon-gamma (IFN-gamma) released from spleen lymphocytes stimulated with purified protein derivative (PPD) were detected with enzyme linked immunosorbent assay (ELISA). The expression of granulysin in tissue was detected with immunohistochemistry. Lungs and spleens were prepared for pathological analysis. RESULTS: The recombinant M. smegmatis group [(4.57 +/- 0.28) in lung, (3.47 +/- 0.25) in spleen] showed significantly reduced number of colony forming units (log CFU/g) compared with the control [(5.77 +/- 0.12) in lung, (4.66 +/- 0.18) in spleen], M. smegmatis [(5.62 +/- 0.14) in lung, (4.65 +/- 0.26) in spleen] and pZM03 [(5.04 +/- 0.22) in lung, (4.25 +/- 0.48) in spleen] group. The expression of granulysin in tissue, and increased level of IL-12 and IgG(2a) in serum were found in recombinant M. smegmatis group. IFN-gamma released from spleen lymphocytes stimulated with PPD in recombinant M. smegmatis group and pZM03 group was higher than that of other groups, but the difference between recombinant M. smegmatis group and pZM03 group was not significant. The pathological changes in lungs of the recombinant M. smegmatis group were localized, while those in the control group were extensive. Significant pathological changes were not found in the spleens of all groups. CONCLUSION: The recombinant M. smegmatis had immunotherapeutic effects, which were associated with a switch to Th1 response and the antibacterial activity of granulysin.

Recombinant M. smegmatis vaccine targeted delivering IL-12/GLS into macrophages can induce specific cellular immunity against M. tuberculosis in BALB/c mice.

In the present study, we constructed a viable therapeutic vaccine of recombinant M. smegmatis mediated IL-12/GLS (granulysin) gene transfer into murine macrophages to exert the immunotherapy effects on the Mycobacterium tuberculosis infection. We tested this recombinant therapeutic vaccine in an in vivo study to determine its capability of stimulating host specific immune responses against M. tuberculosis. BALB/c mice intranasally immunized with the therapeutic vaccine developed an efficient Th1 protective immune response against M. tuberculosis which was equal to that of the BCG strain. Inoculation intranasally with this viable vaccine induced high level of serum IFN-gamma, IL-12 and IgG2a. The viable vaccine was capable of inducing purified protein derivative (PPD) antigen-specific splenocytes proliferation and IFN-gamma production from T cells in spleens of the immunized mice. In addition, intranasally inoculation with the viable vaccine can induce PPD antigen-specific sIgA production in the broncho-alveolar lavage fluid (BALF) of the immunized mice. No change of IL-4 level was found in all groups. The therapeutic mechanism of this viable vaccine against M. tuberculosis infection observed here appeared to be a result of the specific Th1 immune response activated by mycobacterium antigen from M. smegmatis and the expression of sIL-12/GLS in alveolar macrophages via the M. smegmatis-mediated gene transfer method. This research demonstrates that the therapeutic gene can be introduced into a host by viable mycobacteria works to induce the host specific immune response against M. tuberculosis infection in vivo. Since this therapeutic vaccine can strongly induce specific Th1 responses against M. tuberculosis in BALB/c mice and has no obviously harmfulness to the host simultaneously, the recombinant vaccine might be a potential candidate therapeutic vaccine against tuberculosis.

[Inhibitory effects of 10-23 deoxyribozyme targeting ICL gene on the expression of isocitrate lyase and the infection of Mycobacterium tuberculosis in macrophages].

OBJECTIVE: To investigate the inhibitory effects of the 10-23 deoxyribozyme (DRZ) targeting ICL gene on the expression of isocitrate lyase (ICL) and the survival of Mycobacterium tuberculosis in macrophages. METHODS: Five 10-23 DRZ targeting ICL genes (DZ1-DZ5) were designed according to the predicted secondary structure of Mycobacterium tuberculosis ICL mRNA. Their cleavage activity and specificity were identified in cell free conditions. Then Mycobacterium tuberculosis pretreated with DZ4 with or without subinhibitory concentration of isoniazid (INH) were used to infect THP-1 cells. The bacterial burden of the infected THP-1 cells was monitored at indicated times after infection. The effect of 10-23 DRZ on the growth of Mycobacterium tuberculosis in vitro was also assayed by plating Mycobacterium tuberculosis treated with INH alone or DZ4 plus INH on M7H10 agar directly. RESULTS: Four of the five designed 10-23 DRZ, DZ1, DZ3, DZ4 and DZ5 could cleavage ICL mRNA efficiently and specifically. Treating Mycobacterium tuberculosis with 5 micromol/L DZ4 plus subinhibitory concentration of INH decreased the expression of ICL dramatically, by 34.9% - 46.7% (10 microg/L INH, P < 0.01) or 21.9% - 36.9% (5 microg/L INH, P < 0.01) when compared with corresponding concentration of INH alone. The survival of Mycobacterium tuberculosis in THP-1 cells was decreased significantly when Mycobacterium tuberculosis was pretreated with 5 micromol/L DZ4 plus subinhibitory concentration of INH. 4 or 7 days after infection, the bacteria burden in macrophages was decreased from 126.5 x 10(4) CFU, 307.5 x 10(4) CFU to 54.6 x 10(4) CFU, 114.3 x 10(4) CFU (when 10 microg/L INH used) or from 133.0 x 10(4) CFU, 325.4 x 10(4) CFU to 71.7 x 10(4) CFU, 174.4 x 10(4) CFU (when 5 microg/L INH used). 10-23 DRZ showed no obvious effect on the growth of Mycobacterium tuberculosis in M7H10 agar. CONCLUSIONS: INH at the subinhibitory concentration can improve the entry of 10-23 DRZ in Mycobacterium tuberculosis. In the presence of subinhibitory concentration of INH, 10-23 DRZ targeting ICL gene can strongly inhibit the expression of ICL and decrease the survival of Mycobacterium tuberculosis in macrophages.

