卡介苗重组疫苗的构建、免疫原性及抗结核作用的初步研究
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摘要
目的:构建结核分枝杆菌重组卡介苗,研究其免疫原性及抗结核作用,以期获得结核病预防性和治疗性疫苗。
方法:通过基因工程重组技术将结核分枝杆菌保护性抗原ESAT6、MPT64、Ag85A、Ag85B的编码基因分别与穿梭质粒载体PMD31、pYUB295重组,通过电穿孔技术导入卡介苗中,应用PCR扩增、PAGE电泳鉴定重组卡介苗。将重组卡介苗免疫小鼠后,通过ELISA法检测其血清中特异性抗体,用MTS法分析其脾淋巴细胞增殖指数;结核分枝杆菌攻击后,通过小鼠半数死亡时间、一定时间内的死亡率、肺、肝、脾细菌生长最低稀释度等指标评价各重组卡介苗对结核分枝杆菌感染的预防作用和治疗效果。
结果:通过PCR扩增获得esat6、mpt64、ag85a、ag85b基因,与穿梭表达载体PMD31、pYUB295重组后,通过PCR扩增、限制性内切酶酶切、DNA测序鉴定表明成功地构建了esat6、mpt64、ag85a、ag85b基因PMD31、pYUB295重组质粒。
将各重组质粒通过电穿孔导入卡介苗。质粒PMD31重组卡介苗在抗性培养上不能很好生长,质粒pYUB295重组卡介苗在抗性培养基上生长良好。pYUB295-目的基因重组卡介苗基因组DNA的PCR扩增以
Objective: construct esat6 mpt6 ag85a ag85b recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively.Methods: The gene of esat6 mpt64 ag85a ag85b were amplified by PCR with primers designed according to the sequences publicized in Genebank and the vectors used. Those genes were recombined with shuttle plasmids pYUB295 and PMD31 respectively. The recombinant shuttle plasmids were identified by PCR enzyme digestion and DNA sequencing and then transformed into BCG by electroporation. The antibody was detected by ELISA and multiplication of mouse lymph-cell was detected by MTS.The mean time to death and death rate in 2 month et al were used to valued the protection of BCG recombinant vaccines.Results: The gene of esat6 mpt64 ag85a ag85b were amplified by PCR. pYUB295 and PMD31 recombinants of these genes were constructed through recombinant technology and identified them by PCR enzyme digestion and DNA sequencing. The recombinant plasmids were transformed into BCG respectively by electroporation. PMD31
recombinant BCG could not grow very well in kanamycin resistance 7H10 culture medium, then, pYUB295 recombinant BCG was used for further study.The pYUB295 recombinant BCG were identified by amplified recombinant BCG genome DNA and showed pYUB295 recombinant Plasmids were transformed into BCG successfully. The SDS-PAGE results of pYUB295 recombinant BCG culture filtrate showed: the extrinsic esat6 mpt64 ag85a ag85b gene could be expressed in BCG.The weight of the mouse injected with recombinant BCG or BCG had no difference with the negative control and it indicated: recombinant BCG was safe and had no side effect. The antigen-specific antibody levels of mouse immunized with recombinant BCG were evaluation by ELISA using ESAT6 MPT6 Ag85A Ag85B protein and PPD as antigen. The ELISA results indicated: Each extrinsic gene can be express in BCG and can stimulate B cell of mouse to produce antibody; ag85a- recombinant BCG group had the highest antibody level, the antibody level had already improved after immunized 15 days and reached the peak after 45 days.The mouse lymph-cell stimulation test revealed: The lymph-cells of each group multiplicated when stimulated by different antigen such as conA ESAT6, MPT64 . Ag85A Ag85B protein and PPD respectively, all stimulation index reached 2. In other words, each protein could stimulate the mouse lymph-cell of each group obviously. The stimulation
