中国流行株HIV-1gag和hIL-2／hIL-6核酸疫苗构建与实验免疫研究
作者：秦云龙 金宁一 罗坤 李凡 王宏伟 王莉馨 张永生 殷震4sh, http://www.100md.com
单位：秦云龙 罗坤 李凡 张永生(白求恩医科大学病原生物学教研室，吉林 长春 130021)；金宁一 王宏伟 王莉馨 殷震(解放军军需大学研究所病毒室，吉林 长春 130062)4sh, http://www.100md.com
关键词：HIV-1；Gag蛋白；白细胞介素-2；白细胞介素-64sh, http://www.100md.com
免疫学杂志010102 摘 要：目的 探讨中国流行株HIV-1gag与hIL-2/hIL-6共表达重组核酸疫苗质粒的免疫效果。方法 以核酸疫苗质粒pIRES1neo为表达载体，构建重组核酸疫苗质粒pIRES1-gag、pIRES1-gag-hIL-2、pIRES1-gag-hIL-6，通过间接免疫荧光试验、Dot-ELISA检测gag/hIL-2/hIL-6基因的表达产物。另将此重组核酸疫苗质粒免疫Balb/c小鼠，进行淋巴细胞转化试验、CD4+、CD8+T淋巴细胞数量测定、细胞毒性T淋巴细胞(CTL)特异性杀伤作用检测及血清抗体检测，结果 构建的重组质粒转染BHK细胞后可表达目的基因，免疫小鼠后可有效地刺激淋巴细胞增殖、诱导特异性CTL反应，当和hIL-2/hIL-6共表达时免疫效果更加显著。讨论 与Gag蛋白共表达的hIL-2/hIL-6能够进一步增强免疫鼠的细胞免疫与体液免疫水平，构建的重组质粒为HIV-1DNA疫苗的研究提供了一定实验基础。4sh, http://www.100md.com
分类号：R392.7 文献标识码：A4sh, http://www.100md.com
文章编号：1000-8861(2001)01-0005-044sh, http://www.100md.com
Construction of DNA vaccine with HIV-1 gag and hIL-2/hIL-6 and its immunological study4sh, http://www.100md.com
QIN Yun-long(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)4sh, http://www.100md.com
JIN Ning-yi(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)
LUO Kun(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)fe]*f, 百拇医药
LI Fan(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)fe]*f, 百拇医药
WANG Hong-wei(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)fe]*f, 百拇医药
WANG Li-xin(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)fe]*f, 百拇医药
ZHANG Yong-sheng(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)fe]*f, 百拇医药
YIN Zhen(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)fe]*f, 百拇医药
Abstract：Objective To study the immunological effects of recombinant HIV-1 DNA vaccine plasmids in mice.Methods Two DNA vaccine recombinant plasmids pIRES1-gag-hIL-2，pIRES1-gag-hIL-6 were constructed by inserting the HIV-1 gag and hIL-2/hIL-6 gene into a DNA vaccine expression vector(pIRES1neo). The expressed product of gag/hIL-2/hIL-6 gene was examined by Dot-ELISA and indirect immunofluorescent assay. Immune response of Balb/c mice immunized by the two DNA vaccine recombinant plasmids was examined by lymphocyte transformation test(LTT)，cytotoxic T lymphocyte specific killer test，quantity change of T lymphocyte (CD4+， CD8+) and dynamics of specific serum IgG antibody.Results High effective expression was found in BHK cells transfected with pIRES1-gag-hIL-2，pIRES1-gag-hIL-6. Recombinant plasmids could effectively induce the proliferation of effector lymphocytes and the generation of specific lytic activity in vitro. The effects were more significant when co-expressed with human interleukins gene. Conclusion Cytokine hIL-2/hIL-6 can enhance both humoral and cellular immunity in immunized mice. Constructed recombinant plasmids might provide a basis for the development of HIV-1 DNA vaccine.