Therapeutic DNA vaccines against tuberculosis: a promising but arduous task.

OBJECTIVE: To review recent developments in therapeutic DNA vaccines against tuberculosis. DATA SOURCES: The data used in this review were obtained mainly from the studies of therapeutic DNA vaccines against tuberculosis reported from 2000 to 2006. STUDY SELECTION: Relevant articles about studies of therapeutic DNA vaccines against tuberculosis were selected. DATA EXTRACTION: Data were mainly extracted from the 32 articles listed in the reference section of this review. RESULTS: Some DNA vaccines which previously showed to induce protective immunity against infection by Mycobacterium tuberculosis in a prophylactic manner are also surprisingly effective when used therapeutically, including persistent Mycobacterium tuberculosis and multidrug-resistant tuberculosis which are refractory to immune system and antibacterial chemotherapy alone. When used in combination with antibacterial drugs, therapeutic DNA vaccines could effectively eliminate residual bacteria in infected animals and shorten the therapy course of conventional chemotherapy. Detailed studies demonstrated that therapeutic effects of DNA vaccines may at least partly be due to the restoration of the Th(1)/Th(2) balance. Some problems have also emerged along with these exciting results. CONCLUSIONS: Therapeutic DNA vaccine is a promising strategy against tuberculosis, however developing an ideal DNA vaccine for therapy of tuberculosis will require further development.

[Expression of human granulysin in murine macrophages and its effects on cell damage and apoptosis].

AIM: To construct an eukaryotic expression vector pBudCE4.1/GLS encoding human granulysin(GLS) derived from activated CTLs and observe its effects on cell damage and apoptosis when expressed in macrophages of various murine germ lines. METHODS: The gene sequence coding GLS carried on pEGFP-C1/GLS was subcloned into pBudCE4.1 plasmid and then transfected into different murine macrophages. The expression of GLS was detected by RT-PCR and immunocytochemical staining. The impairment and apoptosis of the host cells was assesed by measuring LDH in culture media and fluorescent staining of nuclear DNA, respectively. RESULTS: The pBudCE4.1/GLS recombinant containing accurate ORF of GLS was obtained and successfully expressed in the target cells at 48 h after transfection. No obvious effects on cell damage and apoptosis was detected during this course. CONCLUSION: The gene coding human GLS can be expressed in murine macrophages. The expressed product has no obvious effects on cell damage and apoptosis.

[Therapeutic effects of DNA vaccines in a murine model of Mycobacterium tuberculosis infection].

OBJECTIVE: To study the effects and mechanisms of DNA vaccines encoding Mycobacterium tuberculosis antigens on murine Mycobacterium tuberculosis infection. METHODS: C57BL/J6 mice infected with Mycobacterium tuberculosis were treated with normal saline (NS), pCDNA3.1, psIL-12, pcD85B, pcDMPT64, pcD85B + pcDMPT64, and pcD85B + psIL-12 respectively. The numbers of viable bacteria in the lung and the spleen were counted. The levels of IFN-gamma, IL-4 and TNF-alpha of spleen lymphocytes stimulated with PPD were detected with ELISA. Lungs and spleens were prepared for pathological analysis. RESULTS: The pcD85B group (6.99 +/- 0.40 in lung, 5.17 +/- 0.33 in spleen), the psIL-12 group (7.41 +/- 0.50 in lung, 5.31 +/- 0.21 in spleen)and the pcD85B + psIL-12 group (7.64 +/- 0.28 in lung, 5.49 +/- 0.31 in spleen) showed significantly reduced number of colony forming units (lg(-1) CFU/g, x +/- s) in lungs and spleens compared with the control mice (5.76 +/- 0.16 in saline, 5.88 +/- 0.21 in pCDNA3.1), but the difference between the pcD85B group and the pcD85B + psIL-12 group was not significant. pcD85B vaccination induced high levels of IFN-gamma and TNF-alpha. No change of IL-4 was found in all groups. The pathological changes in lungs of the pcD85B group were localized, while those in the control group were extensive. There was no significant changes in the spleen of all groups. CONCLUSIONS: Ag85B DNA vaccination had immunotherapeutic effects, which were associated with a switch to Th1 response and enhanced production of cytokines TNF-alpha and INF-gamma. psIL-12 alone showed therapeutic effect, but it didn't enhance the therapeutic effect of single Ag85B DNA vaccination.