index of each group had no obviously difference except esat6-recombiant BCG group.The mean time to death of ag85a^ ag85K esat6> mpt64-recombianat BCG^ BCG and saline immuned mouse after injected with M.tuberculosis was 3K 29> >60^ 32> 30 and 13days respectively.The death rate in 2 month of those mouse was 60%, 50%, 30% ^ 50% > 60% and 80% respectively.The results of pathological changes and organ culture indicated: esat6-recombinant BCG group had less burden of M. tuberculosis than other groups. The antibody level showed: the antibody level of ag85a-recombinant BCG group was higher than other groups. The mouse lymph-cell stimulation test revealed: all stimulation indexes of different groups stimulated by different antigen were not obviously different.The mean time to death f ag85a^ ag85b> esat6> mpt64-recombianat BCG^ BCG and saline immuned mouse in the treatment experiment was 24 > 25> 3 K 23 -? 24 and 14days respectively.The death rate in 2 month of those mouse was 60% > 60%^ 50% > 50% > 60% and 70% respectively. Other experiment such as: the pathological changes, the organ culture, the antibody level and the mouse lymph-cell stimulation test were all consistent with their results in the prevention experiment.Conclusion: The esat6^ mpt64> ag85a> ag85b recombinant BCG were constructed successfully. BCG and recombinant BCG can prevent M. tuberculosis infection, but esat6-recombinant BCG was more ffective than
方法:通过基因工程重组技术将结核分枝杆菌保护性抗原ESAT6、MPT64、Ag85A、Ag85B的编码基因分别与穿梭质粒载体PMD31、pYUB295重组,通过电穿孔技术导入卡介苗中,应用PCR扩增、PAGE电泳鉴定重组卡介苗。将重组卡介苗免疫小鼠后,通过ELISA法检测其血清中特异性抗体,用MTS法分析其脾淋巴细胞增殖指数;结核分枝杆菌攻击后,通过小鼠半数死亡时间、一定时间内的死亡率、肺、肝、脾细菌生长最低稀释度等指标评价各重组卡介苗对结核分枝杆菌感染的预防作用和治疗效果。
结果:通过PCR扩增获得esat6、mpt64、ag85a、ag85b基因,与穿梭表达载体PMD31、pYUB295重组后,通过PCR扩增、限制性内切酶酶切、DNA测序鉴定表明成功地构建了esat6、mpt64、ag85a、ag85b基因PMD31、pYUB295重组质粒。
将各重组质粒通过电穿孔导入卡介苗。质粒PMD31重组卡介苗在抗性培养上不能很好生长,质粒pYUB295重组卡介苗在抗性培养基上生长良好。pYUB295-目的基因重组卡介苗基因组DNA的PCR扩增以
Objective: construct esat6 mpt6 ag85a ag85b recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively.Methods: The gene of esat6 mpt64 ag85a ag85b were amplified by PCR with primers designed according to the sequences publicized in Genebank and the vectors used. Those genes were recombined with shuttle plasmids pYUB295 and PMD31 respectively. The recombinant shuttle plasmids were identified by PCR enzyme digestion and DNA sequencing and then transformed into BCG by electroporation. The antibody was detected by ELISA and multiplication of mouse lymph-cell was detected by MTS.The mean time to death and death rate in 2 month et al were used to valued the protection of BCG recombinant vaccines.Results: The gene of esat6 mpt64 ag85a ag85b were amplified by PCR. pYUB295 and PMD31 recombinants of these genes were constructed through recombinant technology and identified them by PCR enzyme digestion and DNA sequencing. The recombinant plasmids were transformed into BCG respectively by electroporation. PMD31
recombinant BCG could not grow very well in kanamycin resistance 7H10 culture medium, then, pYUB295 recombinant BCG was used for further study.The pYUB295 recombinant BCG were identified by amplified recombinant BCG genome DNA and showed pYUB295 recombinant Plasmids were transformed into BCG successfully. The SDS-PAGE results of pYUB295 recombinant BCG culture filtrate showed: the extrinsic esat6 mpt64 ag85a ag85b gene could be expressed in BCG.The weight of the mouse injected with recombinant BCG or BCG had no difference with the negative control and it indicated: recombinant BCG was safe and had no side effect. The antigen-specific antibody levels of mouse immunized with recombinant BCG were evaluation by ELISA using ESAT6 MPT6 Ag85A Ag85B protein and PPD as antigen. The ELISA results indicated: Each extrinsic gene can be express in BCG and can stimulate B cell of mouse to