Objective:To coastruct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2(IL-2) fusion protein(rMS-Hsp65/IL-2) and to explore the effect of this constru...Objective:To coastruct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2(IL-2) fusion protein(rMS-Hsp65/IL-2) and to explore the effect of this construct on lymphocyte function in mice.Methods:The fusion gene encoding Hsp65-hIL-2 was cloned into shuttle vector pSMT3.The recombinant plasmid pSMT3- Hsp65-hIL-2 was transferred to Mycobacterium smegmatis by electroporation.Positive clones were selected by hygromycin and identified by PCR.The expression of fusion protein Hsp65- hIL-2 was verified using indirect immunofluorescence staining.Mice were immunized for two times by subcutaneously injection with 1×10~6 CFU rMS-Hsp65/IL-2 at a three-week interval.Two weeks after the second immunization,mice were sacrificed and the serum samples were collected for determination of anli-Hsp65 specific IgG.Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay.IFN-γand IL-2 in the medium of the treated cells were also determined by ELISA.Results:Successful construction of rMS-Hsp65/IL-2 was verified by PCR and immunofluorescence staining.Compared to the splenic lymphocytes isolated from mice immunized with Bacille Calmette-Guerin or mice immunized with Mycobacterium smegmatis alone,the splenic lymphocytes isolated from mice immunized with rMS-Hsp65/IL-2 showed a marked increase in the proliferation of lymphocytes, together with an increased production of important cytokines such as IFN- 7 and IL-2. Conclusions:rMS-Hsp65/IL-2 markedly enhances lymphocyte function.Therefore,the fusion protein generated by rMS-Hsp65/lL-2 may be of potential value in generating an effective vaccine against tuberculosis.展开更多
目的通过对重组结核杆菌融合蛋白(EC)[该制品名称是国家药典委员会确定的药品中文通用名称,"EC"为重组融合蛋白"结核分枝杆菌早期分泌性抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)"](简称"EC")的免疫特性...目的通过对重组结核杆菌融合蛋白(EC)[该制品名称是国家药典委员会确定的药品中文通用名称,"EC"为重组融合蛋白"结核分枝杆菌早期分泌性抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)"](简称"EC")的免疫特性和临床前安全性研究,探讨其临床应用前景。方法(1)免疫特性研究:取6只雌性豚鼠,使用结核分枝杆菌H37Ra活菌菌液致敏,5周后,皮内注射2.5μg/ml EC原液0.2ml;取4只雌性豚鼠,使用卡介菌活菌菌液致敏,5周后,皮内注射20μg/ml EC原液0.2ml;观察注射部位平均硬结或红晕反应直径[(纵径+横径)/2],≥5mm判为阳性,<5mm判为阴性。(2)急性毒性试验:取80只ICR[(美国)Institute of Cancer Research]小鼠,分为单次肌内和皮内注射组,每组40只,雌雄各半;每组再分为4组,每组10只,雌雄各半。高剂量组(注射EC53.61μg/0.1ml)、低剂量组(注射EC 0.2μg/0.1ml)、溶剂对照组(注射EC稀释液0.1ml)、空白对照组(不给予任何受试物),观察小鼠的外观、运动功能、体质量、各脏器和药物注射部位皮肤等是否有异常。(3)豚鼠全身主动过敏试验:取豚鼠24只,分为4组,每组6只,雌雄各半,各组豚鼠分别隔日腹腔注射高剂量(5μg/kg)EC、低剂量(0.5μg/kg)EC、牛血清白蛋白(60mg)和0.9%NaCl(质量分数为0.9%的NaCl溶液)注射液(2ml),连续3次。致敏结束,末次致敏后第12天静脉快速注射2倍致敏剂量对以上相应的各组致敏豚鼠进行激发。致敏期间,每天观察豚鼠的症状,并于初次和最后一次致敏及激发当日测定每只豚鼠的体质量。(4)皮内刺激试验:取6只新西兰兔,单次皮内注射EC 10μg(0.2ml)/点,每侧5个点,观察皮内注射后的刺激反应情况。结果(1)免疫特性研究结果:EC原液对卡介菌活菌菌液致敏的4只豚鼠的皮肤试验,阳性为0只;EC原液对结核分枝杆菌H37Ra活菌菌液致敏的6只豚鼠的皮肤试验,阳性为6只。