【作者】 杨仕贵；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 目的本课题通过生物信息学手段比较分析A/Anhui/1/2005毒株HA基因抗原的免疫原性和代表性,并整合复制子具有自我复制和高效表达外源抗原特点以及蛋白转导域VP22蛋白的转导功能的优势,构建和包装人禽流感复制子疫苗,以期增强禽流感复制子疫苗诱导的免疫应答,尤其是抗原特异性CD8~+ T淋巴细胞介导的特异性CTL反应,为研制高效、安全及通用性禽流感人疫苗临床应用奠定实验基础。材料与方法首先,检索1997 2008年流感病毒数据库(influenza virus resources)收录全部甲型流感病毒(H5N1亚型)人源分离株的HA基因序列和氨基酸序列。采用系统进化树分析A/Anhui/1/2005与其它各毒株之间亲缘关系。采用DNASTAR、DNAMAN软件及相关在线分析蛋白抗原指数和抗原表位。其次,扩增禽流感病毒(H5N1)人源分离株的HA基因及VP22和EGFP基因的全长编码序列,同时采用剪切重叠延伸PCR(SOE-PCR,splicing by overlapextension-PCR)技术扩增VP22和HA的融合基因(VP22/HA),将HA、VP22、EGFP基因及VP22/HA融合基因分别构建到pSFV载体26S亚基因组下游,构建pSFV-HA、pSFV-VP22、pSFV-EGFP和pSFV-VP22/HA质粒。将构建的质粒进行体外转录,并在BHK-21细胞中包装成VRP-HA、VRP-VP22、VRP-EGFP、VRP-VP22/HA复制子。将包装的各复制子感染BHK-21细胞后,在荧光显微镜下观察EGFP表达情况,采用RT-PCR和间接免疫荧光染色法(IFA)检测HA、VP22及VP22/HA蛋白表达情况。同时,采用Annexin V/PI双染色法检测细胞凋亡情况。最后,分别用不同剂量的VRP-HA及VRP-VP22/HA复制子疫苗及相应的VRP-VP22复制子、VRP-EGFP复制子和生理盐水对照免疫6-8周龄BALB/c雌性小鼠,经右侧大腿肌肉注射初次免疫和3周后加强免疫,加强免疫2周后在乙醚麻醉下处死小鼠分离脾脏,分离淋巴细胞。采用流式细胞仪检测CD4~+T细胞表达IL-4和CD8~+ T细胞表达IFN-γ等细胞内细胞因子表达情况。采用SPSS 13.0统计软件对数据进行统计分析,数据以均数(M)±标准差(SD),表达细胞因子的细胞百分比分析采用近似正态法单因素方差分析(ANOVA),多组两两比较采用SNK法。结果对1997-2008年的232株参考序列进行系统发生树分析发现A/Anhui/1/2005候选株的HA基因与国内及印尼等国的人禽流感病毒分离株亲缘关系最近;该候选株与泰国和越南等国的人禽流感病毒分离株的亲缘关系也相对较近。对各亚系相对构成分析发现,A/Anhui/1/2005候选株所属亚系中同系分离株数量占总分离株数量的70.0%(162/232,当遗传距离为0.0245时),当包含泰国和越南等国的人禽流感病毒分离株时(遗传距离为0.0175),其所属亚系中同源分离株数量占总分离株数量的91.8%(213/232)。对国内14株H5N1禽流感病毒人源分离株HA基因的核苷酸和氨基酸的同源性分析,发现其核苷酸和氨基酸的同源性分别在94.7%-100%和95.3%-100%之间。抗原表位预测表明,疫苗候选株A/Anhui/1/2005(ABD28180)有26个抗原表位,为国内分离株抗原表位最多毒株之一,抗原表位多数都处在亲水区段,且较少处在糖基化位点区段。将构建的表达目的基因的复制子载体进行测序,显示pSFV复制子载体中HA、VP22、EGFP基因及VP22/HA融合基因序列与理论序列完全一致,并位于pSFV载体的亚基因启动子的下游。经琼脂糖变性胶电泳发现pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA和pSFV helper载体的转录产物分别约为9500 bp,8700 bp,8500 bp,10400 bp and 7000左右,并且没有明显拖尾现象。经直接荧光显微镜、RT-PCR和间接免疫荧光检测发现,各复制子感染的细胞均表达其相应目的基因;而且,经间接免疫荧光检测结果发现,VRP-VP22/HA感染的BHK-21细胞比VRP-HA复制子感染的BHK-21细胞产生更强、更密集的荧光强度。凋亡检测发现,各复制子感染的BHK-21细胞均出现明显凋亡现象,尤其是早期凋亡。VRP-HA和VRP-VP22/HA两种复制子疫苗经10~5TU和10~6TU两种剂量的免疫6-8周龄BALB/c雌性小鼠后,发现VRP-HA和VRP-VP22/HA复制子疫苗免疫的小鼠脾脏CD4~+T细胞高水平表达IL-4细胞因子,CD8~+T细胞高水平表达IFN-γ细胞因子,而对照组(VRP-VP22,VRP-EGFP和NS)中两种细胞因子呈现低水平表达;其中,10~6-VRP-HA,10~5-和10~6-VRP-VP22/HA免疫组是显著高于VRP-VP22,VRP-EGFP和NS对照组,差异有显著性(p<0.01)。而10~5TU的VRP-HA疫苗免疫组诱导表达IL-4和IFN-γ与对照组差异无显著性。而且,10~6-VRP-VP22/HA复制子疫苗免疫组高于10~5-VRP-VP22/HA复制子疫苗组,后者又高于10~6-VRP-HA复制子疫苗组。结论采用A/Anhui/1/2005毒株的HA基因构建疫苗具有普遍的代表性,抗原性预测表明该毒株HA基因具有较高抗原性。pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA质粒均已正确构建,并成功包装了VRP-HA、VRP-VP22、VRP-EGFP和VRP-VP22/HA复制子,各复制子均可诱导细胞凋亡。VRP-HA和VRP-VP22/HA复制子疫苗均能诱导IL-4、IFN-γ细胞因子表达,并呈现一种剂量效应关系;VP22基因与HA基因融合的复制子疫苗优于单HA基因的复制子疫苗。更多还原

