Objective This study aimed to characterize the diagnostic and vaccine potential of a novel Mycobacterium tuberculosisantigen Rv0674. Methods To evaluate thediagnostic potential and antigenicity of Rv0674, IgG was eval...Objective This study aimed to characterize the diagnostic and vaccine potential of a novel Mycobacterium tuberculosisantigen Rv0674. Methods To evaluate thediagnostic potential and antigenicity of Rv0674, IgG was evaluated using ELISA and interferon (IFN)-γ was done by using ELISpot assay among TB patients and healthy donors. For immunogenicity evaluation, BALB/c mice were immunized with Rv0674. Cytokine production was determined by cytokine release assay using an ELISA kit, and the antibodies were tested using ELISA. Results The results of serum Elisa tests showed that Rv0674 specific immunoglobulin G (IgG) response was higher in TB patients than negative controls. And Rv0674 had good performance in serological test with sensitivity and specificity of 77.1% and 81.1%, respectively. While it shows poor sensitivity and specificity of 26.23% and 79.69% for IFN-γ tests. In BALB/c mice, Rv0674 adjuvant by DDA/PolyI:C could also induce a high level of IFN-γ, interleukin-2 and interleukin-6 as well as a high IgG titer in both high-and low-dose groups indicating that Rv0674 is essential in humoral and cellular immunity. Moreover, the cytokine profile and IgG isotypecharacterized Rv0674 as a Th1/Th2-mixed-type protective immunity with the predominance of Th1 cytokines. Conclusion Rv0674 may be a good potential candidate for the development of TB serological diagnosis and a new TB vaccine.展开更多
目的运用生物信息学的方法分析结核分枝杆菌潜伏相关蛋白Rv2628的结构与功能。方法自NCBI网站获取Rv2628蛋白的基本信息;利用Protparam、ProtScale预测Rv2628蛋白的理化性质、亲疏水性;应用Signal 6.0 Server、TMHMM Server V.2.0、NLSt...目的运用生物信息学的方法分析结核分枝杆菌潜伏相关蛋白Rv2628的结构与功能。方法自NCBI网站获取Rv2628蛋白的基本信息;利用Protparam、ProtScale预测Rv2628蛋白的理化性质、亲疏水性;应用Signal 6.0 Server、TMHMM Server V.2.0、NLStradamus、PSORTb预测Rv2628蛋白的信号肽、跨膜区域、核定位信号及亚细胞定位;使用NetNGlyc1.0、NetPhos Server v.3.1预测Rv2628蛋白的糖基化位点及磷酸化位点;利用SOPMA分析Rv2628蛋白的二级结构,使用SWISS-MODEL对其进行三级结构同源建模;使用ABCpred、SYFPEITHI预测Rv2628蛋白的相关抗原表位;利用MEGA构建Rv2628蛋白的分子进化树;使用STRING数据库预测与Rv2628蛋白可能发生互作的蛋白;使用autodock实现Rv2628蛋白与化合物的分子对接。结果Rv2628基因全长363 bp,由120个氨基酸构成,分子式为C575H906N178O168S4,等电点(pI)为9.09;Rv2628蛋白亚细胞定位于细胞质,无跨膜结构、信号肽及糖基化位点,存在12个磷酸化位点,包括6个苏氨酸、5个丝氨酸和1个酪氨酸磷酸化位点;该蛋白的二级结构以α-螺旋(41.67%)、无规则卷曲(35.83%)为主;Rv2628蛋白含有10个B细胞、6个Th细胞和6个CTL候选表位,三级结构同源建模的GMQE为0.72;苯基香豆素化合物10可以稳定的与Rv2628蛋白结合并发挥抑制作用;此外,Rv2628蛋白可能与Rv1733c、rip3、hrp1、devR、hspX等蛋白存在相互作用,参与结核分枝杆菌潜伏休眠的过程,诱导Th1免疫反应。结论Rv2628蛋白为亲水性蛋白,存在多个磷酸化位点并能够与多种蛋白相互作用,具有高度免疫原性,包含多个优势B细胞、T细胞抗原表位,可作为结核病诊断的候选蛋白。展开更多
Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was pre...Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.展开更多
基金National Science and Technology Major Project of China [2018ZX10731301-002]
文摘Objective This study aimed to characterize the diagnostic and vaccine potential of a novel Mycobacterium tuberculosisantigen Rv0674. Methods To evaluate thediagnostic potential and antigenicity of Rv0674, IgG was evaluated using ELISA and interferon (IFN)-γ was done by using ELISpot assay among TB patients and healthy donors. For immunogenicity evaluation, BALB/c mice were immunized with Rv0674. Cytokine production was determined by cytokine release assay using an ELISA kit, and the antibodies were tested using ELISA. Results The results of serum Elisa tests showed that Rv0674 specific immunoglobulin G (IgG) response was higher in TB patients than negative controls. And Rv0674 had good performance in serological test with sensitivity and specificity of 77.1% and 81.1%, respectively. While it shows poor sensitivity and specificity of 26.23% and 79.69% for IFN-γ tests. In BALB/c mice, Rv0674 adjuvant by DDA/PolyI:C could also induce a high level of IFN-γ, interleukin-2 and interleukin-6 as well as a high IgG titer in both high-and low-dose groups indicating that Rv0674 is essential in humoral and cellular immunity. Moreover, the cytokine profile and IgG isotypecharacterized Rv0674 as a Th1/Th2-mixed-type protective immunity with the predominance of Th1 cytokines. Conclusion Rv0674 may be a good potential candidate for the development of TB serological diagnosis and a new TB vaccine.
