鸡传染性支气管炎免疫增强型核酸疫苗的构建与免疫保护机理的研究
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摘要
本研究首先对IBV S、IBV S1及ChIL-15基因进行修饰改造,并构建了三段修饰后基因的真核表达载体质粒。在此基础上,利用一段编码(G4S)3多肽的碱基linker将ChIL-15分别与IBV S和IBV S1基因进行连接,并成功构建了两个融合基因真核表达载体质粒,即pcDNA3.1-IBV S1-linker-ChIL-15及pcDNA3.1-IBV S-linker-ChIL-15质粒。两个融合基因质粒的体外瞬时转染试验表明,融合基因能够在细胞内进行表达,同时表达后的融合蛋白可以被ChIL-15单克隆抗体和IBV全病毒粒子多克隆抗体检测出来,进一步证明了融合基因表达的蛋白保留了融合前两段基因各自的生物活性,为后续动物免疫试验提供了理论基础。
在动物免疫试验中设计了7个处理组,即S组(免疫IBV S基因真核表达质粒)、S1组(免疫IBV S1基因真核表达质粒)、S+IL-15组(混合免疫IBV S基因与ChIL-15真核表达质粒)、S1+IL-15组(混合免疫IBV S1基因与ChIL-15真核表达质粒)、S-IL-15组(免疫IBV S与ChIL-15融合基因真核表达质粒)、S1-IL-15组(免疫IBV S1与ChIL-15融合基因真核表达质粒),以及1个空质粒对照组组(C组)。同时,利用淋巴细胞转化试验、酶联免疫吸附试验、实时荧光定量PCR技术及流式细胞仪技术对不同处理龄雏鸡的各项免疫指标进行了检测,最后应用病毒攻击试验进一步比较了不同质粒之间保护率的差别。
试验结果表明,6组免疫组各日龄多数免疫指标都高于C组,其中有ChIL-15基因参与的S+IL-15、S1+IL-15、S-IL-15及S1-IL-15四组免疫组免疫效果尤为突出。具体表现为:T、B淋巴细胞增殖功能,CD4+、CD8+、、TCRαβ+及TCRγδ+T细胞数量,外周血液中抗IBV特异性抗体水平以及免疫器官中IL-2 mRNA的表达量与C组相比较都有明显的提高,而且以S1-IL-15组最为显著。对S组及S1组比较时发现,S1组的最明显优势在于其可以较早地诱导机体产生细胞免疫及体液免疫应答。
攻毒后对各试验组IBV靶器官进行了病理组织学研究,同时还对不同免疫组之间的免疫保护率进行了比较,结果进一步证明了ChIL-15能够很好地提高机体的免疫保护能力,尤其是S1-IL-15抵御病毒感染的能力更为突出,这也表明在linker帮助下融合基因能够在机体内更好地发挥二者的生物学协同作用,而且ChIL-15能够更有效地展示其免疫增强作用的优势。
综上所述,通过本试验研究表明,ChIL-15基因在配合IBV抗原基因免疫过程中,不但能够促进机体的细胞免疫应答能力,而且能够提高机体体液免疫应答的能力,进而促进机体产生较强的免疫力以抵抗病毒的攻击。本试验首次构建的ChIL-15基因与病毒抗原基因融合基因质粒以及试验结果为进一步研究ChIL-15的生物活性提供了试验依据,同时为探讨开发研制新型免疫增强型抗病毒疫苗提供了理论基础。
In this research, IBV S, IBV S1 and ChIL-15 gene were modified and reconstructed firstly, then these genes were inserted into a eukaryotic expression vector, pcDNA3.1(+), to construct three new plasmids. Furthermore, a flexible linker which could express the (G4S)3 polypeptide was used to link the ChIL-15 gene with IBV S and S1 gene to construct two fusion gene plasmid of eukaryotic expression vetor-pcDNA3.1-IBV S1-linker-ChIL-15 and pcDNA3.1-IBV S-linker-ChIL-15. The results of the transient transfection of these two fusion plasmids in vitro showed that the fused gene could express together in Vero cells and could be located by specific anti-ChIL-15 monoclonal antibody and anti-IBV polyclonal antibody, therefore, it proved that the protein expressed by fusion gene could retain the bioactivities of two proteins, it also provided the theoretical foundation for the following immunization research in vivo.
