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@#Objective To develop and verify a reversed phase high-performance liquid chromatography method for the determination of the purity of recombinant Mycobacterium tuberculosis(Mtb)Ag85b protein stock solution.Methods Fourfactor,three-level orthogonal test was designed,with the area,trailing factor,peak area and peak area RSD as the evaluation indexes to explore the optimal detection conditions. The methodology verification of specificity,linear range,precision and durability was conducted in accordance with the general principles of Chinese Pharmacopoeia(Volume Ⅳ,2020 edition)9101.Results The results of all the evaluation indexes were good when the elution ratio of organic phase was30% ～ 95%,the detection temperature was 35 ℃,the sample volume was 3 μg,and the elution time of 95% organic phase was 15 min. The method had the linear correlation coefficient(R2)of 0. 998 5,the linear range of 1. 8 ～ 4. 2 μg,the reproducibility RSD of 0. 01%,and the intermediate precision RSD of 0. 16%,with good durability under slight changes of column temperature and flow rate.Conclusion The reversed phase high-performance liquid chromatography method for the purity determination of recombinant Mtb Ag85b protein stock solution was developed,which has good specificity,precision and durability,and can be used for the quality control of recombinant Mtb Ag85b protein stock solution.
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@#Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function. Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E. coli BL21(DE3),and induced by IPTG. The expressed His-Acr2 protein was purified by Ni-NTA chromatography and SuperdexTM200 10/300 GL gel filtration chromatography to obtain Acr2 protein. The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay. Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturationrenaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃. Conclusion The Acr2protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.
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@#Abstract： Objective To clone PE_PGRS35 gene of Mycobacterium tuberculosis（MTB），construct recombinant vector pET28a⁃PE_PGRS35，express and purify the PE_PGRS35 protein of MTB H37Rv heterologously，and explore a new target against MTB after bioinformatics analysis. Methods The PE_PGRS35 coding gene was amplified by PCR and used to construct the expression vector pET28a⁃PE_PGRS35 by recombinant cloning technology，which was transformed to E. coli BL21（DE3）after successful sequencing and induced by using IPTG. The obtained PE_PGRS35 protein was purified by Ni column affinity chromatography and analyzed by bioinformatics. Results The pET28a⁃PE_PGRS35 prokaryotic expression vector was constructed correctly as identified by sequencing. The PE_PGRS35 protein was mainly expressed in the form of inclusion bodies，with a relative molecular mass of about 53 000 and a purity of 90%. Bioinformatics analysis showed that PE_PGRS35 protein was an acid⁃labile protein，with main secondary structure of β⁃sheet and random coil，and no transme⁃ mbrane region，which was presumed to be an extramembrane protein with 39 phosphorylation sites and two conserved domains. Total 10 proteins，including Rv1769，PPE8，PPE64，PPE54，PPE24，PPE16，PPE35，PPE6，PPE28 and PE2， interacted with PE_PGRS35 protein. Conclusion PE_PGRS35 protein with high purity was successfully obtained，which provided a reference for the further development of new targets for drugs against MTB.
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Objective:To research the anti-inflammatory effect of LL-37 in mycobacterium tuberculosis ( Mtb ) infected-macrophages,as well as its influence on the secretion of inflammatory cytokines. Methods:( 1 ) THP-1 cells were cultured and incubated with phorbol 12-myristate 13-acetate (PMA) to transform into an adherent macrophage-like state (macrophage,Mφ). Then the THP-1 cell derived macrophages were infected with mycobacterium tuberculosis,and then stimulated with different concentrations of LL-37. (2)The experiment was divided into following groups: ① Control group:THP-1+normal saline (NS);② Mtb group:THP-1+Mtb;③Mtb+LL-37 5 μg/ml group:THP-1+Mtb+5 μg/ml LL-37;④Mtb+LL-37 10 μg/ml group:THP-1+Mtb+10 μg/ml LL-37;⑤Mtb+LL-37 20 μg/ml group:THP-1+Mtb+20 μg/ml LL-37. (3) The mRNA expression levels of IL-12p40,TNF-α,IL-4,and IL-10 will be determined by Real-time PCR respectively at 6,12,24 and 48 hours. The secreted levels of IL-12p40,TNF-α,IL-4,and IL-10 will be determined by ELISA analysis respectively at 6,12,24 and 48 hours. Results:The mRNA expression levels and secreted levels of IL-12p40,TNF-α,IL-4 and IL-10 were increased in Mtb group than those in control group. The mRNA expression levels and secreted levels of pro-inflammatory cytokines IL-12p40 and TNF-α were decreased in the LL-37 groups than those in Mtb group. However,anti-inflammatory cytokines IL-4 and IL-10 mRNA expression levels and secreted levels were increased in the LL-37 groups than those in Mtb group. Conclusion:Exogenous LL-37 inhibited the secretion of inflammatory cytokines in macrophages during mycobacterium tu-berculosis infection. The effect is related to the concentrations of LL-37 and the stimulated time of macrophages which were infected with mycobacterium tuberculosis. The results will provide new insights into the treatment of Mtb infection.
