结核分枝杆菌Ag85B-MPT64融合基因疫苗对鼠结核杆菌感染的保护作用
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摘要
目的：
本研究拟从结核分枝杆菌H37Rv基因组中扩增出ag85b、mpt64以及引入甘氨酸接头连接的Ag85B-MPT64融合基因（AM），构建DNA疫苗并探讨其在小鼠体内诱导的免疫应答和在小鼠结核感染模型中的保护作用，为新型结核病疫苗的研制奠定基础，同时也为结核病的免疫防治提供理论和实验依据。
方法：
1、用PCR和基因克隆技术从结核分枝杆菌H37Rv基因组中扩增出Ag85B、MPT64编码基因以及通过甘氨酸接头连接的Ag85B-MPT64融合基因（AM），构建真核表达质粒pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM，用酶切和双向DNA测序进行鉴定；
2、将MPT64、Ag85B编码基因亚克隆入pET32a中，构建pET/Ag85B和pET/MPT64原核表达质粒，使其在大肠杆菌中表达并鉴定。用重组的MPT64和Ag85B蛋白免疫BABL/c小鼠， 制备MPT64
与Ag85B抗血清。
3、将pcDNA3.1(+)、pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM转染COS-7细胞，用RT-PCR、ELISA和斑点印迹的方法鉴定融合基因的表达。
4、用质粒pcDNA3.1(+)、pcDNA/Ag85B、pcDNA/MPT64、pcDNA/AM和pcDNA/BM（经过限制性内切酶消化后粘粘连接的Ag85B-MPT64融合基因）免疫C57BL/6小鼠，采用 ELISA法测量免疫后相应的PPD特异性抗体和脾淋巴细胞IL-4 以及IFN-γ分泌水平；MTT法检测PPD特异性脾淋巴细胞增殖能力。
5、以重组质粒免疫C57BL/6小鼠，末次免疫5周, BCG免疫100天后，用经过动物体内恢复毒力的1×106CFU MTb.H37Rv标准株由尾静脉注射攻击，以PBS组和空质粒组作阴性对照、BCG组作阳性对照；6周后分批处死动物，测量PPD特异性抗体、PPD特异性脾淋巴细胞IL-4和IFN-γ分泌水平；观察肺和脾组织器官荷菌量、组织抗酸染色、病理学改变以及小鼠存活时间。
结果：
1、经过限制性内切酶酶切及DNA双向测序证实 pcDNA/MPT64、pcDNA/Ag85B序列与理论值一致，pcDNA/AM碱基突变率为0.11%（2/1707），突变为无意义突变。
2、将MPT64、Ag85B编码基因亚克隆入pET32a原核表达质粒中，构建的pET/ MPT64和pET/Ag85B在大肠杆菌中表达了具有生物学活性的重组MPT64和Ag85B蛋白。用重组蛋白制备的鼠抗
MPT64与Ag85B血清效价分别为1:32和1:64。
3、重组质粒转染COS-7细胞后，分别用MPT64、Ag85B多克隆抗体通过ELISA法检测细胞培养上清液，抗MPT64与pcDNA/MPT64、抗Ag85B与pcDNA/Ag85B呈阳性反应，pcDNA/AM组与抗MPT64和抗Ag85B均呈阳性反应；对照组均为阴性反应。
4、将PBS、pcDNA3.1、pcDNA/Ag85B、pcDNA/MPT64、pcDNA/AM和pcDNA/BM免疫C57BL/6小鼠，末次免疫4周后，实验组PPD特异性抗体水平均有不同程度增高，以pcDNA/Ag85B+pcDNA/MPT64、pcDNA/AM组最高（P<0.05）；脾淋巴细胞经过PPD刺激后， IFN-γ分泌水平以及脾淋巴细胞增殖能力pcDNA/Ag85B+pcDNA/MPT64、pcDNA/AM组和pcDNA/BM组明显高于其他组（P<0.05），pcDNA/AM组高于pcDNA/BM组（P<0.05），pcDNA/Ag85B+pcDNA/MPT64组与pcDNA/AM组之间差异无显著性（P>0.05）。
5、用H37Rv标准株攻击小鼠后6周，BCG、pcDNA/Ag85B、pcDNA/MPT64、pcDNA/Ag85B+pcDNA/MPT64、pcDNA/BM和pcDNA/AM组IFN-γ分泌水平以及脾淋巴细胞增殖能力均有不同程度增高，以BCG组最高，pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组次之，pcDNA3.1和PBS组最低，但pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组的差异无显著性（P>0.05）。各组均未检测到IL-4的分泌。
6、肺、脾荷菌量以BCG组最低（P<0.01），混合质粒组和AM免疫
组次之；但混合质粒组和AM免疫组之间差异无显著性（P>0.05），AM与BM DNA免疫组之间有显著性差异（P<0.05）。