【Abstract】 By analysing the antigenic peptides and homogeneity of the HA sequences（human isolates of the influenza A virus H5N1 subtype） and exploring a novel alphavirus replicon system of VP22 fused with HA,this study assesses whether the immunogenicity of an HA-based replicon vaccine could be induced and augmented by fusing with VP22.Materials and methodsThe first step in developing an efficient gene vaccine against human-avian H5N1 influenza is to select a representative and highly immunogenic HA gene.A total of 232 reference sequences of the H5N1 human isolates during 1997-2008 retrieved from the influenza database（influenza virus resources） were used in this study.Phylogenetic trees and antigenicity analysis were constructed using neighbour-joining programs included in DNASTAR and DNAMAN software.The HA,VP22 and EGFP genes were amplified and cloned into the unique Xho I and Not I restriction sites of plasmid pSFV,resulting in the plasmid pSFV-HA、pSFV-VP22、pSFV-EGFP.To construct pSFV-VP22-HA,the VP22-HA fusion gene inserting five glycine-coding sequences at the fusion site was firstly produced,by SOE-PCR（i.e.,splicing by overlap extension PCR）.This fusion gene was then subcloned into the unique BamHI and NotI restriction sites of the pSFV vector.The pSFV-HA,pSFV-VP22,pSFV-EGFP and pSFV-VP22-HA vectors were linearised and transcribed into the RNA transcripts in vitro.Then,VRP-HA,VRP-VP22,VRP-EGFP and VRP-VP22/HA were assembled in BHK-21 cells by using In vitro-transcribed RNAs of pSFV helper vectors and that of pSFV-HA,pSFV-VP22,pSFV-EGFP and pSFV-VP22-HA,respectively.EGFP expression was observed under a fluorescence microscope after the replicons infecting the BHK-21 cells,while RT-PCR and immunofluorescence staining were performed to detect HA,VP22 and VP22-HA expressions.The percentages of apoptotic and necrotic BHK-21 cells,which were infected by VRP-HA,VRP-VP22,VRP-EGFP and VRP-VP22/HA,were analysed by using a flow cytometry with annexin V apoptosis detection kitsSix- to eight-week-old BALB/c mice were immunised intramuscularly（i.m.; 200μl） at zero weeks and boosted at three weeks with VRP-HA and VRP-VP22/HA, respectively,and VRP-VP22,VRP-EGFP or normal saline（NS） was also used as controls.Lymphocytes were separated from the spleens of mice and detected for specific cell-mediated responses.IL-4 expression of CD4⁺ T cells and IFN-γexpression of CD8⁺ T cells were assayed by means of intracellular cytokine staining（ICCS） with flow cytometry.The percentage of cells expressing