produce antibody; ag85a- recombinant BCG group had the highest antibody level, the antibody level had already improved after immunized 15 days and reached the peak after 45 days.The mouse lymph-cell stimulation test revealed: The lymph-cells of each group multiplicated when stimulated by different antigen such as conA ESAT6, MPT64 . Ag85A Ag85B protein and PPD respectively, all stimulation index reached 2. In other words, each protein could stimulate the mouse lymph-cell of each group obviously. The stimulation
index of each group had no obviously difference except esat6-recombiant BCG group.The mean time to death of ag85a^ ag85K esat6> mpt64-recombianat BCG^ BCG and saline immuned mouse after injected with M.tuberculosis was 3K 29> >60^ 32> 30 and 13days respectively.The death rate in 2 month of those mouse was 60%, 50%, 30% ^ 50% > 60% and 80% respectively.The results of pathological changes and organ culture indicated: esat6-recombinant BCG group had less burden of M. tuberculosis than other groups. The antibody level showed: the antibody level of ag85a-recombinant BCG group was higher than other groups. The mouse lymph-cell stimulation test revealed: all stimulation indexes of different groups stimulated by different antigen were not obviously different.The mean time to death f ag85a^ ag85b> esat6> mpt64-recombianat BCG^ BCG and saline immuned mouse in the treatment experiment was 24 > 25> 3 K 23 -? 24 and 14days respectively.The death rate in 2 month of those mouse was 60% > 60%^ 50% > 50% > 60% and 70% respectively. Other experiment such as: the pathological changes, the organ culture, the antibody level and the mouse lymph-cell stimulation test were all consistent with their results in the prevention experiment.Conclusion: The esat6^ mpt64> ag85a> ag85b recombinant BCG were constructed successfully. BCG and recombinant BCG can prevent M. tuberculosis infection, but esat6-recombinant BCG was more ffective than
引文
[1] 全国结核病流行病学抽样调查技术指导组。全国结核病流行病学抽样调查报告,中国防痨杂志,2002,24(2):65-108。
[2] Sqrensen AL, Nagai S, Houen G, et al. purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun, 1995;63(5):1710.
[3] Sambrook J,Fristsch E F,Maniatist.金冬雁,黎孟岚,张德政,等译。分子克隆实验指南。第2版。北京科学出版社,1959,19-59。
[4] 路艳艳,皇莆永穆。电穿孔法对分枝杆菌进行高效转化,北京医科大学学报,2000,32(2):178-181。
[5] M.kumar, A K, Behera, H Matsuse, et al. a recombinant BCG vaccine generates a Th1-like response and hibits IgE synthesis in BALB/c mice. Immunology 1999, 97: 515-521.
[6] Harth G,Lee BY, Wang J, et al. Novel insights into the genetics, biochemistry,and immunocytochemistry of the 30-kilodalton major extracellular protein of mycobacterium tuberculosis. Infect Immun,1996,64(8):3038.
[7] Feng CG,Palendira U, Demangel C, et al. Priming by DNA immunization augments protective efficacy of mycobacterium bovis Bacille Calmette_Guerin against tuberculosis. Infect Immun,2001,69(6):4174-4176.
[8] Bruibunt M,lefevre P,de wit L, et al.A mycobacterium tuberculosis gene cluster encoding proteins of a phosphate homulogous to the E.Coli Pst system.Gene, 1996,176:171-176.
[9] Zhou A T, Mu W L, Zhang P Y,et al. Detection of pulmonary and extrapumonary patients with the 38 kDa antigen from mycobacterium tuberculosis in a rapid membrane based assay. Clinical diagnostic laboratory immunology, 1996, 3: 337-341.
[10] Havlir D V, Willas R S, Boom W H, et al. Human immune response to mycobacterium tuberculosis antigens. Infect immune, 1991, 59: 665-670.