7、用H37Rv标准株攻击小鼠后6周，肺组织病理改变：阴性对照组的改变，主要为大量的浆液纤维素性和少量的淋巴细胞渗出以及不同程度的肺组织坏死；阳性对照组主要表现为大量的淋巴细胞、组织细胞和类上皮细胞浸润以及结核肉芽肿的形成；以混合质粒组和AM免疫组病变和阳性对照组最为接近，其他各组介于BM组和阴性对照之间。脾组织的病理改变主要是炎性浸润和淋巴细胞的增生，阴性对照组主要表现为炎性浸润，阳性对照组主要表现为淋巴细胞的增生，混合质粒组和AM免疫组病变与BCG组相似，其余各组介于BM与阴性对照组之间。
8、小鼠存活时间以BCG组最长，混合质粒组和pcDNA/AM组次之，pcDNA3.1和PBS组最短。
结论：
1、pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM DNA疫苗构建成功；pET/Ag85B、pET/MPT64能在大肠杆菌中表达，并分离纯化出rAg85B、rMPT64重组蛋白。
2、pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM能在COS-7细胞中分泌表达，且表达的蛋白具有免疫反应性。
3、本研究构建的DNA疫苗均可诱导小鼠产生特异性体液免疫和细胞免疫，其诱导能力以pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组最强，而pcDNA/AM组优于pcDNA/BM组。
4、免疫小鼠经H37Rv标准株攻击后，PPD特异性抗体以pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组最高； IFN-γ分泌水平以及脾淋巴细胞增殖能力以BCG、pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组最高。提示DNA疫苗接种后可以通过提高特异性体液免疫和细胞免疫水平发挥抗感染作用。
5、组
Objective
The study aimed to construct the fusion gene DNA vaccine encoding Ag85B and MPT64 linked with (Gly4Ser)3 linker, amplified from M.tuberculosis H37Rv chromosomal DNA, and, to explore the immune responses and protective efficacy of the DNA vaccine in mice infected with MTb. It would lay a foundation for a new tuberculosis vaccine and provide data for immunoprophylaxis and immunotherapy.
Methods
1. Construction of eukaryotic expression plasmids
The genes encoding Ag85B(ag85b), MPT64(mpt64) and AM[Ag85B and MPT64 fusion gene linked with (Gly4Ser)3 linker] were obtained from M.tuberculosis H37Rv chromosomal DNA by PCR
amplification,with primers designed to generate HindIII and BamHI restriction sites at the 5'and 3'ends of the amplified fragments respectively. The genes were cloned into unique HindIII and BamHI sites of the pcDNA3.1(+).The recombinant plasmids were identified with DNA sequence and endonucleases.
2. Expression and purification of Ag85B and MPT64 recombinant proteins in E.coli BL21, and fabrication of anti-Ag85B and anti- MPT64 antibodies.
The ag85b and mpt64 gene fragements were isolated from the pcDNA/Ag85B and pcDNA/MPT64 by restriction endonucleases, and subcloned into prokaryotic expression vector pET32a, then recombinant plasmids pET/Ag85B and pET/MPT64 were constructed. The recombinant plasmids were expressed in E.cli BL21, and its expressive products were purified. BABL/c mice were immunizated with the purified proteins.
