结核DNA疫苗的构建、免疫功能及保护效果研究
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摘要
结核病仍对人类健康构成重要威胁。全球现有TB患者近3000万，每年病死数近300万。结核病唯一可用的免疫预防制剂是BCG。它的免疫保护力介于0-80％之间，WHO的调查研究表明其保护力也仅为50％。成年人及老年人并不能得到BCG的有效保护。BCG对热敏感，在运输、贮存中需要低温保存。因此寻找新型高效，效果稳定的疫苗一直是相关领域研究人员关注的热点。
本研究的目的，是设计和构建真核表达结核菌蛋白的载体即DNA疫苗，并对其免疫功能及保护力进行研究。所设计构建的10种结核DNA疫苗依次为TB-V1，表达结核菌Mtb8.4；TB-V2，表达结核菌Ag85B蛋白；TB-V3，表达Mtb8.4、38kDa的Th表位和CTL表位以及Ag85B的融合蛋白；TB-V4，表达GST和Mtb8.4的融合蛋白；TB-V5，表达GST与Ag85B的融合蛋白；TB-V6，表达含hIL-2信号肽的Mtb8.4；TB-V7，表达含信号肽的Ag85B；TB-V8，表达含信号肽的Mtb8.4、38kDa蛋白的CTL／Th表位以及Ag85B成熟蛋白的融合蛋白；TB-V9，表达信号肽的Mtb8.4与GST的融合蛋白；TB-V10，表达含信号肽的Ag85B与GST的融合蛋白。TB-V6-TB-V10等5种载体，均在表达结核蛋白的N末端插入hIL-2的信号肽。p-hIL2质粒表达hIL-2；p-hIL2s质粒仅表达hIL-2的信号肽，作为对照。
所构建的12种质粒DNA，经PCR、酶谱鉴定后，并对插入表达目的的DNA片段进行了测序分析，表明所构建的12种载体质粒与构建设计准确无误。
p-hIL2、p-IL2s、TB-V6、TB-V7、TB-V8、TB-V9与TB-V10质粒DNA，以0.1M PBS将其分别稀释成1mg／mL溶液，除菌，然后分别在0d、15d和30d，分3次在8-10周龄的雌性C57BL／6小鼠四肢肌肉各注射0.1mL(100 μg／只小鼠)。对照组为等剂量的pVAX1、D-IL2s、PBS溶液。阳性对照组为BCG，以10~5CFU注射小鼠皮内，与其他小鼠初免时一同免疫，该组每只小鼠只免疫1次。疫苗免疫组、对照组共12组，分别是TB-V6～TB-V10等5组，p-IL2质粒注射组，TB-V6+p-hIL2
2作者:华南热带农业大学博士研究生朱中元，导师:张春发
和TB一7+，hILZ联合免疫2组(每种质粒各50 ug免疫)，PBs，BcG，
pVAXI和尸LZs共4个对照组。每组10只小鼠。
在最后一次DNA疫苗加强免疫15天后，杀死每组的其中5只小鼠，
取脾磨成匀浆，并用10%FCS RPMll64O培养基将脾淋巴细胞配成10V
mL浓度，加入到24孔培养板中，200 pl吼，5%Cq条件下培养72h，
上清经ELIsA法测定鼠白细胞介素一(mIL一2)、而L-6、而L一10和鼠7
干扰素(mIFN斗)。处死小鼠时采血，用于测定hIL一2。
在DNA疫苗最后一次加强免疫的21天，将结核菌H37RLv株培养约
20天的细菌磨成悬液，取106/0.llnL菌经尾静脉注射实验感染每组剩下
的5只小鼠。感染15天后，杀死所有小鼠，取脾、肝和肺分别称重，制
成匀浆，接种0.lmL于改良罗氏培养基(L一)培养瓶，置37C培养20一30
天，计数。并根据器官重量和稀释倍数，换算出每种器官培养物的
CFUlg。每个小鼠的每种器官各接种3支L一培养，取其均数作为最后
结果。
检测各免疫组、对照组的上清中的而L一2，而L一6，而L一10和
而FN斗以及血清中hIL一2的含量，显示TB一v6，TB一V7，TB一vs，
TB一vg，TB一V10，TB一v6+P扣L2，TB一v7+p扣L2及p一hILZ等8个免疫
组的mIFN斗及mIL一显著高于pVAXI，PBs和p一LZs质粒对照组，与
BCG免疫组的水平稍低或相当。其中p.hiLZ注射组，TB一v6+p-hiLZ
以及犯一7+p一址LZ联合免疫组小鼠血清hIL一2水平显著升高。而这8组
的体外淋巴细胞培养上清中的nilL一6及mIL一10的含量不升高或升高不
明显。表明犯J6~TB一vlo等5种DNA疫苗及p扣LZ表达载体能够刺
激预防结核病所需的几1型免疫应答。
TB一6~TB一V10等5种疫苗免疫的5只小鼠的脾结核菌载量分别为
44 1 68、28400、1 8597、34703和22954CFIJ/g。TB一v6+P一LZ和
TB一V7+P一L2联合免疫组和p一hILZ注射组的脾结核菌载量分别为
12471、34x57和51339 CFU/g。对照组邓S、pVAXI、p一LZs及BCG
的脾细胞悬液的结核菌计数结果均数分别是:61012、60716、69395
和1 1 164CFU/g。表明本研究中构建的，IB一6~TB一v10等疫苗具有减少
免疫鼠经H37Rv攻击后脾脏结核菌载量，且TB一vs疫苗，TB一v6+P一砚
联合免疫，产生的保护力与BCG免疫产生的保护力非常接近。为进一
步研究这些疫苗防止大动物感染结核菌H37Rv的实验研究奠定了基
础。
我们在结核DNA疫苗的构建设计中使用了hIL一2信号肤序列，并采
