为研制更为安全有效的疫苗用于禽传染性支气管炎的防制。本研究选取IBV的M基因，通过PCR扩增在两端引入BamHⅠ酶切位点，经酶切插入“自杀性”DNA疫苗载体pSCA1 CMV IE启动子之下的克隆位点，构建了“自杀性”DNA疫苗表达质粒pSCA1-M。经由菌落PCR、酶切及测序鉴定目的基因被成功克隆至载体pSCA1，且方向正确。分别取等量的重组质粒pSCA1-M、常规IBV M基因DNA疫苗pcDNAM及空载体pSCA1转染Vero细胞。48h后经间接免疫荧光试验观察发现，pSCA1-M以及pcDNAM转染组细胞浆均可见特异性亮绿色荧光，pSCA1空载体组无荧光为阴性。这证实“自杀性”DNA疫苗载体能成功表达M基因。而pSCA1-M组所发荧光细胞数比pcDNAM组少，说明的“自杀性”DNA疫苗转染率低于常规DNA疫苗。
为评价IBV “自杀性”DNA疫苗的免疫效果，分别用构建的“自杀性”DNA疫苗pSCA1-M、常规DNA疫苗pcDNAM、空载体pSCA1和IBV灭活疫苗免疫非免疫健康雏鸡，并设对照组注射等量的PBS液。自免疫后每隔一周各组鸡只采血分离血清持续五周，采用流式细胞仪(FACS)和间接ELISA试验分别对宿主外周血T淋巴细胞亚类CD3+、CD4+、CD8+和特异性抗IBV血清IgG的动态变化进行检测，并在第五周后以IBV强毒株进行攻毒保护实验。间接ELISA试验检测结果表明，我们构建的“自杀性”DNA疫苗能刺激机体产生针对IBV的特异性血清抗体，免疫后第三周上升到峰值，随后开始下降，其抗体水平与常规IBV DNA疫苗相当，低于灭活疫苗。T淋巴细胞亚类动态变化结果显示，“自杀性”DNA疫苗pSCA1-M组CD3+、CD4+、CD8+各T淋巴细胞数量均高于其余各组，表现为差异显著（P＜0.01或P＜0.05）。但在第4周左右有一个明显的下降，之后又略有回升。这说明“自杀性”DNA疫苗能诱导机体产生高于常规DNA疫苗和灭活疫苗的细胞免疫，但随着宿主细胞的凋亡迅速下降。攻毒保护试验结果统计，pSCA1-M组鸡只发病率为12%，保护率为45%；pcDNAM组鸡只发病率为13%，保护率略低于pSCA1-M，为40%；空载体组发病率（75%）＜对照组（85%），攻毒保护率两组相同为25%；灭活苗组无鸡只发病死亡。对强毒的抵抗力单基因的“自杀性”DNA疫苗与常规DNA疫苗相当（40%）＜灭活苗（100%）。
本研究采用IBV M基因构建“自杀性”DNA疫苗对鸡进行免疫，并对其免疫效果进行评价，在国内外还未见报道。可为研制IBV “自杀性”DNA疫苗提供了基础材料和科学依据。
万分感谢！！！
The M gene is one of infectious bronchitis virus important immunity original genes, the M gene possibly has the important antigen decision bunch, also has highly the conservative nature in the evolution process. This research selects the M gene of IBV , on the foundation of researching the IBV structure protein gene, expands the PCR increases introduces the BamH I enzyme in the beginnings and ends to cut the position spot, cuts the insertion after the enzyme "the suicide" DNA vaccine carrier pSCA1 CMV the IE start under clone position spot, constructed "the suicide" the DNA vaccine to express material particle pSCA1-M. By the way of colony PCR, the enzyme cut and measured the foreword appraisal goal gene succeeds the clone to carrier pSCA1, also the direction is correct. Separately takes the isometric reorganization material particle pSCA1-M, conventional IBV M gene DNA vaccine pcDNAM and the spatial carrier pSCA1 extension dyes the Vero cell. After 48h, by the indirect immunity fluorescence experiment observation we discovery that pSCA1-M as well as the pcDNAM extension dyes the group 细胞浆 obviously specificity bright green fluorescence, the pSCA1 spatial carrier group does not have the fluorescence is the negative. This confirmed "the suicide" DNA vaccine carries can express the M gene successfully. But the pSCA1-M group institute luminescence cell number are less than the pcDNAM group which shows "the suicide" DNA vaccine extension dyeing rate to be lower than the conventional DNA vaccine.