3. Expression of the DNA vaccines in COS-7 cell lines.
COS-7 cell lines were transfected with plasmid vectors using cationic liposome respectively. 48 hours later, the fusion protein, Ag85B and MPT64 proteins expressed in COS-7 cell lines were determined by RT-PCR, ELISA and dot blotting.
4. Immunogenicity of the DNA vaccines.
C57BL/6 mice were intramuscularly immunized with the saline,
plasmid vector, pcDNA/Ag85B, pcDNA/MPT64, pcDNA/AM and pcDNA/BM respectively. The specific antibody levels were determined by ELISA, and spleen lymphocyte proliferation and IFN-γ levels in response to antigen restimulation was measured by MTT and ELISA in vaccinated mice respectively.
5. The evaluation of the protective effects of DNA vaccines against M. tuberculosis.
C57BL/6 mice were intramuscularly immunized with the DNA vaccines or BCG (i.d.). The mice were challenged with 106CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-γ,IL-4 and the survival time in all of the mice were evaluated .
Results
1.Recombinant pcDNA/Ag85B, pcDNA/MPT64, pcDNA/AM, pcDNA/BM were constructed and the inserted target genes were confirmed by restriction enzyme analysis and DNA sequencing. The fusion gene was sequenced, the mutation rate was 0.11%(2/1707) and mutation was nonsense.
2.The pET/Ag85B and pET/MPT64 were expressed in E.coli BL21, and its expressive products were analyzed by SDS-PAGE and identified by Western-blot. Antibody titer of anti-Ag85B and anti-MPT64 was 1:64 and 1:32 respectively.
3.The supernatant of COS-7 cell cultures transfected with pcDNA/AM showed positive reaction to both Ag85B antibody and MPT64 antibody by ELISA.
4.The antibody titer against PPD, antigen-specific lymphocyte proliferation and IFN-γproduction in Ag85B, MPT64 and AM DNA vaccine groups were higher than that of other groups (p<0.05).
5.The sera from immunized C57BL/6 mice were examined for PPD antibody by ELISA at 6 weeks after challenge with M.tuberculosis H37Rv. Antibody titer of pcDNA/Ag85B+pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (p<0.05). Spleen lymphocytes from immunized mice were restimulated in vitro with PPD.The level of IFN-γproduced by spleen lymphoc
本研究拟从结核分枝杆菌H37Rv基因组中扩增出ag85b、mpt64以及引入甘氨酸接头连接的Ag85B-MPT64融合基因（AM），构建DNA疫苗并探讨其在小鼠体内诱导的免疫应答和在小鼠结核感染模型中的保护作用，为新型结核病疫苗的研制奠定基础，同时也为结核病的免疫防治提供理论和实验依据。
方法：
1、用PCR和基因克隆技术从结核分枝杆菌H37Rv基因组中扩增出Ag85B、MPT64编码基因以及通过甘氨酸接头连接的Ag85B-MPT64融合基因（AM），构建真核表达质粒pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM，用酶切和双向DNA测序进行鉴定；
2、将MPT64、Ag85B编码基因亚克隆入pET32a中，构建pET/Ag85B和pET/MPT64原核表达质粒，使其在大肠杆菌中表达并鉴定。用重组的MPT64和Ag85B蛋白免疫BABL/c小鼠， 制备MPT64
与Ag85B抗血清。