(2)急性毒性试验结果:所有小鼠未出现明显的急性中毒反应和显示急性中毒靶器官。小鼠皮内注射EC前、注射EC后1d、3d、5d、7d、8d、10d、12d、14d高剂量组、低剂量组小鼠平均体质量分别为(20.6±1.3)g^(24.9±2.1)g、(20.5±1.6)g^(26.0±3.1)g;注射后不同观察时间各组平均体质量与空白对照组[(21.0±1.1)g^(25.3±2.3)g]比较差异均无统计学意义(t值分别为0.571~0.392,0.695~0.615;P值分别为0.575~0.700,0.496~0.546);小鼠肌内注射EC前、注射EC后1d、3d、5d、7d、8d、10d、12d、14d高剂量组、低剂量组小鼠平均体质量分别为(21.0±1.5)g^(26.2±1.9)g、(20.5±2.1)g^(25.8±3.8)g;注射后不同观察时间各组平均体质量与空白对照组[(21.2±1.7)g^(25.8±3.1)g]比较差异均无统计学意义(t值分别为0.360~0.318,0.900~0.006;P值分别为0.723~0.754,0.380~0.995)。(3)豚鼠全身主动过敏试验结果:豚鼠无过敏反应。高剂量组、低剂量组豚鼠首次致敏、末次致敏、激发的平均体质量分别为(327.5±24.3)g、(347.2±32.7)g、(402.2±34.9)g;(331.3±26.7)g、(346.2±32.0)g、(411.3±38.9)g,与相应观察时间注射0.9%NaCl的阴性对照组[(329.5±27.4)g、(348.3±27.0)g、(399.4±25.4)g]比较,差异均无统计学意义(t值分别为0.328、0.181、0.284,0.474、0.366、0.875;P值分别为0.757、0.864、0.788,0.656、0.730、0.422)。(4)兔皮内刺激试验结果:单次皮内注射10μg(0.2ml)/点EC,无明显刺激反应。结论EC能够鉴别结核感染与卡介苗免疫,临床前动物试验安全性好,有望应用于人群结核感染的诊断与鉴别诊断。展开更多
[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the ...[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.展开更多
基金funded by the National Natural Science Eoundation of China(Grant ID:30972767)
文摘Objective:To coastruct a strain of recombinant Mycobacterium smegmatis expressing the heat shock protein 65(Hsp65) and human interleukin 2(IL-2) fusion protein(rMS-Hsp65/IL-2) and to explore the effect of this construct on lymphocyte function in mice.Methods:The fusion gene encoding Hsp65-hIL-2 was cloned into shuttle vector pSMT3.The recombinant plasmid pSMT3- Hsp65-hIL-2 was transferred to Mycobacterium smegmatis by electroporation.Positive clones were selected by hygromycin and identified by PCR.The expression of fusion protein Hsp65- hIL-2 was verified using indirect immunofluorescence staining.Mice were immunized for two times by subcutaneously injection with 1×10~6 CFU rMS-Hsp65/IL-2 at a three-week interval.Two weeks after the second immunization,mice were sacrificed and the serum samples were collected for determination of anli-Hsp65 specific IgG.Splenic lymphocytes were isolated and treated with the rMS-Hsp65/IL-2 to determine lymphocytic proliferation activity by MTT assay.IFN-γand IL-2 in the medium of the treated cells were also determined by ELISA.Results:Successful construction of rMS-Hsp65/IL-2 was verified by PCR and immunofluorescence staining.Compared to the splenic lymphocytes isolated from mice immunized with Bacille Calmette-Guerin or mice immunized with Mycobacterium smegmatis alone,the splenic lymphocytes isolated from mice immunized with rMS-Hsp65/IL-2 showed a marked increase in the proliferation of lymphocytes, together with an increased production of important cytokines such as IFN- 7 and IL-2. Conclusions:rMS-Hsp65/IL-2 markedly enhances lymphocyte function.Therefore,the fusion protein generated by rMS-Hsp65/lL-2 may be of potential value in generating an effective vaccine against tuberculosis.