In order to appraise IBV "the suicide" DNA vaccine immunity effect, we use constructly "the suicide" DNA vaccine pSCA1-M, conventional DNA vaccine pcDNAM, spatial carrier pSCA1 and the IBV 灭活 vaccine separately immunity non- immunity health young chicken, and supposes the comparison group to inject the isometric PBS fluid. Since every week after immunization group chickens blood serum separation sustained five weeks. Using FACS and indirect ELISA test regarding 宿主外周 T lymphocyte subpopulations CD 3 +, CD4 +, CD8 + and specificity of serum IgG anti-IBV dynamic change detection separately, after the fifth week take the IBV strains strong attacking drug protection experiment. The indirect ELISA experiment examination result indicated that, we construct "the suicide" DNA vaccine be able to stimulate the organism to produce in view of the IBV specificity blood serum immune body. The third week after the immunity rise the peak value, afterwards starts to drop, its immune body level quite to conventional IBV and the DNA vaccine, is lower than the 灭活 vaccine. The T lymphocyte subgroup dynamic change result showed that, "the suicide" DNA vaccine pSCA1-M group and CD3+, CD4+, CD8+ each T lymphocyte quantity is higher than others. It displays difference remarkable (P＜0.01 or P＜0.05). But it has an obvious drop about at the 4th week, and slightly afterwards. It indicated that "the suicide" DNA vaccine can induce the organism to produce higher cellular immunity than the conventional DNA vaccine and 灭活 vaccine , but rapid drop along with the death of 宿主 cell. The attacks poisonous protection test result statistics that, the pSCA1-M group chicken disease incidence's rate is 12%, the protection's rate is 50%; The pcDNAM group chicken disease incidence's rate is 13%, the protection's rate is 40%, which is slightly lower than pSCA1-M; The spatial carrier group disease incidence's rate (75%) ＜ the comparison group (85%), two groups of the attacks poisonous protection's rate are in the same as 25%; 灭活苗组无鸡 only arises the death. 对强毒的抵抗力单基因的“自杀性”DNA疫苗与常规DNA疫苗相当（40%）＜灭活苗（100%）。
This research uses the IBV M gene to construct "the suicide" DNA vaccine to immunity the chicken, and carries on the appraisal to its immunity effect. It has not been reported at home and in the world . It can provide the foundation material and the scientific basis for researching IBV "the suicide"DNA vaccine.
有句不会翻译~
In order to appraise IBV "the suicide" DNA vaccine immunity effect, we use constructly "the suicide" DNA vaccine pSCA1-M, conventional DNA vaccine pcDNAM, spatial carrier pSCA1 and the IBV 灭活 vaccine separately immunity non- immunity health young chicken, and supposes the comparison group to inject the isometric PBS fluid. Since every week after immunization group chickens blood serum separation sustained five weeks. Using FACS and indirect ELISA test regarding 宿主外周 T lymphocyte subpopulations CD 3 +, CD4 +, CD8 + and specificity of serum IgG anti-IBV dynamic change detection separately, after the fifth week take the IBV strains strong attacking drug protection experiment. The indirect ELISA experiment examination result indicated that, we construct "the suicide" DNA vaccine be able to stimulate the organism to produce in view of the IBV specificity blood serum immune body. The third week after the immunity rise the peak value, afterwards starts to drop, its immune body level quite to conventional IBV and the DNA vaccine, is lower than the 灭活 vaccine. The T lymphocyte subgroup dynamic change result showed that, "the suicide" DNA vaccine pSCA1-M group and CD3+, CD4+, CD8+ each T lymphocyte quantity is higher than others. It displays difference remarkable (P＜0.01 or P＜0.05). But it has an obvious drop about at the 4th week, and slightly afterwards. It indicated that "the suicide" DNA vaccine can induce the organism to produce higher cellular immunity than the conventional DNA vaccine and 灭活 vaccine , but rapid drop along with the death of 宿主 cell. The attacks poisonous protection test result statistics that, the pSCA1-M group chicken disease incidence's rate is 12%, the protection's rate is 50%; The pcDNAM group chicken disease incidence's rate is 13%, the protection's rate is 40%, which is slightly lower than pSCA1-M; The spatial carrier group disease incidence's rate (75%) ＜ the comparison group (85%), two groups of the attacks poisonous protection's rate are in the same as 25%; 灭活苗组无鸡 only arises the death. 对强毒的抵抗力单基因的“自杀性”DNA疫苗与常规DNA疫苗相当（40%）＜灭活苗（100%）。
This research uses the IBV M gene to construct "the suicide" DNA vaccine to immunity the chicken, and carries on the appraisal to its immunity effect. It has not been reported at home and in the world . It can provide the foundation material and the scientific basis for researching IBV "the suicide"DNA vaccine.