用表达hIL.2的质粒与TB DNA疫苗联合免疫及构建表达融合蛋白
(GST)的疫苗，并获得与BCG非常接近的保护力。均为TB DNA疫
结核D队疫苗的构建、免疫功能及保护效果研究」
苗研究首次报道。取得了预期的研究结果，对其他疫苗的研究有指导
参考价值。
结论:
1.所构建的表达结核菌蛋白或融合蛋白的载体:邓一1、TB一2、
TB~V3、TB.V4、TB~VS、TB一V6、TB~V7、TB一VS、TB一Vg、TB一V10、
表达bIL.2的载体卜拍LZ和表达hiL一2信号肤的载体尸LZs等经抗性筛
选、酶谱鉴定及DNA序列分析，结果表明12种DNA疫苗表达载体质粒
的构建均与设计要求准确无误。
2.TB一6、TB一V7、TB一7、TB一VS、TB一Vg与TB一V105种DNA疫苗
单独免疫，或者
Tuberculosis was an ancient, but still is a chronic consumptive disease that threatens the human lives globally. There are 30 million cases of TB in the world and 3 million TB cases die annually. BCG is the only available vaccine for prevention of tuberculosis. Investigation results showed that the protective efficacy of the yaccine was from -36% to 80%. A well-designed and controlled survey indicated the BCG efficacy was about 50%. The goal of this study is to characterize and compare the immune responses and protective efficacy in mice induced by varied plasmid DNA vaccines that express the surface antigens of Mtb8.4, Ag85B etc of Mtuberculosis and BCG
Ten plasmid DNA vaccines that express M tuberculosis surface antigens or fusion proteins as well as human interleukin-2 (hIL-2) using pVAXl as a vector have been constructed. Among them, TB plasmid DNA vaccine TB-V1, TB-V2, TB-V3, TB-V4 and TB-V5 expressed Mtb8.4, Ag85B, Mtb8.4-38kDa CTL/Th epitopes-Ag85B, Mtb8.4-GST and Ag85B-GST antigens or fusion proteins respectively; TB-V6 and TB-V7 plasmid expressed M tuberculosis Mtb8.4 and Ag85B protein with ML-2 signal peptide and TB-V8 expressed Mtb8.4, 38kDa CTL/Th epitope and Ag85B fusion protein with hIL-2 signal peptide; TB-V9 and TB-V10 expressed Mtb8.4-GST, Ag85B-GST fusion proteins with hIL-2 signal peptide respectively. The plasmid of p-IL2s was also constructed as a negative control since hIL-2 signal peptide was inserted into the vector only. The DNA fragments containing the sequences encoding the antigens of Mtb8.4, Ag85B, 38 kDa CTL/Th epitope were generated from M tuberculosis H37Rv genomic DNA by PCR and hIL-2 and GST cDNA
were sub-cloned from the plasmid of pGex-2T-hIL-2 respectively. The constructs of the plasmid DNA vaccine were transformed into bacteria DH5a cells and the plasmid DNA were verified by restriction endonucleases mapping and sequence analysis.