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Objective To investigate secreting pulmonary surfactant-associated protein B (SP-B) and apoptosis in human lung adenocarcinoma cell line (A549) cells,treated with heat-resistant antigen of Mycobacterium tuberculosis (Mtb-HAg).Methods Different concentrations of Mtb-HAg were used to culture A549 cells for 24 h and 48 h respectively,and the blank control groups(control group 1 and control group 2) and positive control groups [lipopolysaccharide (LPS) group and curcumin group] were set up.SP-B in the culture supernatant,was assessed by ELISA in control group 1,LPS group and experimental group 1 (A549 cells were respectively induced by 2 μg/ml,3 μg/ml Mtb-HAg to culture for 24 h and 48 h).For control group 1,LPS group and experimental group 1,the relative expression of SFTPB gene was quantified with quantitative real-time fluorescence quantitative PCR (qRT-PCR).The apoptosis rate of control group 2,curcumin group and experimental group 2(A549 cells were respectively induced by 2 μg/ml,3 μg/ml Mtb-HAg to culture for 24 h) in A549 cells were detected by flow cytometry.Results SP-B expression in the experimental group 1 was significantly lower than the control group 1,the difference was statistically significant (P ＜ 0.05).The difference of SP-B expression in the experimental group 1 was not obvious with the prolongation of the same concentration.At the same incubation time,the expression of SFTPB in the experimental group 1 decreased obviously with the increasing concentration of Mtb-HAg,the difference was statistically significant (P ＜ 0.05).The change with time was not significant.The apoptosis rate of curcumin group and experimental group 2 were significantly higher than that in control group 2 in A549 cells (P ＜ 0.05).Conclusion Mtb-HAg inhibited the expression of SP-B in A549 cells significantly,and induced apoptosis of A549 cells.
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Objective: To predict and identify HLA-A * 0201 restricted CD8+ CTL epitopes in Mycobacterium tuberculosis (Mtb) antigen Ag85C, so as to provide evidence for epitope-based study for tuberculosis (TB) vaccine. Methods: The online database SYFPEITHI was applied to predict the potential HLA-A * 0201 restricted epitopes from Ag85C, an antigen of Mycobacterium tuberculosis. T2 cell line was used to assay the affinity between the predicted peptides and HLA-A * 0201 molecules. The specific CTL lines were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A * 0201 positive TB patients and PPD+ healthy donors by peptides with high binding affinity to HLA-A * 0201 molecules. IFN-γ production, in vitro proliferation and cytotoxicity of peptide-induced CTL were determined to screen HLA-A * 0201 restricted CD8+ CTL epitopes from those candidates. Results: Fourteen potential epitopes were identified from the SYFPEITHI database. After binding affinity assay, 3 of the 14 peptides (170-178 aa, 317-325 aa, and 144-153 aa) were found to have high binding affinity to HLA-A * 0201 molecules. However, only one peptide (144-153 aa) stimulated its specific CTL to release IFN-γ, proliferate in vitro and produce specific cytotoxicity. Conclusion: We have successfully identified a HLA-A * 0201 restricted CD8+ CTL epitope of Mtb Ag85C-FLTREMPAWL(144-153 aa), which might be a candidate epitope for TB vaccine designing. Our findings provides a basis for developing novel and effective anti-TB vaccine.