cytokines was analysed by using the ANOVA test with SPSS software,and all data are presented as mean±SD values.ResultsA phylogenetic analysis of the 232 reference sequences of the H5N1 human isolates during 1997-2008 revealed that the genetic relationships among the A/Anhui/1/2005 isolates were similar to most of the 1997-2008 H5N1 human isolates (i.e.,70%at the genetic distance of 7×10^-6 and 90%at the genetic distance of 7×10^-5). An antigenicity analysis revealed that the four isolates（A/Anhui/1/2005, A/Beijing/01/2003,A/China/2006 and A/human/China/GD02/2006） had 26 antigenic peptides,and A/Anhui/1/2005 isolates had frequent antigenic peptides.The pSFV-HA,pSFV-VP22,pSFV-EGFP and pSFV-VP22-HA vectors were confirmed by sequencing.The integrity and quantity of RNA transcripts of the pSFV-HA,pSFV-VP22,pSFV-EGFP,pSFV-VP22-HA and pSFV helper vectors were determined via denaturing gel electrophoresis.Observation under a fluorescence microscope revealed EGFP expression in the infected BHK-21 cells,especially in the nuclei.HA,VP22 and VP22-HA expression in the infected BHK-21 cells was confirmed by RT-PCR and immunofluorescence staining.Furthermore,all the BHK-21 cells infected with any of the replicons showed high levels of apoptosis,but at significantly higher-than-normal levels-especially those of early apoptosis.Four groups of mice immunised with VRP-HA（10⁵ TU and 10⁶ TU） and VRP-VP22/HA（10⁵ TU and 10⁶ TU） presented high percentages of cells expressing IL-4 and IFN-γ,while the three control groups that received 10⁵ TU of VRP-VP22, VRP-EGFP or NS presented low percentages of expression.The ANOVA-SNK analysis revealed that the IL-4 expressions in three groups（10⁶ TU VRP-HA,and 10⁵ TU and 10⁶ TU VRP-VP22/HA） were significantly higher than those of the control groups（VRP-VP22,VRP-EGFP and NS）（p < 0.01）.A dose titration effect for IL-4 expression,as well as for IFN-γexpression,was observed both in the VRP-HA-vaccinated mice and in the VRP-VP22/HA-vaccinated mice.ConclusionsOur results revealed that the HA gene of A/Anhui/1/2005 isolates presented with comparatively representative and high immunogenicity.VRP-HA、VRP-VP22、VRP-EGFP and VRP-VP22/HA expressing the target proteins had be successfully assembled.And,VRP-HA、VRP-VP22、VRP-EGFP and VRP-VP22/HA could induce infected cells apoptosis.Our results revealed that both VRP-VP22/HA and VRP-HA replicon particles presented a promising approach for developing vaccines against human-avian influenza, and VP22 could increase the immunogenicity of the HA antigens to which it is fused.更多还原