Effective siRNA targets screening for human telomerase reverse transcriptase.

AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence. METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4 sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000. At 48-72 h after siRNAs transfection, MTT assay, RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively. RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2 cells. CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method, synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.

[Construction and expression of the eukaryotic coexpression plasmid containing Ag85B gene of Mycobacterium tuberculosis and IL-12 gene].

AIM: To construct an eukaryotic coexpression plasmid containing Mycobacterium tuberculosis (MTB) Ag85B and IL-12 genes. METHODS: MTB Ag85B gene and IL-12 gene were cloned into pBudCE4.1 which has multiple promoters to construct recombinant plasmid pBud85B-IL12. The recombinant plasmid was transfected into COS-7 cells and the expression of target genes was assessed by RT-PCR and ELISA. RESULTS: The expressions of Ag85B and IL-12 could be detected in COS-7 cells. CONCLUSION: The recombinant plasmid pBud85B-IL12 was constructed and expressed successfully, which lays the foundation for further development of DNA vaccine against tuberculosis.

[A study of the protective effect of the DNA vaccine encoding tubercle antigen 85B with MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To explore the protective effect of the DNA vaccine encoding Mycobacterium tuberculosis Ag85B with MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: Fifty four C57BL/6 mice were randomized into six groups and subjected to the following treatments respectively: intramuscularly immunized with PBS, pcDNA3.1, BCG, pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/Ag85B + pcDNA/MPT64 on three occasions at 3-week intervals. The BCG group received a single subcutaneous injection of 1 x 10(6) CFU BCG. The mice were challenged with 10(6) CFU H(37)Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed, and the number of CFU in organs and histopathologic changes were determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: The number of bacterial colonies in the lungs and spleens were lg(-1) (7.854 +/- 0.003) CFU/g and lg(-1) (7.190 +/- 0.016) CFU/g in PBS group, lg(-1) (7.700 +/- 0.016) CFU/g and lg(-1) (7.072 +/- 0.068) CFU/g in pcDNA3.1 group, lg(-1) (6.449 +/- 0.002) CFU/g and lg(-1) (5.436 +/- 0.042) CFU/g in BCG group, lg(-1) (7.370 +/- 0.002) CFU/g and lg(-1) (6.430 +/- 0.009) CFU/g in pcDNA/Ag85B group, lg(-1) (7.547 +/- 0.003) CFU/g and lg(-1) (6.784 +/- 0.002) CFU/g in pcDNA/MPT64 group, and lg(-1) (6.918 +/- 0.002) CFU/g and lg(-1) (6.079 +/- 0.004) CFU/g in pcDNA/Ag85B + pcDNA/MPT64 group respectively, which showed significant decrease at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/Ag85B group was higher than that of the other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B + pcDNA/MPT64 group was higher than that of the other groups (P < 0.05). No IL-4 was detected in all groups. The pulmonary histopathological changes were observed 6 weeks later following challenge with Mycobacterium tuberculosis H(37)Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B group was characterized by serofibrous inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in the lung. The survival time of BCG vaccinated mice after challenge with Mycobacterium tuberculosis H(37)Rv was longer than that of the other groups. The survival time of pcDNA/Ag85B + pcDNA/MPT64 group was longer than that of other DNA vaccine groups. CONCLUSION: The protective effect of BCG was more significant than the other groups, while the effect of pcDNA/Ag85B + pcDNA/MPT64 was better than other DNA vaccines.

[Construction and expression of fusion gene expression plasmid HBV preS2S-rhGM-CSF].

AIM: To construct GM-CSF and preS2 fusion gene expression plasmid to enhance the immunogenicity of hepatitis B DNA vaccine. METHODS: HBV preS2S gene(846 bp) and rhGM-CSF gene(384 bp) were amplified by PCR, respectively. The eukaryotic expression plasmid pcDNA3.1-S2S-rhGM-CSF was constructed by means of T-A clone and directional gene cloning techniques, and then the recombinant plasmid was expressed in HepG2 cells. RESULTS: Restriction enzyme digestion analysis, PCR amplification and/or DNA sequencing proved that the recombinant plasmid was constructed successfully. The transcription of target gene was confirmed by RT-PCR. The fusion protein expressed in HepG2 cells could reacted to the monoclonal antibodies (mAbs) against HBsAg, preS2 and GM-CSF, respectively. CONCLUSION: The successful construction and expression of pcDNA3.1-S2S-rhGM-CSF lay the foundation for further study of HBV DNA vaccine.