有句不会翻译~
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第1个回答 2007-04-26
In order to develop more effective and safe vaccine for the avian infectious bronchitis control. The selection of the M gene isolates by PCR in both the introduction of BamH Ⅰ, amplified digested by the words "suicide" DNA vaccine vector pSCA1 CMV IE promoter under the cloning site, Construction of the "suicide" expression plasmid DNA vaccine pSCA1-M. Colony by PCR, enzyme and gene sequencing has been successfully cloned pSCA1 vector and the right direction. Admit contour pSCA1-M the recombinant plasmid, IBV M conventional DNA vaccines pcDNAM and empty vector transfected Vero pSCA1. cells 48 hours after indirect immunofluorescence assay observed, pSCA1-M and pcDNAM transfected cells were found specific slurry bright green fluorescence, pSCA1 empty vector group fluorescent negative. This confirms "suicide" DNA vaccine vector successful M gene expression. PSCA1-M Group and the fluorescence of cells in less than pcDNAM group, Note the "suicide" DNA vaccine transfection rate lower than conventional DNA vaccines. IBV for the evaluation of the "suicide" DNA Vaccine, were used to construct the "suicide" DNA vaccine pSCA1-M conventional DNA vaccines pcDNAM. empty vector pSCA1 IBV and non-inactivated vaccine immune health chicks, and the control group injected contour of PBS. Since every week after immunization group chickens blood serum separation sustained five weeks. using flow cytometry (FACS) and indirect ELISA test were to host T lymphocyte subpopulations CD 3 +, CD4 +, CD8 + and specificity of serum IgG anti-IBV dynamic change detection, in the fifth week after IBV strains strong attacking drug protection experiment. Indirect ELISA test results show that We construct the "suicide" DNA vaccines can stimulate the body to produce against IBV specific serum antibodies, Immunity after the third week rose to the peak and then begin to decline, the level of antibodies and conventional DNA vaccines quite IBV, lower than the inactivated vaccine. T lymphocyte subpopulations dynamic change results showed that the "suicide" DNA vaccine pSCA1-M group CD3 +, CD4 + and CD8 + T lymphocytes of the volume is higher than the other groups, showed significant differences (P <0.01 or P<0.05). However, in about 4 weeks a significant decrease, followed by a slight rebound. This shows that "suicide" DNA vaccine can induce higher than conventional DNA vaccines and vaccine immune cells. But with the host cell apoptosis rapidly declining. The attack protect the test results statistics, pSCA1-M Group chickens incidence rate of 12%, the protection rate of 45% pcDNAM Group chickens incidence rate of 13%, slightly lower than the rate of protection pSCA1-M to 40% Air carrier group incidence rate (75%) <control group (85%), infection rates of the two groups the same protection for 25% Vaccine group of chickens died of infection. Right-drug resistance genes "suicide" DNA vaccine with conventional DNA vaccines very (40%) <Vaccine (100%). This study IBV M gene construct "suicide" DNA vaccine immunization for chicken, and their immune effects evaluation at home and abroad, has not been reported. IBV for the development of "suicide" DNA vaccines provide basic materials and scientific basis.