The experimental chicken were divided into 7 groups, the S group inoculated with the pcDNA3.1-IBV S, S1 group inoculated with the pcDNA3.1-IBV S1, the S+IL-15 group inoculated with the pcDNA3.1-IBV S and pcDNA3.1-Ch IL-15, the S1+IL-15 group inoculated with the pcDNA3.1-IBV S1 and pcDNA3.1- Ch IL-15, the S1-IL-15 group inoculated wiht the pcDNA3.1-IBV S1-linker-ChIL-15, the S-IL-15 group inoculate with the pcDNA3.1-IBV S-linker-ChIL-15 and the control group with empty pcDNA3.1 vetor. Thereafter, lymphocyte proliferative assay, enzyme linkered immunosorbent assay, Real Time RT-PCR and flow cytometric technique were used to detect the immune response of different groups. Finally chickens in different treatment groups were challenged with IBV Beaudette strain to compare the difference of the protection rate.
The results showed that most of immune response of six inoculated groups are higher than the control group, especially, the S+IL-15, S1+IL-15, S-IL-15 and S1-IL-15 group which involved ChIL-15. The details were as follows: the T, B lymphocyte proliferative function; the number of CD4+, CD8+, TCRαβ+ and TCRγδ+T cell; the levels of special anti-IBV antibody in peripheral blood and IL-2 mRNA expression in thymus and spleen of immune groups were all higher than those of C group. And the S1-IL-15 group was the most significant among those groups. Meanwhile, when the S group was compared with the S1 group, the most obvious predominance of the S1 group was that S1 gene could induce cellular immunity and humoral immunity earlier than the S gene.
The histopathological change of IBV target organs of each group were analyzed after challenge and the immunoprotection rates were compared among every group. The results proved that ChIL-15 could enhance the immunoprotection of the host to resist the virus infection. It also showd that the fusion genes could well display their bioactivities, and ChIL-15 could effectively reveal its immunologic enhancement with the help of the“linker”.
Above all, the results of this experiment showed that when the antigen genes of IBV were inoculated with ChIL-15, it could improve the host immune response and higher protection rate could be observed against the IBV chanllenge. Furthermore, IBV antigen genes and ChIL-15 fusion plasmid was first constructed in this experiment and the results observe here could provide solid scientific ground to the further study of ChIL-15 bioactivities, and the theoretical basis to the dicovery of new type immune-enhancing vaccine to virual diseases.
在动物免疫试验中设计了7个处理组,即S组(免疫IBV S基因真核表达质粒)、S1组(免疫IBV S1基因真核表达质粒)、S+IL-15组(混合免疫IBV S基因与ChIL-15真核表达质粒)、S1+IL-15组(混合免疫IBV S1基因与ChIL-15真核表达质粒)、S-IL-15组(免疫IBV S与ChIL-15融合基因真核表达质粒)、S1-IL-15组(免疫IBV S1与ChIL-15融合基因真核表达质粒),以及1个空质粒对照组组(C组)。同时,利用淋巴细胞转化试验、酶联免疫吸附试验、实时荧光定量PCR技术及流式细胞仪技术对不同处理龄雏鸡的各项免疫指标进行了检测,最后应用病毒攻击试验进一步比较了不同质粒之间保护率的差别。
试验结果表明,6组免疫组各日龄多数免疫指标都高于C组,其中有ChIL-15基因参与的S+IL-15、S1+IL-15、S-IL-15及S1-IL-15四组免疫组免疫效果尤为突出。具体表现为:T、B淋巴细胞增殖功能,CD4+、CD8+、、TCRαβ+及TCRγδ+T细胞数量,外周血液中抗IBV特异性抗体水平以及免疫器官中IL-2 mRNA的表达量与C组相比较都有明显的提高,而且以S1-IL-15组最为显著。对S组及S1组比较时发现,S1组的最明显优势在于其可以较早地诱导机体产生细胞免疫及体液免疫应答。
攻毒后对各试验组IBV靶器官进行了病理组织学研究,同时还对不同免疫组之间的免疫保护率进行了比较,结果进一步证明了ChIL-15能够很好地提高机体的免疫保护能力,尤其是S1-IL-15抵御病毒感染的能力更为突出,这也表明在linker帮助下融合基因能够在机体内更好地发挥二者的生物学协同作用,而且ChIL-15能够更有效地展示其免疫增强作用的优势。
综上所述,通过本试验研究表明,ChIL-15基因在配合IBV抗原基因免疫过程中,不但能够促进机体的细胞免疫应答能力,而且能够提高机体体液免疫应答的能力,进而促进机体产生较强的免疫力以抵抗病毒的攻击。本试验首次构建的ChIL-15基因与病毒抗原基因融合基因质粒以及试验结果为进一步研究ChIL-15的生物活性提供了试验依据,同时为探讨开发研制新型免疫增强型抗病毒疫苗提供了理论基础。
