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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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PURPOSE: In the present study, the protective immunological markers in serum and peripheral blood mononuclear cells (PBMCs) of bacillus Calmette-Guerin (BCG) vaccinated and unvaccinated children were evaluated after vaccination. Further, PBMCs of children with low protective levels were boosted with BCG, Ag85B, and Ag85B peptides to study their booster effects to increase waning BCG induced immunity. MATERIALS AND METHODS: Fifty children from 1 month to 18 years of age were randomized for the study. Blood samples were collected from 27 participants with/without BCG vaccination. Immunological markers (anti-BCG, interferon gamma [IFN-gamma], and adenosine deaminase activity) were assessed in both serum and PBMCs of children. Children with low levels of protective immunological markers were further recruited and their PBMCs were boosted with BCG, Ag85B, and Ag85B peptides. RESULTS: Children in age group of 4-6 years were associated with significantly (p<0.05) higher BCG-specific IgG and IFN-gamma levels compared to those in age group greater than 10 years. Vaccinated children had greater repertoire of immunological memory which on in vitro stimulation with BCG showed increase in BCG-specific response compared to unvaccinated controls. Assessment of booster effects of BCG, Ag85B, and Ag85B peptides in PBMCs of children revealed greater potential of peptides to boost BCG induced immunity compared to BCG and Ag85B. CONCLUSION: To conclude, children within age 4-6 years are associated with high immunological markers which eventually diminish with age thereby suggesting need for booster dose in later years. Mycobacterium tuberculosis peptides along with BCG may be used as attractive candidates to boost such waning BCG induced immunity in children.
Subject(s)
Child , Humans , Adenosine Deaminase , Bacillus , BCG Vaccine , Immunoglobulin G , Immunologic Memory , Interferon-gamma , Interferons , Mycobacterium bovis , Mycobacterium tuberculosis , Peptides , VaccinationABSTRACT
Objective: To use in vivo fluorescence image analysis system for evaluating the efficacies of pVAX1-Ag85A and pVAX1-Ag85B DNA vaccines in treatment of bladder cancer cell-implanted tumors in mice. Methods: Discosomasp red fluorescent protein (DsRed) stably transfected bladder cancer BTT cell line (BTT-DsRed) was established and BTT-DsRed cell-implanted mouse model was constructed. Six days later, 24 BTT-DsRed-bearing mice were randomly divided into pVAX1-Ag85A DNA vaccine group, pVAX1-Ag85B DNA vaccine group, and saline group through injecting the pVAX1-Ag85A, pVAX1-Ag85B, and saline into the right hind limbs of mice, respectively. The growth and metastasis of implanted BTT-DsRed tumors were examined by in vivo fluorescence image analysis system. Results: BTT cell line stably transfected with DsRed (BTT-DsRed) was successfully established. Fluorescence visible mouse model was successfully es-tablished by inoculating BTT-DsRed cells into hind limbs of mice. After treatment with pVAX1-Ag85A or pVAX1-Ag85B for 2 weeks, the in vivo tumor fluorescence intensity in pVAX1-Ag85B group was significantly lower than that in the saline group (P <0.05). After 3 weeks, tumor fluorescence intensities in both pVAX1-Ag85A and pVAX1-Ag85B groups were significantly lower than that in the saline group (P < 0.01). But the efficacies of pVAX1-Ag85A and pVAX1-Ag85B groups were similar (P > 0.05). The distant lymphatic metastasis rate in pVAX1-Ag85B group was significantly lower than those in the saline (25.0% vs 87.5%) and pVAX1-Ag85A groups (25.0% vs 62.5%) (P <0.05). Conclusion: In vivo fluorescence image analysis system can dynamically, sensitively and visually evaluate the anti-tumor effects of DNA vaccines against bladder cancer cell-implanted tumors. Both pVAX1-Ag85A and pVAX1-Ag85B DNA vaccines have anti-tumor effects for bladder cancers.
