呼吸道合胞病毒G蛋白候选疫苗对小鼠免疫功能的影响
发布时间：2019-06-17 14:35
【摘要】：呼吸道合胞病毒(Respiratory syncytial virus, RSV)是一种能导致婴幼儿下呼吸道感染的主要病原体。感染后出现急性支气管炎、肺炎、严重者甚至死亡。截止目前还没有一种安全有效的疫苗得到应用。本研究通过将截短的RSV G 130-230位氨基酸(包含有CX3C模序)与GCTL(用CTL表位替换了CX3C)或辅以黏膜佐剂后免疫Balb/c小鼠,来分析和评价疫苗对小鼠免疫功能的影响。 Gcx3c蛋白和GCTL蛋白在大肠杆菌中表达。表达的蛋白,应用His-Tag与Co2+的亲和力对蛋白进行纯化。将纯化后的Gcx3c蛋白、GCTL蛋白分别与无毒型大肠杆菌不耐热肠毒素(Heat-labil enterotoxin, LT)混合后免疫Balb/c小鼠,设立Gcx3c组；GCTL组；GCTL组；GCTL+LT组；LT组；PBS组,共6组,分别在0,1,4周免疫。免疫结束后,针对血清中的IgG、IgA、IgG2a/IgG1、呼吸道sIgA、组胺、嗜酸性粒细胞以及淋巴细胞体外增殖、CTL应答效应等免疫指标分别通过ELISA、HE染色以及MTT染色法等进行测定。用107 pfu/ml RSV滴鼻攻击免疫后的小鼠,连续3天。攻毒结束后处死小鼠,计数嗜酸性粒细胞,检测组胺水平,并制备组织切片。结果使用SPSS16.0软件进行分析。 Gcx3c蛋白和GCTL蛋白在大肠杆菌中以包涵体的形式大量表达,在37℃下诱导的包涵体占到总蛋白的40%以上。应用His-tag与金属离子的亲和力纯化后纯度达到95%以上,Western blotting结果证明所表达的是目的蛋白。 免疫检测结果表明：血清IgG在第二次免疫后,GCTL、GCTL+LT组抗体滴度显著高于Gcx3c+LT及Gcx3c(P0.05),且以上四组均高于LT及PBS组(P0.001)：血清IgA在第二次免疫后,GCTL、GCTL+LT组抗体滴度显著高于Gcx3c及Gcx3c+LT(P0.05),且以上四组均高于LT及PBS组(P0.05)；而在sIgA中,加了LT佐剂的两个实验组的sIgA抗体滴度显著高于Gcx3c、GCTL、LT、PBS组(P0.05)；血清IgG2a/IgG1中GCTL组平衡效果优于GCTL+LT、GCX3C+LT、GCX3C组(P0.05),GCTL组在用CTL表位替换了CX3C模序后,Th1/Th2趋于平衡,并且还发现加入了佐剂LT后能发挥一个协调Th1/Th2的生理作用,有调节免疫反应极化的情况；淋巴细胞体外增殖实验中,抗原刺激后,只有相应的Gcx3c和GCTL蛋白组细胞出现了增殖,对照组和佐剂组都没有增殖,且蛋白组细胞增殖量显著高于对照组(P0.05)；比较Gcx3C、GcTL组以及PBS组的组胺,GCX3C组胺含量明显高于其他两组(P0.05),GcTL与PBS组间无显著差异(P0.05)；Gcx3c+LT组的嗜酸性粒细胞数量高于GCTL、GCTL+LT、PBS组(P0.05),而后三组之间数量无显著差异(P0.05)；组织切片结果也显示,GCTL蛋白组小鼠肺部没有出现炎症反应,而GCX3C蛋白组则有明显的炎症反应；RSV特异性CTL应答结果表明,GCTL组诱导了显著的RSV特异性CTL杀伤活性,而GCX3C在针对病毒时也存在一定程度的杀伤。 攻毒检测结果表明：攻毒后GCTL组嗜酸性粒细胞数量显著低于GCX3C组、PBS组和LT组(P0.05)；GCX3C组的组胺含量显著高于GCTL组(P0.05)；肺表和组织切片结果显示,GCTL蛋白组小鼠肺没有明显病变,而GCX3C组、PBS组和LT组则出现了严重的病变。 本实验成功的表达纯化了GCX3C、GCTL和LT蛋白,并就疫苗对小鼠免疫功能的影响和攻毒后的保护能力进行了测定。实验结果显示,混以LT佐剂后,提高了黏膜部的sIgA的量。针对GCX3C进行的改造是成功有效的,GCTL增强了对RSV特异性的CTL应答反应,并且有效的降低了呼吸道组胺和嗜酸性粒细胞的数量,避免了再次感染的过激反应。攻毒后的部分实验结果也从一个侧面体现了GCTL在保护性方面发挥了应有的作用。呼吸道合胞病毒G蛋白亚单位疫苗的小鼠实验,为进一步的研究RSV致病机理及RSV疫苗的研发奠定了基础。
[Abstract]:Respiratory syncytial virus (RSV) is a major pathogen that can lead to lower respiratory tract infection in infants. Acute bronchitis, pneumonia, and even death after infection. The application of a safe and effective vaccine is not yet available. The effects of the vaccine on the immune function of the mice were analyzed and evaluated by comparing the truncated RSV G 130-230 amino acids (including the CX3C mold sequence) with the GCTL (replacing the CX3C with the CTL epitope) or by immunizing the Balb/ c mice with a mucosal adjuvant. Gcx3c protein and GCTL protein in Escherichia coli D. expressed protein, and the affinity of His-Tag and Co2 + is used for the purification of the protein. The purified Gcx3c