文摘目的运用生物信息学的方法分析结核分枝杆菌潜伏相关蛋白Rv2628的结构与功能。方法自NCBI网站获取Rv2628蛋白的基本信息;利用Protparam、ProtScale预测Rv2628蛋白的理化性质、亲疏水性;应用Signal 6.0 Server、TMHMM Server V.2.0、NLStradamus、PSORTb预测Rv2628蛋白的信号肽、跨膜区域、核定位信号及亚细胞定位;使用NetNGlyc1.0、NetPhos Server v.3.1预测Rv2628蛋白的糖基化位点及磷酸化位点;利用SOPMA分析Rv2628蛋白的二级结构,使用SWISS-MODEL对其进行三级结构同源建模;使用ABCpred、SYFPEITHI预测Rv2628蛋白的相关抗原表位;利用MEGA构建Rv2628蛋白的分子进化树;使用STRING数据库预测与Rv2628蛋白可能发生互作的蛋白;使用autodock实现Rv2628蛋白与化合物的分子对接。结果Rv2628基因全长363 bp,由120个氨基酸构成,分子式为C575H906N178O168S4,等电点(pI)为9.09;Rv2628蛋白亚细胞定位于细胞质,无跨膜结构、信号肽及糖基化位点,存在12个磷酸化位点,包括6个苏氨酸、5个丝氨酸和1个酪氨酸磷酸化位点;该蛋白的二级结构以α-螺旋(41.67%)、无规则卷曲(35.83%)为主;Rv2628蛋白含有10个B细胞、6个Th细胞和6个CTL候选表位,三级结构同源建模的GMQE为0.72;苯基香豆素化合物10可以稳定的与Rv2628蛋白结合并发挥抑制作用;此外,Rv2628蛋白可能与Rv1733c、rip3、hrp1、devR、hspX等蛋白存在相互作用,参与结核分枝杆菌潜伏休眠的过程,诱导Th1免疫反应。结论Rv2628蛋白为亲水性蛋白,存在多个磷酸化位点并能够与多种蛋白相互作用,具有高度免疫原性,包含多个优势B细胞、T细胞抗原表位,可作为结核病诊断的候选蛋白。
基金supported by the National Key Program for Infectious Diseases of China (2009ZX10004-4001)
文摘Objective LcrV is an important component for the development of a subunit vaccine against plague.To reduce immunosuppressive activity of LcrV,a recombinant LcrV variant lacking amino acids 271 to 326 （rV270） was prepared by different methods in this study.Methods A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a,or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a.After Co2＋ affinity chromatography,a purification strategy was developed by cleavage of His tag on column,following Sephacryl S-200HR column filtration chromatography.Results Removal of His tag by thrombin,enterokinase and factor Xa displayed a yield of 99.5%,32.4% and 15.3%,respectively.Following Sephacryl S-200HR column filtration chromatography,above 97% purity of rV270 protein was obtained.Purified rV270 that was adsorbed to 25% （v/v） Al（OH）3 adjuvant in phosphate-buffered saline （PBS） induced very high titers of antibody to rV270 in BALB/c mice and protected them （100% survival） against subcutaneous challenge with 106 CFU of Y.pestis virulent strain 141.Conclusion The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa,but they exhibited extremely low cleavage activity to the corresponding recognition site.Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy.The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.