[11] Lyashchnko K, Colangeli R,Houde M, et al. Heterogeneous antibodys responses in tuberculosis. Infect Immune, 1998,66:3936-3940.
[12] Peter A, Dorthe A, Lene L, et al. Protein released from M. tuberculosis during growth. Infection and immunity, 1991,59(6)1905-1910.
[13] Martin J E, Thomas O, Peter A, et al. Delayed-type hypersensitivity response to ESAT6 and MPT64 in the guinea pig. Infection and immunity, 1998,66(12):3454-3456.
[14] 张灵霞,吴雪琼,史迎昌,等。结核分枝杆菌重组ESAT6蛋白诱发豚鼠迟发型超敏反应的研究。中华结核和呼吸杂志,2001,24(6):345—346。
[15] 程继忠,皇铺永穆,重组卡介苗疫苗的研究进展[J].微生物学通报,1996,16(4):47-49
[16] 程继忠,皇铺永穆,海涛.外源基因在耻垢分枝杆菌中表达效率的研究[J].生物化学与生物物理进展,1997,24(3):249-253.
[17] 路艳燕,冯作化,皇铺永穆,等.新型大肠杆菌-分枝杆菌穿梭载体的构建及卡那酶素抗性基因表达的研究[J].同济医科学学报,1998,27(2):89-93.
[18] 程继忠,郑波,肖红,等.结核杆菌HSP70在耻垢分枝杆菌中的表达及其免疫原性研究[J].中华微生物和免疫学杂志,1998,18(5):337-341
[19] 李文桂,石佑恩,汪燕鸣,等.血吸虫重组卡介苗Sj26GST疫苗接种后保护力的观察[J].同济医科大学学报 1999,28(1)13-16.
[20] 程继忠,郑波,海涛,等.大肠杆菌-分枝穿梭表达质粒P卡介苗-2100的构建及应用[J].生物工程学报,1999,15(2):225-229.
[21] 路艳艳,皇铺永穆,电穿孔法对分枝杆菌进行高效基因转化[J].北京医科大学学报,2000,32(2):178-181.
[22] 邱洪宇,鲍朗,宴菊芳,等钩端螺旋体外膜蛋白基因重组载体构建及在卡介菌中的表达[J].中国生物制品学杂志,2000,13(3):136-139.
[23] 戴五星,皇铺永穆,郑波,等,血吸虫重组卡介苗-Sj26GST疫苗的构建和免疫原性的研究[J].中华医学杂志,2000,80(6):407-410.
[24] 戴五星,皇铺永穆,郑波,等日本血吸虫重组卡介苗Sj26GST疫苗对小鼠免疫应答的影响[J].同济医科大学学报,2000,29(1):1-3.
[25] 肖红,郑波,祁学忠,等.日本血吸虫32ku抗原基因的克隆及其在耻垢分枝杆菌中的表达[J].同济医科大学学报,2000,39(1)4-7
[26] 曾星,梅玉屏,黄羽,等rBCG IL-2对肿瘤生长抑制作用和免疫刺激作用的研究[J].肿瘤防治研究,2000,27(6):425-427.
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[48] Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G, Leclerc C, Cole ST. Recombinant BCG
[2] Sqrensen AL, Nagai S, Houen G, et al. purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun, 1995;63(5):1710.
[3] Sambrook J,Fristsch E F,Maniatist.金冬雁,黎孟岚,张德政,等译。分子克隆实验指南。第2版。北京科学出版社,1959,19-59。
[4] 路艳艳,皇莆永穆。电穿孔法对分枝杆菌进行高效转化,北京医科大学学报,2000,32(2):178-181。
[5] M.kumar, A K, Behera, H Matsuse, et al. a recombinant BCG vaccine generates a Th1-like response and hibits IgE synthesis in BALB/c mice. Immunology 1999, 97: 515-521.
[6] Harth G,Lee BY, Wang J, et al. Novel insights into the genetics, biochemistry,and immunocytochemistry of the 30-kilodalton major extracellular protein of mycobacterium tuberculosis. Infect Immun,1996,64(8):3038.