Keywords：HIV-1；Gag protein；hIL-2/hIL-6\a, 百拇医药
基金项目：国家自然科学基金(3977066)和国家杰出青年基金(39770661)资助项目\a, 百拇医药
作者简介：秦云龙(1972-)，男，黑龙江双鸭山市人，助教，硕士，主要从事分子病毒学的研究。Tel：(0431)7981365；E-mail：QinYL 72＠263.net\a, 百拇医药
参考文献：\a, 百拇医药
[1]金宁一.HIV基因结构及其疫苗研究[J]. 中国病毒学， 1997，12(4)：285-294.\a, 百拇医药
[2]Hanke T， Neumann VC， Blanchard TJ， et al. Effective induction of HIV-specific CTL by multiepitope using gene gun in a combined vaccination regime [J]. Vaccine， 1999，17(6)：589-596.\a, 百拇医药
[3]Kim JJ， Tsai A， Nottingham LK，et al. Intracellular adhesion molecule-1 modulates beta-chemokines and directly co-stimulates T cells in vivo [J]. J Clin Invest， 1999，103(6)：869-877.\a, 百拇医药
[4]Ayyavoo V， Nagashunmugam T，Phung MT， et al. Construction of attenuated HIV-1 accessory gene immunization cassettes [J]. Vaccine，1999，16(19)：1 872-1 879.\a, 百拇医药
[5]Bagarazzi ML， Boyer JD， Ugen KE， et al. Safety and immunogenicity of HIV-1 DNA constructs in chimpanzees [J]. Vaccine，1998，16(19)：1 836-1 841.\a, 百拇医药
[6]Peet NM， Mckeating JA， Souza JB， et al. The effect of low-profile serine substitutions in the V3 loop of HIV-1 gp120 IIIB/LAT on the immunogenicity of the envelope protein [J]. Virology，1998，251(1)：59-70.\a, 百拇医药
[7]Ugen KE， Nylanl SB， Boyer JD， et al. DNA vaccination with HIV-1 expressing constructs elicits immune responses in human [J]. Vaccine，1998，16(19)：1 818-1 821.\a, 百拇医药
[8]Winchell JM， Routray S， Betts PW， et al. Mucosal and systemic antibody responses to a C4/V3 construct following DNA vaccination of rabbits via the Peyer’s patch [J]. J Infect Dis，1998，178(3)：850-853.\a, 百拇医药
收稿日期：2000年9月20日\a, 百拇医药
修稿日期：2000年10月25日\a, 百拇医药
出版日期：2001年1月15日(秦云龙 金宁一 罗坤 李凡 王宏伟 王莉馨 张永生 殷震)
单位：秦云龙 罗坤 李凡 张永生(白求恩医科大学病原生物学教研室，吉林 长春 130021)；金宁一 王宏伟 王莉馨 殷震(解放军军需大学研究所病毒室，吉林 长春 130062)4sh, http://www.100md.com
关键词：HIV-1；Gag蛋白；白细胞介素-2；白细胞介素-64sh, http://www.100md.com
免疫学杂志010102 摘 要：目的 探讨中国流行株HIV-1gag与hIL-2/hIL-6共表达重组核酸疫苗质粒的免疫效果。方法 以核酸疫苗质粒pIRES1neo为表达载体，构建重组核酸疫苗质粒pIRES1-gag、pIRES1-gag-hIL-2、pIRES1-gag-hIL-6，通过间接免疫荧光试验、Dot-ELISA检测gag/hIL-2/hIL-6基因的表达产物。另将此重组核酸疫苗质粒免疫Balb/c小鼠，进行淋巴细胞转化试验、CD4+、CD8+T淋巴细胞数量测定、细胞毒性T淋巴细胞(CTL)特异性杀伤作用检测及血清抗体检测，结果 构建的重组质粒转染BHK细胞后可表达目的基因，免疫小鼠后可有效地刺激淋巴细胞增殖、诱导特异性CTL反应，当和hIL-2/hIL-6共表达时免疫效果更加显著。讨论 与Gag蛋白共表达的hIL-2/hIL-6能够进一步增强免疫鼠的细胞免疫与体液免疫水平，构建的重组质粒为HIV-1DNA疫苗的研究提供了一定实验基础。4sh, http://www.100md.com
分类号：R392.7 文献标识码：A4sh, http://www.100md.com
文章编号：1000-8861(2001)01-0005-044sh, http://www.100md.com
Construction of DNA vaccine with HIV-1 gag and hIL-2/hIL-6 and its immunological study4sh, http://www.100md.com
QIN Yun-long(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)4sh, http://www.100md.com
JIN Ning-yi(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)
LUO Kun(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)fe]*f, 百拇医药
LI Fan(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)fe]*f, 百拇医药
WANG Hong-wei(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)fe]*f, 百拇医药
WANG Li-xin(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)fe]*f, 百拇医药