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Objective To construct protective immunity to Mycobncterium tuberculosis latent infection, a novel fusion protein consisting of HspX, the 190 to 198 peptide of Mpt64 and Ag85B, which were confirmed to be the effective protective antigens mainly expressed in the dormancy and exponential phase of growth, was constructed and its immunogenicity was investigated. Methods Ag85B and Mpt64190-198-HspX sequences were amplified by PCR and cloned into plasmids pET-28a. The fusion protein, Ag85BMpt64190-198-HspX (AMH) was expressed in E. coli BL21 and purified with Ni-NTA resins. C57BL/6 mice were immunized three times at 2-week intervals subcutaneously with AMH formulated with the adjuvant composed of dimethyl-dioctyldecyl ammonium bromide (DDA) and BCG polysaccharide nucleic acid (BCGPSN). Humoral and cell-mediated immunity responses were analyzed at five weeks after the last injection. Results AMH was expressed stably in E. coli and could be purified well by Ni-NTA affinity chromatography. C57BL/6 mice immunized with AMH subunit vaccine generated specific cellular and humoral immunologic response to the stimulation of Ag85B, Mpt64190-198 and HspX. Conclusion It suggested that AMH was a promising candidate antigen of tuberculosis subunit vaccine.
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BACKGROUND: IFN-gamma is the main effector mediator of the host immune response against Mycobacterium tuberculosis. Evaluating the IFN-gamma gene expression in response to M. tuberculosis antigens may help in elucidating the host defense mechanism against M. tuberculosis and in the development of a vaccine. METHODS: The IFN-gamma mRNA expression in the lymphocytes obtained from pleural effusions from tuberculous pleurisy patients (TB-PLC) after in vitro stimulation with whole cell M. tuberculosis(H37Rv), purified protein derivatives(PPD), man-lipoarabinamman (man-LAM), ara-LAM and Antigen 85B(Ag85B) were evaluated. The degree of IFN-gamma mRNA expression was determined by a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. RESULTS: M. tuberculosis induced the expression of IFN-gamma mRNA in the TB-PLC in time and dose dependent manners. The PPD and Ag85B induced high levels of IFN-gamma mRNA expression in the TB-PLC. However, man-LAM inhibited IFN-gamma mRNA expression in the TB-PLC, while ara-LAM did not. CONCLUSION: IFN-gamma mRNA expression in TB-PLC is stimulated by PPD and Ag85B, but inhibited by man-LAM.
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Humans , Gene Expression , Interferon-gamma , Lymphocytes , Mycobacterium tuberculosis , Pleural Effusion , RNA, Messenger , Tuberculosis , Tuberculosis, PleuralABSTRACT
Objective To study the anti-tumor effect of mycobacterium Ag85B/IL-2 fusion protein on syngenic mice bearing bladder cancer. Methods After being implanted BTT739 bladder transitional carcinoma cells from tumor bearing mice, 80 T739 mice were divided into Ag85B protein, BCG, IL-2 and normal saline groups (n=20). Mycobacterium Ag85B/IL-2 protein was locally injected to the mice at the site of implanting tumor in the trial group, and Ag85B protein, BCG, IL-2 and normal saline to the control group mice. The weight and volume of tumor and the survival period were recorded after treatment. Results The average weight of Ag85B/IL-2 fusion protein treatment group was significantly lower than those of Ag85B, BCG,IL-2 and normal saline group (P
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Objective:To construct an eukaryotic coexpression plasmid containing Mycobacterium tuberculosis Ag85B and MPT64 gene.Methods:Mycobacterium tuberculosis Ag85B and MPT64 gene were cloned into pBudCE4.1 to construct recombinant plasmid pBud85B-MPT.The recombinant plasmid was transfected into COS-7 cells,and its expression of Ag85B and MPT64 was assesed by RT-PCR.Results:Mycobacterium tuberculosis Ag85B and MPT64 were detected in transfected COS-7 cells.Conclusion:The recombinant plasmid pBud85B-MPT can express Ag85B and MPT simultaneously in COS-7 cells which provides the basis for further investigation of DNA vaccine against tuberculosis.