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Objective:To reconstruct Mycobacterium shuttle expression plasmid with Mycobacterium tuberculosis HSP70 promoter based on shuttle plasmid pJEM11.Methods:The cDNA fragment of 229bp promoter and its correlative sequence was amplified by PCR.Multiple clone sites was added to the end of amplified fragment,and then it was cloned into the pJEM11 to construct the plasmid pJCH02.The pJCH02 was identified by digestion with restriction endonuclease and sequence analysis.The lhp-esat6 fusion gene was cloned into the pJCH02,then its expression in BCG was confirmed by SDS-PAGE.Results:The sequence of HSP70 promoter and its correlative sequence was in accordance with the predicted sequence.The lhp-esat6 fusion gene was expressed in BCG.Conclusion:The shuttle expression plasmid pJCH02 is constructed successfully.The study provides the basis for BCG polyvaccine.
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Objective:To construct prokaryotic expression vector carrying esat6 gene and express in E. coli. Methods: The MTb esat6 gene was amplified by PCR,then cloned into pQE30 plasmid, sequenced and then cloned into pET32a( + ) plasmid. Thus two kinds of prokaryotic expression vectors were constructed. Results: After being transformed into the E. coli and inducted with 1mM IPTG,no protein was expressed in the pQE30 - ESAT6 system, but a recombinant protein, about 19 kDa,was expressed in the pET32a( + ) - ESAT6 system. In the presence of 1mM IPTG for 4h,the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 42% total protein of E. coli. It's antigenicity was confirmed by Westem blotting. The protein was purified through the Ni - NTA resin and the purity reached 92 % . Conclusion : The prokaryotic expression vector (pET32a( + ) - ESAT6) was constructed successfully,and the rESAT6 was obtained,providing an experimental basis for application of rESAT6.
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Objective:To construct lhp-esat6 fusion gene and its prokaryotic expression vector and express it in E.coli.Methods:By Gene SOEing techniques,a fusion gene was constructed by splicing lhp gene and esat6 gene,then cloned into pQE30 plasmid and expressed in DH5a.Results:The fusion gene was identified by DNA sequencing.A fusion protein about 26kDa was expressed in the E.coli.In presence of 1mM IPTG for 4h,the fusion protein was expressed to the maximum.The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E.coli.Its antigenicity was confirmed by Western blotting.The fusion protein was purified through the Ni-NTA resin and the purity reached 98%.Conclusion:The lhp-esat6 fusion gene and the prokaryotic expression vector (pQE30-CFP10-ESAT6) were constructed successfully,and the fusion protein CFP10-ESAT6 was obtained,so that it can provide an experimental basis for application of the recombinant CFP10-ESAT6.
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Objective:To predict and identify HLA-A * 0201 restricted CD8+ CTL epitopes in Mycobacterium tuberculosis (Mtb) antigen Ag85C, so as to provide evidence for epitope-based study for tuberculosis (TB) vaccine. Methods: The online database SYFPEITHI was applied to predict the potential HLA-A * 0201 restricted epitopes from Ag85C, an antigen of Mycobacterium tuberculosis. T2 cell line was used to assay the affinity between the predicted peptides and HLA-A * 0201 molecules. The specific CTL lines were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A * 0201 positive TB patients and PPD+ healthy donors by peptides with high binding affinity to HLA-A * 0201 molecules. IFN-?production, in vitro proliferation and cytotoxicity of peptide-induced CTL were determined to screen HLA-A * 0201 restricted CD8+ CTL epitopes from those candidates. Results: Fourteen potential epitopes were identified from the SYFPEITHI database. After binding affinity assay, 3 of the 14 peptides (170-178 aa, 317-325 aa, and 144-153 aa) were found to have high binding affinity to HLA-A* 0201 molecules. However, only one peptide (144-153 aa) stimulated its specific CTL to release IFN-y, proliferate in vitro and produce specific cytotoxicity. Conclusion: We have successfully identified a HLA-A * 0201 restricted CD8+ CTL epitope of Mtb Ag85C-FLTREMPAWL( 144-153 aa) , which might be a candidate epitope for TB vaccine designing. Our findings provides a basis for developing novel and effective anti-TB vaccine.