For the immunization experiments, C57BL/6 mice were used as test animal and they were divided into 12 groups with 10 mice each. The C57BL/6 mice were immunized by injecting into legs muscles with lOOjig plasmid DNA 3 times at 15 days intervals. Groups No 1 to Group No 5 were immunized with TB-V6~TB-V10, and Groups No. 6 to No. 8 were immunized with p-hIL2, p-WL2+TB-V6 and p-hIL2+TB-V7 (SO^ig/each) respectively. Groups No 9 to No 12 were as controls that were immunized with PBS, plasmid DNA of pVAXl, p-IL2s and BCG. BCG was introdermally injected at the dosage of 105 CPUs only once.
15 days after the last boost immunization, 5 mice in each tested group were killed and the spleen lymphocyte suspensions were harvested and cultured in 24-well plates for 72hr at 37. The supernatants were collected and assayed for murine interleukin-2(mIL-2) , IL-6 , IL-10 and murine interferon gamma (mIFN-) activities. A quantitative ELISA assay was used to identify the hIL-2 activity for the serum from the eyes of the mice. 21 days after the last boost immunization, the remaining 5 mice were challenged by injection of 0.1 ml of M tuberculosis H37Rv strain suspension (10 CPUs). 15 days post challenges, the mice were killed and their lungs, livers and spleens were homogenized respectively. The immune response and protective efficacy assay were performed with three repeating by couture 0.1 ml of the each suspension onto the Lowenstein-Jensen slants and the CPUs were counted after 20-30 days culture. The bacterial loads were calculated according to the organ's weight and dilutions.
The results showed that the average activities of mIL-2, INF- Y in the speenocyte culture supernatants of the immunized group of TB-V6-TB-V10, p-WL2, TB-V6+p-hIL2 and TB-V7+p-hIL2 were significant different with those of PBS, pVAXl and p-IL2s controls, but were almost as high as BCG immunized controls. However, the activity of mIL-6 and mIL-10 was low both in tested or control groups which indicated that Thl immune response can be induced by the 5 plasmid DNA vaccines TB-V6-TB-V10 which is required for immunity against M tuberculosis. High hIL-2 activity in the sera of the p-hIL2, TB-V6+p-hIL2 an
本研究的目的，是设计和构建真核表达结核菌蛋白的载体即DNA疫苗，并对其免疫功能及保护力进行研究。所设计构建的10种结核DNA疫苗依次为TB-V1，表达结核菌Mtb8.4；TB-V2，表达结核菌Ag85B蛋白；TB-V3，表达Mtb8.4、38kDa的Th表位和CTL表位以及Ag85B的融合蛋白；TB-V4，表达GST和Mtb8.4的融合蛋白；TB-V5，表达GST与Ag85B的融合蛋白；TB-V6，表达含hIL-2信号肽的Mtb8.4；TB-V7，表达含信号肽的Ag85B；TB-V8，表达含信号肽的Mtb8.4、38kDa蛋白的CTL／Th表位以及Ag85B成熟蛋白的融合蛋白；TB-V9，表达信号肽的Mtb8.4与GST的融合蛋白；TB-V10，表达含信号肽的Ag85B与GST的融合蛋白。TB-V6-TB-V10等5种载体，均在表达结核蛋白的N末端插入hIL-2的信号肽。p-hIL2质粒表达hIL-2；p-hIL2s质粒仅表达hIL-2的信号肽，作为对照。
所构建的12种质粒DNA，经PCR、酶谱鉴定后，并对插入表达目的的DNA片段进行了测序分析，表明所构建的12种载体质粒与构建设计准确无误。
p-hIL2、p-IL2s、TB-V6、TB-V7、TB-V8、TB-V9与TB-V10质粒DNA，以0.1M PBS将其分别稀释成1mg／mL溶液，除菌，然后分别在0d、15d和30d，分3次在8-10周龄的雌性C57BL／6小鼠四肢肌肉各注射0.1mL(100 μg／只小鼠)。对照组为等剂量的pVAX1、D-IL2s、PBS溶液。阳性对照组为BCG，以10~5CFU注射小鼠皮内，与其他小鼠初免时一同免疫，该组每只小鼠只免疫1次。疫苗免疫组、对照组共12组，分别是TB-V6～TB-V10等5组，p-IL2质粒注射组，TB-V6+p-hIL2
2作者:华南热带农业大学博士研究生朱中元，导师:张春发
和TB一7+，hILZ联合免疫2组(每种质粒各50 ug免疫)，PBs，BcG，
pVAXI和尸LZs共4个对照组。每组10只小鼠。