3、将pcDNA3.1(+)、pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM转染COS-7细胞，用RT-PCR、ELISA和斑点印迹的方法鉴定融合基因的表达。
4、用质粒pcDNA3.1(+)、pcDNA/Ag85B、pcDNA/MPT64、pcDNA/AM和pcDNA/BM（经过限制性内切酶消化后粘粘连接的Ag85B-MPT64融合基因）免疫C57BL/6小鼠，采用 ELISA法测量免疫后相应的PPD特异性抗体和脾淋巴细胞IL-4 以及IFN-γ分泌水平；MTT法检测PPD特异性脾淋巴细胞增殖能力。
5、以重组质粒免疫C57BL/6小鼠，末次免疫5周, BCG免疫100天后，用经过动物体内恢复毒力的1×106CFU MTb.H37Rv标准株由尾静脉注射攻击，以PBS组和空质粒组作阴性对照、BCG组作阳性对照；6周后分批处死动物，测量PPD特异性抗体、PPD特异性脾淋巴细胞IL-4和IFN-γ分泌水平；观察肺和脾组织器官荷菌量、组织抗酸染色、病理学改变以及小鼠存活时间。
结果：
1、经过限制性内切酶酶切及DNA双向测序证实 pcDNA/MPT64、pcDNA/Ag85B序列与理论值一致，pcDNA/AM碱基突变率为0.11%（2/1707），突变为无意义突变。
2、将MPT64、Ag85B编码基因亚克隆入pET32a原核表达质粒中，构建的pET/ MPT64和pET/Ag85B在大肠杆菌中表达了具有生物学活性的重组MPT64和Ag85B蛋白。用重组蛋白制备的鼠抗
MPT64与Ag85B血清效价分别为1:32和1:64。
3、重组质粒转染COS-7细胞后，分别用MPT64、Ag85B多克隆抗体通过ELISA法检测细胞培养上清液，抗MPT64与pcDNA/MPT64、抗Ag85B与pcDNA/Ag85B呈阳性反应，pcDNA/AM组与抗MPT64和抗Ag85B均呈阳性反应；对照组均为阴性反应。
4、将PBS、pcDNA3.1、pcDNA/Ag85B、pcDNA/MPT64、pcDNA/AM和pcDNA/BM免疫C57BL/6小鼠，末次免疫4周后，实验组PPD特异性抗体水平均有不同程度增高，以pcDNA/Ag85B+pcDNA/MPT64、pcDNA/AM组最高（P<0.05）；脾淋巴细胞经过PPD刺激后， IFN-γ分泌水平以及脾淋巴细胞增殖能力pcDNA/Ag85B+pcDNA/MPT64、pcDNA/AM组和pcDNA/BM组明显高于其他组（P<0.05），pcDNA/AM组高于pcDNA/BM组（P<0.05），pcDNA/Ag85B+pcDNA/MPT64组与pcDNA/AM组之间差异无显著性（P>0.05）。
5、用H37Rv标准株攻击小鼠后6周，BCG、pcDNA/Ag85B、pcDNA/MPT64、pcDNA/Ag85B+pcDNA/MPT64、pcDNA/BM和pcDNA/AM组IFN-γ分泌水平以及脾淋巴细胞增殖能力均有不同程度增高，以BCG组最高，pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组次之，pcDNA3.1和PBS组最低，但pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组的差异无显著性（P>0.05）。各组均未检测到IL-4的分泌。
6、肺、脾荷菌量以BCG组最低（P<0.01），混合质粒组和AM免疫
组次之；但混合质粒组和AM免疫组之间差异无显著性（P>0.05），AM与BM DNA免疫组之间有显著性差异（P<0.05）。
7、用H37Rv标准株攻击小鼠后6周，肺组织病理改变：阴性对照组的改变，主要为大量的浆液纤维素性和少量的淋巴细胞渗出以及不同程度的肺组织坏死；阳性对照组主要表现为大量的淋巴细胞、组织细胞和类上皮细胞浸润以及结核肉芽肿的形成；以混合质粒组和AM免疫组病变和阳性对照组最为接近，其他各组介于BM组和阴性对照之间。脾组织的病理改变主要是炎性浸润和淋巴细胞的增生，阴性对照组主要表现为炎性浸润，阳性对照组主要表现为淋巴细胞的增生，混合质粒组和AM免疫组病变与BCG组相似，其余各组介于BM与阴性对照组之间。
8、小鼠存活时间以BCG组最长，混合质粒组和pcDNA/AM组次之，pcDNA3.1和PBS组最短。
结论：
1、pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM DNA疫苗构建成功；pET/Ag85B、pET/MPT64能在大肠杆菌中表达，并分离纯化出rAg85B、rMPT64重组蛋白。
2、pcDNA/Ag85B、pcDNA/MPT64和pcDNA/AM能在COS-7细胞中分泌表达，且表达的蛋白具有免疫反应性。
3、本研究构建的DNA疫苗均可诱导小鼠产生特异性体液免疫和细胞免疫，其诱导能力以pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组最强，而pcDNA/AM组优于pcDNA/BM组。
4、免疫小鼠经H37Rv标准株攻击后，PPD特异性抗体以pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组最高； IFN-γ分泌水平以及脾淋巴细胞增殖能力以BCG、pcDNA/Ag85B+pcDNA/MPT64和pcDNA/AM组最高。提示DNA疫苗接种后可以通过提高特异性体液免疫和细胞免疫水平发挥抗感染作用。
5、组
Objective
The study aimed to construct the fusion gene DNA vaccine encoding Ag85B and MPT64 linked with (Gly4Ser)3 linker, amplified from M.tuberculosis H37Rv chromosomal DNA, and, to explore the immune responses and protective efficacy of the DNA vaccine in mice infected with MTb. It would lay a foundation for a new tuberculosis vaccine and provide data for immunoprophylaxis and immunotherapy.