protein and the GCTL protein were mixed with the non-toxic E. coli heat-labile enterotoxin (LT) to immunize the Balb/ c mice to set up the Gcx3c group, the GCTL group, the GCTL group, the GCTL + LT group, the LT group, the PBS group, the total 6 groups, respectively, at 0,1 and 4 weeks. The immune indexes such as IgG, IgA, IgG2a/ IgG1, sIgA, histamine, eosinophils and lymphocytes in the serum were measured by ELISA, HE staining and MTT staining. Determination. Mice immunized with 107 pfu/ ml RSV nasal attack, continuous 3 d. The mice were sacrificed after the challenge, the eosinophils were counted, the histamine level was detected, and the tissue was prepared The results are separated using the SPSS16.0 software The Gcx3c protein and the GCTL protein were expressed in the form of inclusion bodies in E. coli, and the inclusion bodies induced at 37 鈩，
本文编号：2501057
[Abstract]:Respiratory syncytial virus (RSV) is a major pathogen that can lead to lower respiratory tract infection in infants. Acute bronchitis, pneumonia, and even death after infection. The application of a safe and effective vaccine is not yet available. The effects of the vaccine on the immune function of the mice were analyzed and evaluated by comparing the truncated RSV G 130-230 amino acids (including the CX3C mold sequence) with the GCTL (replacing the CX3C with the CTL epitope) or by immunizing the Balb/ c mice with a mucosal adjuvant. Gcx3c protein and GCTL protein in Escherichia coli D. expressed protein, and the affinity of His-Tag and Co2 + is used for the purification of the protein. The purified Gcx3c protein and the GCTL protein were mixed with the non-toxic E. coli heat-labile enterotoxin (LT) to immunize the Balb/ c mice to set up the Gcx3c group, the GCTL group, the GCTL group, the GCTL + LT group, the LT group, the PBS group, the total 6 groups, respectively, at 0,1 and 4 weeks. The immune indexes such as IgG, IgA, IgG2a/ IgG1, sIgA, histamine, eosinophils and lymphocytes in the serum were measured by ELISA, HE staining and MTT staining. Determination. Mice immunized with 107 pfu/ ml RSV nasal attack, continuous 3 d. The mice were sacrificed after the challenge, the eosinophils were counted, the histamine level was detected, and the tissue was prepared The results are separated using the SPSS16.0 software The Gcx3c protein and the GCTL protein were expressed in the form of inclusion bodies in E. coli, and the inclusion bodies induced at 37 鈩，
本文编号：2501057
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2501057.html
最近更新
教材专著