在最后一次DNA疫苗加强免疫15天后，杀死每组的其中5只小鼠，
取脾磨成匀浆，并用10%FCS RPMll64O培养基将脾淋巴细胞配成10V
mL浓度，加入到24孔培养板中，200 pl吼，5%Cq条件下培养72h，
上清经ELIsA法测定鼠白细胞介素一(mIL一2)、而L-6、而L一10和鼠7
干扰素(mIFN斗)。处死小鼠时采血，用于测定hIL一2。
在DNA疫苗最后一次加强免疫的21天，将结核菌H37RLv株培养约
20天的细菌磨成悬液，取106/0.llnL菌经尾静脉注射实验感染每组剩下
的5只小鼠。感染15天后，杀死所有小鼠，取脾、肝和肺分别称重，制
成匀浆，接种0.lmL于改良罗氏培养基(L一)培养瓶，置37C培养20一30
天，计数。并根据器官重量和稀释倍数，换算出每种器官培养物的
CFUlg。每个小鼠的每种器官各接种3支L一培养，取其均数作为最后
结果。
检测各免疫组、对照组的上清中的而L一2，而L一6，而L一10和
而FN斗以及血清中hIL一2的含量，显示TB一v6，TB一V7，TB一vs，
TB一vg，TB一V10，TB一v6+P扣L2，TB一v7+p扣L2及p一hILZ等8个免疫
组的mIFN斗及mIL一显著高于pVAXI，PBs和p一LZs质粒对照组，与
BCG免疫组的水平稍低或相当。其中p.hiLZ注射组，TB一v6+p-hiLZ
以及犯一7+p一址LZ联合免疫组小鼠血清hIL一2水平显著升高。而这8组
的体外淋巴细胞培养上清中的nilL一6及mIL一10的含量不升高或升高不
明显。表明犯J6~TB一vlo等5种DNA疫苗及p扣LZ表达载体能够刺
激预防结核病所需的几1型免疫应答。
TB一6~TB一V10等5种疫苗免疫的5只小鼠的脾结核菌载量分别为
44 1 68、28400、1 8597、34703和22954CFIJ/g。TB一v6+P一LZ和
TB一V7+P一L2联合免疫组和p一hILZ注射组的脾结核菌载量分别为
12471、34x57和51339 CFU/g。对照组邓S、pVAXI、p一LZs及BCG
的脾细胞悬液的结核菌计数结果均数分别是:61012、60716、69395
和1 1 164CFU/g。表明本研究中构建的，IB一6~TB一v10等疫苗具有减少
免疫鼠经H37Rv攻击后脾脏结核菌载量，且TB一vs疫苗，TB一v6+P一砚
联合免疫，产生的保护力与BCG免疫产生的保护力非常接近。为进一
步研究这些疫苗防止大动物感染结核菌H37Rv的实验研究奠定了基
础。
我们在结核DNA疫苗的构建设计中使用了hIL一2信号肤序列，并采
用表达hIL.2的质粒与TB DNA疫苗联合免疫及构建表达融合蛋白
(GST)的疫苗，并获得与BCG非常接近的保护力。均为TB DNA疫
结核D队疫苗的构建、免疫功能及保护效果研究」
苗研究首次报道。取得了预期的研究结果，对其他疫苗的研究有指导
参考价值。
结论:
1.所构建的表达结核菌蛋白或融合蛋白的载体:邓一1、TB一2、
TB~V3、TB.V4、TB~VS、TB一V6、TB~V7、TB一VS、TB一Vg、TB一V10、
表达bIL.2的载体卜拍LZ和表达hiL一2信号肤的载体尸LZs等经抗性筛
选、酶谱鉴定及DNA序列分析，结果表明12种DNA疫苗表达载体质粒
的构建均与设计要求准确无误。
2.TB一6、TB一V7、TB一7、TB一VS、TB一Vg与TB一V105种DNA疫苗
单独免疫，或者
Tuberculosis was an ancient, but still is a chronic consumptive disease that threatens the human lives globally. There are 30 million cases of TB in the world and 3 million TB cases die annually. BCG is the only available vaccine for prevention of tuberculosis. Investigation results showed that the protective efficacy of the yaccine was from -36% to 80%. A well-designed and controlled survey indicated the BCG efficacy was about 50%. The goal of this study is to characterize and compare the immune responses and protective efficacy in mice induced by varied plasmid DNA vaccines that express the surface antigens of Mtb8.4, Ag85B etc of Mtuberculosis and BCG
Ten plasmid DNA vaccines that express M tuberculosis surface antigens or fusion proteins as well as human interleukin-2 (hIL-2) using pVAXl as a vector have been constructed. Among them, TB plasmid DNA vaccine TB-V1, TB-V2, TB-V3, TB-V4 and TB-V5 expressed Mtb8.4, Ag85B, Mtb8.4-38kDa CTL/Th epitopes-Ag85B, Mtb8.4-GST and Ag85B-GST antigens or fusion proteins respectively; TB-V6 and TB-V7 plasmid expressed M tuberculosis Mtb8.4 and Ag85B protein with ML-2 signal peptide and TB-V8 expressed Mtb8.4, 38kDa CTL/Th epitope and Ag85B fusion protein with hIL-2 signal peptide; TB-V9 and TB-V10 expressed Mtb8.4-GST, Ag85B-GST fusion proteins with hIL-2 signal peptide respectively. The plasmid of p-IL2s was also constructed as a negative control since hIL-2 signal peptide was inserted into the vector only. The DNA fragments containing the sequences encoding the antigens of Mtb8.4, Ag85B, 38 kDa CTL/Th epitope were generated from M tuberculosis H37Rv genomic DNA by PCR and hIL-2 and GST cDNA