Methods
1. Construction of eukaryotic expression plasmids
The genes encoding Ag85B(ag85b), MPT64(mpt64) and AM[Ag85B and MPT64 fusion gene linked with (Gly4Ser)3 linker] were obtained from M.tuberculosis H37Rv chromosomal DNA by PCR
amplification,with primers designed to generate HindIII and BamHI restriction sites at the 5'and 3'ends of the amplified fragments respectively. The genes were cloned into unique HindIII and BamHI sites of the pcDNA3.1(+).The recombinant plasmids were identified with DNA sequence and endonucleases.
2. Expression and purification of Ag85B and MPT64 recombinant proteins in E.coli BL21, and fabrication of anti-Ag85B and anti- MPT64 antibodies.
The ag85b and mpt64 gene fragements were isolated from the pcDNA/Ag85B and pcDNA/MPT64 by restriction endonucleases, and subcloned into prokaryotic expression vector pET32a, then recombinant plasmids pET/Ag85B and pET/MPT64 were constructed. The recombinant plasmids were expressed in E.cli BL21, and its expressive products were purified. BABL/c mice were immunizated with the purified proteins.
3. Expression of the DNA vaccines in COS-7 cell lines.
COS-7 cell lines were transfected with plasmid vectors using cationic liposome respectively. 48 hours later, the fusion protein, Ag85B and MPT64 proteins expressed in COS-7 cell lines were determined by RT-PCR, ELISA and dot blotting.
4. Immunogenicity of the DNA vaccines.
C57BL/6 mice were intramuscularly immunized with the saline,
plasmid vector, pcDNA/Ag85B, pcDNA/MPT64, pcDNA/AM and pcDNA/BM respectively. The specific antibody levels were determined by ELISA, and spleen lymphocyte proliferation and IFN-γ levels in response to antigen restimulation was measured by MTT and ELISA in vaccinated mice respectively.
5. The evaluation of the protective effects of DNA vaccines against M. tuberculosis.
C57BL/6 mice were intramuscularly immunized with the DNA vaccines or BCG (i.d.). The mice were challenged with 106CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-γ,IL-4 and the survival time in all of the mice were evaluated .
Results
1.Recombinant pcDNA/Ag85B, pcDNA/MPT64, pcDNA/AM, pcDNA/BM were constructed and the inserted target genes were confirmed by restriction enzyme analysis and DNA sequencing. The fusion gene was sequenced, the mutation rate was 0.11%(2/1707) and mutation was nonsense.
2.The pET/Ag85B and pET/MPT64 were expressed in E.coli BL21, and its expressive products were analyzed by SDS-PAGE and identified by Western-blot. Antibody titer of anti-Ag85B and anti-MPT64 was 1:64 and 1:32 respectively.