[Preparation and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 fusion protein].

OBJECTIVE: To prepare the recombinant CFP10-ESAT-6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis. METHODS: The lhp-ESAT-6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid. The recombinant CFP10-ESAT-6 fusion protein was expressed and purified. Its antigenicity was confirmed by Western blot. Animal models infected with M. tuberculosis H(37)Rv strain and M. bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis. RESULTS: The sequence of recombinant plasmid pQE30-CFP10-ESAT-6 was identical to the predicted sequence. The recombinant protein (rCFP10-ESAT-6), about 26 000, existed in the cytoplasm of DH5alpha in soluble form and represented 40% of the total bacterial protein. The purity and concentration of the final product was 98% and 1.2 g/L, respectively. Western blot showed that the rCFP10-ESAT-6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT-6 respectively. The positive cutoff value was A(490) plus 2 standard deviation from negative guinea pig sera detected by ELISA. Serological reactivity to rCFP10-ESAT-6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG. CONCLUSIONS: The rCFP10-ESAT-6 fusion protein was highly expressed in soluble form in E. coli. It had antigenicity of both CFP10 and ESAT-6, and could be used to differentiate infection with M. tuberculosis H(37)Rv strain from immunization with M. bovis BCG. The study provided experimental data for potential application of rCFP10-ESAT-6 in the diagnosis of tuberculosis.

[The estimation of protective efficacy of the fusion gene vaccine encoding tubercle antigen 85B and MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To evaluate the protective efficacy of the fusion DNA vaccine (AM) encoding tubercle Ag85B and MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: C57BL/6 mice were intramuscularly immunized with the DNA vaccines. The mice were challenged with 10(6) CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B, pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). No IL-4 was found in all groups. The number of bacterial colonies in the lungs and spleens was significantly decreased at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). The pulmonary histopathological changes were observed 6 weeks later following challenge with M. tuberculosis H37Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by seroplastic inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B groups was characterized by seroplastic inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in lung. The survival time of BCG vaccinated mice after challenge with M. tuberculosis H37Rv was longer than that of other groups. The survival time of AM group was longer than that of other DNA vaccine groups. CONCLUSION: The pcDNA/AM can improve the protective efficacy in immunized mice against M. tuberculosis.

[Construction of the prokaryotic expression vector of MTB lhp gene and its expression].

AIM: To construct prokaryotic expression vector carrying lhp gene and express it in E.coli. METHODS: The MTB lhp gene was amplified by PCR and then cloned into plasmid pQE30. After sequencing, the gene was cloned into plasmid pET32a(+) to construct recombinant prokaryotic expression vectors pQE30-CFP10 and pET32a(+)-CFP10. RESULTS: After transformation of the E.coli and induction with 1 mmol/L of IPTG, no additional protein was expressed in pQE30-CFP10 system, but recombinant target protein with M(r) 20 000 or so was expressed in pET32a(+)-CFP10 system, and the expressed protein was maximum when induced with IPTG for 4 h. The expressed protein existed in cytoplasm in soluble form and amounted to 38% of total protein of E.coli. Western blot analysis showed that the protein had good antigenicity. The purity of the protein purified through the Ni-NTA resin reached 93%. CONCLUSION: The prokaryotic expression vector pET32a(+)-CFP10 was constructed successfully and the rCFP10 protein was obtained, which laid the foundation for application of the rCFP10.

[Immunogenicity of DNA vaccine encoding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64].

AIM: To study the immunogenicity of a DNA vaccine encoding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64. METHODS: C57BL/6 mice were intramuscularly injected respectively with PBS(A group), plasmid vector (B group), pcDNA/Ag85B(C group), pcDNA/MPT64(D group), and pcDNA/Ag85B MPT64 (E group) for three times with 2 week intervals. The mice were sacrificed 4 weeks after the final immunization. The total specific antibody levels were detected by ELISA and simultaneously spleen lymphocyte proliferation response to PPD and IFN-gamma level in culture supernatant of the PPD stimulated spleen lymphocytes was measured by MTT colorimetry and ELISA, respectively. RESULTS: The Ag85B, MPT64 and fusion gene Ag85B MPT64 DNA vaccines could induce anti-PPD antibody production as well as antigen specific lymphocyte proliferation and high levels of IFN-gamma in immunized mice. CONCLUSION: The Ag85B MPT64 fusion gene can induce specific cellular immunity and humoral immunity in immunized mice.