In this research, IBV S, IBV S1 and ChIL-15 gene were modified and reconstructed firstly, then these genes were inserted into a eukaryotic expression vector, pcDNA3.1(+), to construct three new plasmids. Furthermore, a flexible linker which could express the (G4S)3 polypeptide was used to link the ChIL-15 gene with IBV S and S1 gene to construct two fusion gene plasmid of eukaryotic expression vetor-pcDNA3.1-IBV S1-linker-ChIL-15 and pcDNA3.1-IBV S-linker-ChIL-15. The results of the transient transfection of these two fusion plasmids in vitro showed that the fused gene could express together in Vero cells and could be located by specific anti-ChIL-15 monoclonal antibody and anti-IBV polyclonal antibody, therefore, it proved that the protein expressed by fusion gene could retain the bioactivities of two proteins, it also provided the theoretical foundation for the following immunization research in vivo.
The experimental chicken were divided into 7 groups, the S group inoculated with the pcDNA3.1-IBV S, S1 group inoculated with the pcDNA3.1-IBV S1, the S+IL-15 group inoculated with the pcDNA3.1-IBV S and pcDNA3.1-Ch IL-15, the S1+IL-15 group inoculated with the pcDNA3.1-IBV S1 and pcDNA3.1- Ch IL-15, the S1-IL-15 group inoculated wiht the pcDNA3.1-IBV S1-linker-ChIL-15, the S-IL-15 group inoculate with the pcDNA3.1-IBV S-linker-ChIL-15 and the control group with empty pcDNA3.1 vetor. Thereafter, lymphocyte proliferative assay, enzyme linkered immunosorbent assay, Real Time RT-PCR and flow cytometric technique were used to detect the immune response of different groups. Finally chickens in different treatment groups were challenged with IBV Beaudette strain to compare the difference of the protection rate.
The results showed that most of immune response of six inoculated groups are higher than the control group, especially, the S+IL-15, S1+IL-15, S-IL-15 and S1-IL-15 group which involved ChIL-15. The details were as follows: the T, B lymphocyte proliferative function; the number of CD4+, CD8+, TCRαβ+ and TCRγδ+T cell; the levels of special anti-IBV antibody in peripheral blood and IL-2 mRNA expression in thymus and spleen of immune groups were all higher than those of C group. And the S1-IL-15 group was the most significant among those groups. Meanwhile, when the S group was compared with the S1 group, the most obvious predominance of the S1 group was that S1 gene could induce cellular immunity and humoral immunity earlier than the S gene.
The histopathological change of IBV target organs of each group were analyzed after challenge and the immunoprotection rates were compared among every group. The results proved that ChIL-15 could enhance the immunoprotection of the host to resist the virus infection. It also showd that the fusion genes could well display their bioactivities, and ChIL-15 could effectively reveal its immunologic enhancement with the help of the“linker”.
Above all, the results of this experiment showed that when the antigen genes of IBV were inoculated with ChIL-15, it could improve the host immune response and higher protection rate could be observed against the IBV chanllenge. Furthermore, IBV antigen genes and ChIL-15 fusion plasmid was first constructed in this experiment and the results observe here could provide solid scientific ground to the further study of ChIL-15 bioactivities, and the theoretical basis to the dicovery of new type immune-enhancing vaccine to virual diseases.
引文
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