were sub-cloned from the plasmid of pGex-2T-hIL-2 respectively. The constructs of the plasmid DNA vaccine were transformed into bacteria DH5a cells and the plasmid DNA were verified by restriction endonucleases mapping and sequence analysis.
For the immunization experiments, C57BL/6 mice were used as test animal and they were divided into 12 groups with 10 mice each. The C57BL/6 mice were immunized by injecting into legs muscles with lOOjig plasmid DNA 3 times at 15 days intervals. Groups No 1 to Group No 5 were immunized with TB-V6~TB-V10, and Groups No. 6 to No. 8 were immunized with p-hIL2, p-WL2+TB-V6 and p-hIL2+TB-V7 (SO^ig/each) respectively. Groups No 9 to No 12 were as controls that were immunized with PBS, plasmid DNA of pVAXl, p-IL2s and BCG. BCG was introdermally injected at the dosage of 105 CPUs only once.
15 days after the last boost immunization, 5 mice in each tested group were killed and the spleen lymphocyte suspensions were harvested and cultured in 24-well plates for 72hr at 37. The supernatants were collected and assayed for murine interleukin-2(mIL-2) , IL-6 , IL-10 and murine interferon gamma (mIFN-) activities. A quantitative ELISA assay was used to identify the hIL-2 activity for the serum from the eyes of the mice. 21 days after the last boost immunization, the remaining 5 mice were challenged by injection of 0.1 ml of M tuberculosis H37Rv strain suspension (10 CPUs). 15 days post challenges, the mice were killed and their lungs, livers and spleens were homogenized respectively. The immune response and protective efficacy assay were performed with three repeating by couture 0.1 ml of the each suspension onto the Lowenstein-Jensen slants and the CPUs were counted after 20-30 days culture. The bacterial loads were calculated according to the organ's weight and dilutions.
The results showed that the average activities of mIL-2, INF- Y in the speenocyte culture supernatants of the immunized group of TB-V6-TB-V10, p-WL2, TB-V6+p-hIL2 and TB-V7+p-hIL2 were significant different with those of PBS, pVAXl and p-IL2s controls, but were almost as high as BCG immunized controls. However, the activity of mIL-6 and mIL-10 was low both in tested or control groups which indicated that Thl immune response can be induced by the 5 plasmid DNA vaccines TB-V6-TB-V10 which is required for immunity against M tuberculosis. High hIL-2 activity in the sera of the p-hIL2, TB-V6+p-hIL2 an
引文
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2. Andersen,A.B., Ljungqvist, L. and Olsen,M. 1990. Evidence that protein antigen b of Mycohacterium tuberculosis is involved in phosphate metabolism. J. Gen. Microbiil.136, 477-480.
3. Anonymous. 2001. Global tuberculosis control. World Health Organization report. WHO/CDS/TB/2001.287.
4. Baldwin,S. L., C. D'Souza, A.D. Roberts, B. P. Kelly, A. A. Frank, M. A. Lin,J. B. Ulmer, K. Huygen, D. M.. Me Murray, and I. M. Orme. 1998. Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect Immun. 66:2951-2959.
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