[7] Feng CG,Palendira U, Demangel C, et al. Priming by DNA immunization augments protective efficacy of mycobacterium bovis Bacille Calmette_Guerin against tuberculosis. Infect Immun,2001,69(6):4174-4176.
[8] Bruibunt M,lefevre P,de wit L, et al.A mycobacterium tuberculosis gene cluster encoding proteins of a phosphate homulogous to the E.Coli Pst system.Gene, 1996,176:171-176.
[9] Zhou A T, Mu W L, Zhang P Y,et al. Detection of pulmonary and extrapumonary patients with the 38 kDa antigen from mycobacterium tuberculosis in a rapid membrane based assay. Clinical diagnostic laboratory immunology, 1996, 3: 337-341.
[10] Havlir D V, Willas R S, Boom W H, et al. Human immune response to mycobacterium tuberculosis antigens. Infect immune, 1991, 59: 665-670.
[11] Lyashchnko K, Colangeli R,Houde M, et al. Heterogeneous antibodys responses in tuberculosis. Infect Immune, 1998,66:3936-3940.
[12] Peter A, Dorthe A, Lene L, et al. Protein released from M. tuberculosis during growth. Infection and immunity, 1991,59(6)1905-1910.
[13] Martin J E, Thomas O, Peter A, et al. Delayed-type hypersensitivity response to ESAT6 and MPT64 in the guinea pig. Infection and immunity, 1998,66(12):3454-3456.
[14] 张灵霞,吴雪琼,史迎昌,等。结核分枝杆菌重组ESAT6蛋白诱发豚鼠迟发型超敏反应的研究。中华结核和呼吸杂志,2001,24(6):345—346。
[15] 程继忠,皇铺永穆,重组卡介苗疫苗的研究进展[J].微生物学通报,1996,16(4):47-49
[16] 程继忠,皇铺永穆,海涛.外源基因在耻垢分枝杆菌中表达效率的研究[J].生物化学与生物物理进展,1997,24(3):249-253.
[17] 路艳燕,冯作化,皇铺永穆,等.新型大肠杆菌-分枝杆菌穿梭载体的构建及卡那酶素抗性基因表达的研究[J].同济医科学学报,1998,27(2):89-93.
[18] 程继忠,郑波,肖红,等.结核杆菌HSP70在耻垢分枝杆菌中的表达及其免疫原性研究[J].中华微生物和免疫学杂志,1998,18(5):337-341
[19] 李文桂,石佑恩,汪燕鸣,等.血吸虫重组卡介苗Sj26GST疫苗接种后保护力的观察[J].同济医科大学学报 1999,28(1)13-16.
[20] 程继忠,郑波,海涛,等.大肠杆菌-分枝穿梭表达质粒P卡介苗-2100的构建及应用[J].生物工程学报,1999,15(2):225-229.
[21] 路艳艳,皇铺永穆,电穿孔法对分枝杆菌进行高效基因转化[J].北京医科大学学报,2000,32(2):178-181.
[22] 邱洪宇,鲍朗,宴菊芳,等钩端螺旋体外膜蛋白基因重组载体构建及在卡介菌中的表达[J].中国生物制品学杂志,2000,13(3):136-139.
[23] 戴五星,皇铺永穆,郑波,等,血吸虫重组卡介苗-Sj26GST疫苗的构建和免疫原性的研究[J].中华医学杂志,2000,80(6):407-410.
[24] 戴五星,皇铺永穆,郑波,等日本血吸虫重组卡介苗Sj26GST疫苗对小鼠免疫应答的影响[J].同济医科大学学报,2000,29(1):1-3.
[25] 肖红,郑波,祁学忠,等.日本血吸虫32ku抗原基因的克隆及其在耻垢分枝杆菌中的表达[J].同济医科大学学报,2000,39(1)4-7
[26] 曾星,梅玉屏,黄羽,等rBCG IL-2对肿瘤生长抑制作用和免疫刺激作用的研究[J].肿瘤防治研究,2000,27(6):425-427.
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