3.The supernatant of COS-7 cell cultures transfected with pcDNA/AM showed positive reaction to both Ag85B antibody and MPT64 antibody by ELISA.
4.The antibody titer against PPD, antigen-specific lymphocyte proliferation and IFN-γproduction in Ag85B, MPT64 and AM DNA vaccine groups were higher than that of other groups (p<0.05).
5.The sera from immunized C57BL/6 mice were examined for PPD antibody by ELISA at 6 weeks after challenge with M.tuberculosis H37Rv. Antibody titer of pcDNA/Ag85B+pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (p<0.05). Spleen lymphocytes from immunized mice were restimulated in vitro with PPD.The level of IFN-γproduced by spleen lymphoc
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14. Tang D, Devit M, Jonston SA, et al. Genetic immunization is a simple method for eliciting an immune response. Nature, 1992,356:252-154
Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection
15. against influenza by injection of DNA encoding e viral protein. Science, 1993; 259: 1745-1749.
16. Lowrie DE, Tascon RE, Colston Md, et al. Towards a DNA vaccine against tuberculosis. Vaccine, 1994; 12(i6): 1537-1540.
17. Silva C L & Lowrie D B. A single mycobacterial protein (hsp 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis. Immunology, 1994; 82:244-248.
18. Lowrie D B, Silva CL, Colston MJ, et al, Protection against tuberculosis by a plasmid DNA Vaccine. Vaccine, 1997: 15 (8): 834-838.
19. Bonato V L, Lima V M, Tascon R E, et al. Identification and characterization of protective T cell In hsD65 DNA-Vaccined and M. tuberculosis-infected mice. Infect Immun, 1998: 66(1): 168-175.
20. Huygen K, Content J, Denis O, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med, 1996; 2(8): 857-859.
21. Wiker HG, Nagai S, Harboe M, et al. A family of cross-reacting proteins secreted by Mycobacterium tuberculosis. Scand J Immunol. 1992; 36(2): 307-319
22. Li H, Ulstrup JC, Jonassen TO, et al. Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG. Infect Immun. 1993; 61(5): 1730-1734
23. Haga S, Yamaguchi R, Nagai S, et al. Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG. J Leukoc Biol 1995; 57(2): 221-225
24. Kamath AT, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect immun, 1999; 67:1702-1707.
25. Delogu 0, Howard A, Collins F M, et al. DNA Vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial Immunity. Infect Immun, 2000; 68(6): 3097-3102.
26. Kamath AT, Hanke T, Briscoe H, et al. Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis. Immunology, 1999;96(4):511-516
27. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosisfrom the complete genome sequence. Nature, 1998; 393:537-544.
28. Montgomery DL. Tuberculosis vaccine design: influence of the completed genome sequence. Brief Bioinform 2000; 1(3): 289-296
Li Z, Howard A, Kelley C, et al. Immunogenicity of DNA vaccines
29. expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect Immun, 1999; 67(9): 4780-4786
30. Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tuberculosis. Vaccine 2000; 18 (20): 2155-2163
31. Huston JS, Mccarteny J, Tal ME, et al. Medical application of single chain antibodies. Intern Rev Immuno, 1993,10:195.