ZHANG Yong-sheng(Department of Microbiology, Norman Bethune University of Medical Sciences，Changchun 130021，China)fe]*f, 百拇医药
YIN Zhen(Viral Laboratory，the Quartermaster University of PLA，Changchun 130062，China)fe]*f, 百拇医药
Abstract：Objective To study the immunological effects of recombinant HIV-1 DNA vaccine plasmids in mice.Methods Two DNA vaccine recombinant plasmids pIRES1-gag-hIL-2，pIRES1-gag-hIL-6 were constructed by inserting the HIV-1 gag and hIL-2/hIL-6 gene into a DNA vaccine expression vector(pIRES1neo). The expressed product of gag/hIL-2/hIL-6 gene was examined by Dot-ELISA and indirect immunofluorescent assay. Immune response of Balb/c mice immunized by the two DNA vaccine recombinant plasmids was examined by lymphocyte transformation test(LTT)，cytotoxic T lymphocyte specific killer test，quantity change of T lymphocyte (CD4+， CD8+) and dynamics of specific serum IgG antibody.Results High effective expression was found in BHK cells transfected with pIRES1-gag-hIL-2，pIRES1-gag-hIL-6. Recombinant plasmids could effectively induce the proliferation of effector lymphocytes and the generation of specific lytic activity in vitro. The effects were more significant when co-expressed with human interleukins gene. Conclusion Cytokine hIL-2/hIL-6 can enhance both humoral and cellular immunity in immunized mice. Constructed recombinant plasmids might provide a basis for the development of HIV-1 DNA vaccine.
Keywords：HIV-1；Gag protein；hIL-2/hIL-6\a, 百拇医药
基金项目：国家自然科学基金(3977066)和国家杰出青年基金(39770661)资助项目\a, 百拇医药
作者简介：秦云龙(1972-)，男，黑龙江双鸭山市人，助教，硕士，主要从事分子病毒学的研究。Tel：(0431)7981365；E-mail：QinYL 72＠263.net\a, 百拇医药
参考文献：\a, 百拇医药
[1]金宁一.HIV基因结构及其疫苗研究[J]. 中国病毒学， 1997，12(4)：285-294.\a, 百拇医药
[2]Hanke T， Neumann VC， Blanchard TJ， et al. Effective induction of HIV-specific CTL by multiepitope using gene gun in a combined vaccination regime [J]. Vaccine， 1999，17(6)：589-596.\a, 百拇医药
[3]Kim JJ， Tsai A， Nottingham LK，et al. Intracellular adhesion molecule-1 modulates beta-chemokines and directly co-stimulates T cells in vivo [J]. J Clin Invest， 1999，103(6)：869-877.\a, 百拇医药
[4]Ayyavoo V， Nagashunmugam T，Phung MT， et al. Construction of attenuated HIV-1 accessory gene immunization cassettes [J]. Vaccine，1999，16(19)：1 872-1 879.\a, 百拇医药
[5]Bagarazzi ML， Boyer JD， Ugen KE， et al. Safety and immunogenicity of HIV-1 DNA constructs in chimpanzees [J]. Vaccine，1998，16(19)：1 836-1 841.\a, 百拇医药
[6]Peet NM， Mckeating JA， Souza JB， et al. The effect of low-profile serine substitutions in the V3 loop of HIV-1 gp120 IIIB/LAT on the immunogenicity of the envelope protein [J]. Virology，1998，251(1)：59-70.\a, 百拇医药
[7]Ugen KE， Nylanl SB， Boyer JD， et al. DNA vaccination with HIV-1 expressing constructs elicits immune responses in human [J]. Vaccine，1998，16(19)：1 818-1 821.\a, 百拇医药
[8]Winchell JM， Routray S， Betts PW， et al. Mucosal and systemic antibody responses to a C4/V3 construct following DNA vaccination of rabbits via the Peyer’s patch [J]. J Infect Dis，1998，178(3)：850-853.\a, 百拇医药
收稿日期：2000年9月20日\a, 百拇医药
修稿日期：2000年10月25日\a, 百拇医药
出版日期：2001年1月15日(秦云龙 金宁一 罗坤 李凡 王宏伟 王莉馨 张永生 殷震)