文摘目的通过对重组结核杆菌融合蛋白(EC)[该制品名称是国家药典委员会确定的药品中文通用名称,"EC"为重组融合蛋白"结核分枝杆菌早期分泌性抗原靶6(ESAT-6)和培养滤液蛋白10(CFP-10)"](简称"EC")的免疫特性和临床前安全性研究,探讨其临床应用前景。方法(1)免疫特性研究:取6只雌性豚鼠,使用结核分枝杆菌H37Ra活菌菌液致敏,5周后,皮内注射2.5μg/ml EC原液0.2ml;取4只雌性豚鼠,使用卡介菌活菌菌液致敏,5周后,皮内注射20μg/ml EC原液0.2ml;观察注射部位平均硬结或红晕反应直径[(纵径+横径)/2],≥5mm判为阳性,<5mm判为阴性。(2)急性毒性试验:取80只ICR[(美国)Institute of Cancer Research]小鼠,分为单次肌内和皮内注射组,每组40只,雌雄各半;每组再分为4组,每组10只,雌雄各半。高剂量组(注射EC53.61μg/0.1ml)、低剂量组(注射EC 0.2μg/0.1ml)、溶剂对照组(注射EC稀释液0.1ml)、空白对照组(不给予任何受试物),观察小鼠的外观、运动功能、体质量、各脏器和药物注射部位皮肤等是否有异常。(3)豚鼠全身主动过敏试验:取豚鼠24只,分为4组,每组6只,雌雄各半,各组豚鼠分别隔日腹腔注射高剂量(5μg/kg)EC、低剂量(0.5μg/kg)EC、牛血清白蛋白(60mg)和0.9%NaCl(质量分数为0.9%的NaCl溶液)注射液(2ml),连续3次。致敏结束,末次致敏后第12天静脉快速注射2倍致敏剂量对以上相应的各组致敏豚鼠进行激发。致敏期间,每天观察豚鼠的症状,并于初次和最后一次致敏及激发当日测定每只豚鼠的体质量。(4)皮内刺激试验:取6只新西兰兔,单次皮内注射EC 10μg(0.2ml)/点,每侧5个点,观察皮内注射后的刺激反应情况。结果(1)免疫特性研究结果:EC原液对卡介菌活菌菌液致敏的4只豚鼠的皮肤试验,阳性为0只;EC原液对结核分枝杆菌H37Ra活菌菌液致敏的6只豚鼠的皮肤试验,阳性为6只。(2)急性毒性试验结果:所有小鼠未出现明显的急性中毒反应和显示急性中毒靶器官。小鼠皮内注射EC前、注射EC后1d、3d、5d、7d、8d、10d、12d、14d高剂量组、低剂量组小鼠平均体质量分别为(20.6±1.3)g^(24.9±2.1)g、(20.5±1.6)g^(26.0±3.1)g;注射后不同观察时间各组平均体质量与空白对照组[(21.0±1.1)g^(25.3±2.3)g]比较差异均无统计学意义(t值分别为0.571~0.392,0.695~0.615;P值分别为0.575~0.700,0.496~0.546);小鼠肌内注射EC前、注射EC后1d、3d、5d、7d、8d、10d、12d、14d高剂量组、低剂量组小鼠平均体质量分别为(21.0±1.5)g^(26.2±1.9)g、(20.5±2.1)g^(25.8±3.8)g;注射后不同观察时间各组平均体质量与空白对照组[(21.2±1.7)g^(25.8±3.1)g]比较差异均无统计学意义(t值分别为0.360~0.318,0.900~0.006;P值分别为0.723~0.754,0.380~0.995)。(3)豚鼠全身主动过敏试验结果:豚鼠无过敏反应。高剂量组、低剂量组豚鼠首次致敏、末次致敏、激发的平均体质量分别为(327.5±24.3)g、(347.2±32.7)g、(402.2±34.9)g;(331.3±26.7)g、(346.2±32.0)g、(411.3±38.9)g,与相应观察时间注射0.9%NaCl的阴性对照组[(329.5±27.4)g、(348.3±27.0)g、(399.4±25.4)g]比较,差异均无统计学意义(t值分别为0.328、0.181、0.284,0.474、0.366、0.875;P值分别为0.757、0.864、0.788,0.656、0.730、0.422)。(4)兔皮内刺激试验结果:单次皮内注射10μg(0.2ml)/点EC,无明显刺激反应。结论EC能够鉴别结核感染与卡介苗免疫,临床前动物试验安全性好,有望应用于人群结核感染的诊断与鉴别诊断。
基金Supported by Project of Science and Technology Development of Jilin Province(20140204018YY)
文摘[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.