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4. Doherty TM, Andersen P. Tuberculosis vaccines: developmental work and the future. Curr Opin Pulm Med 2000; 6(3): 203-208
5. Enserink M. Driving a stake into resurgent TB. Science, 2001; 293 (5528): 234-235
6. von Reyn CF, Vuola JM.New vaccines for the prevention of tuberculosis.Clin Infect Dis 2002 ;35(4):465-474
7. Martin E, Triccas JA, Kamath AT, et al. Comparative protective effects of recombinant DNA and Mycobacterium bovis bacille Calmette-Guerin vaccines against M. avium infection. Clin Exp Immunol 2001; 126 (3): 482-487
8. McShane H, Brookes R, Gilbert SC, et al. Enhanced immunogenicity of CD4 (+) t-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis. Infect Immun .2001; 69(2): 681-686
9. Jackson M, Phalen SW, Lagrandenrie M, et al. Presistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect Immun, 1999,67:2867-2873
10. Harth G, Lee BY, Horwitz MA. High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect Immune, 1997; 65(6): 2321-2328
11. Wards BJ, De Lisle GW, Collin DM.An ESAT-6 knockout mutant of Mycobacterium bovis produced by homologous recombination will contribute to the development of a live tuberculosis vaccine. Tuber Lung Dis, 2000,80:185-189
12. Wolf JA, Malone RW, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science, 1990,247: 1465-1468
13. Williams RS, Johnston SA, Riedy M, et al. Introduction of foreign genes into tissues of living mice by DNA-coated microprojectiles. Proc Natl Acad Sci USA, 1991,88(7): 2726-2730
14. Tang D, Devit M, Jonston SA, et al. Genetic immunization is a simple method for eliciting an immune response. Nature, 1992,356:252-154
Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection
15. against influenza by injection of DNA encoding e viral protein. Science, 1993; 259: 1745-1749.
16. Lowrie DE, Tascon RE, Colston Md, et al. Towards a DNA vaccine against tuberculosis. Vaccine, 1994; 12(i6): 1537-1540.
17. Silva C L & Lowrie D B. A single mycobacterial protein (hsp 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis. Immunology, 1994; 82:244-248.
18. Lowrie D B, Silva CL, Colston MJ, et al, Protection against tuberculosis by a plasmid DNA Vaccine. Vaccine, 1997: 15 (8): 834-838.
19. Bonato V L, Lima V M, Tascon R E, et al. Identification and characterization of protective T cell In hsD65 DNA-Vaccined and M. tuberculosis-infected mice. Infect Immun, 1998: 66(1): 168-175.
20. Huygen K, Content J, Denis O, et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med, 1996; 2(8): 857-859.
21. Wiker HG, Nagai S, Harboe M, et al. A family of cross-reacting proteins secreted by Mycobacterium tuberculosis. Scand J Immunol. 1992; 36(2): 307-319
22. Li H, Ulstrup JC, Jonassen TO, et al. Evidence for absence of the MPB64 gene in some substrains of Mycobacterium bovis BCG. Infect Immun. 1993; 61(5): 1730-1734
23. Haga S, Yamaguchi R, Nagai S, et al. Delayed-type hypersensitivity to a recombinant mycobacterial antigen, MPB64, in guinea pigs sensitized to Mycobacterium tuberculosis or Mycobacterium bovis BCG. J Leukoc Biol 1995; 57(2): 221-225
24. Kamath AT, Feng CG, Macdonald M, et al. Differential protective efficacy of DNA vaccines expressing secreted proteins of Mycobacterium tuberculosis. Infect immun, 1999; 67:1702-1707.
25. Delogu 0, Howard A, Collins F M, et al. DNA Vaccination against tuberculosis: expression of a ubiquitin-conjugated tuberculosis protein enhances antimycobacterial Immunity. Infect Immun, 2000; 68(6): 3097-3102.
26. Kamath AT, Hanke T, Briscoe H, et al. Co-immunization with DNA vaccines expressing granulocyte-macrophage colony-stimulating factor and mycobacterial secreted proteins enhances T-cell immunity, but not protective efficacy against Mycobacterium tuberculosis. Immunology, 1999;96(4):511-516
27. Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosisfrom the complete genome sequence. Nature, 1998; 393:537-544.
28. Montgomery DL. Tuberculosis vaccine design: influence of the completed genome sequence. Brief Bioinform 2000; 1(3): 289-296
Li Z, Howard A, Kelley C, et al. Immunogenicity of DNA vaccines
29. expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect Immun, 1999; 67(9): 4780-4786
30. Morris S, Kelley C, Howard A, et al. The immunogenicity of single and combination DNA vaccines against tuberculosis. Vaccine 2000; 18 (20): 2155-2163
31. Huston JS, Mccarteny J, Tal ME, et al. Medical application of single chain antibodies. Intern Rev Immuno, 1993,10:195.