基于蛋白转导域的人禽流感复制子疫苗的构建和诱导细胞免疫应答研究
发布时间：2019-06-14 22:43
【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(p0.01)。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。
[Abstract]:Purpose In this paper, the immunogenicity and representativeness of the HA gene antigen of A/ Anhua/1/2005 strain are compared and analyzed by means of bioinformatics, and the whole replicon has the characteristics of self-replication and high-efficiency expression of the foreign antigen and the transduction function of the protein transduction domain VP22 protein. The invention has the advantages that the human avian influenza replicon vaccine is constructed and packaged with the aim of enhancing the immune response induced by the avian influenza replicon vaccine, in particular the specific CTL reaction mediated by the antigen-specific CD8 + T lymphocyte, and laying an experiment for the development of a high-efficiency, safe and general-purpose avian influenza vaccine clinical application. The foundation. Materials and methods First, the HA gene of all human isolates of influenza A virus (H5N1 subtype) was retrieved from the influenza virus database in 2008. Sequence and amino acid sequence. A/ Anhua/1/2005 and others are analyzed using the phylogenetic tree The genetic relationship among the strains was determined by the DNA STAR, DNMAN software and the related on-line analysis protein. Next, the full length coding sequence of the HA gene and the VP22 and the EGFP gene of the human isolated strain of the avian influenza virus (H5N1) is amplified, and the fusion gene (VP22/ H) of the VP22 and the HA is amplified by using a shear overlap extension PCR (SOE-PCR) technology. A) constructing pSFV-HA, pSFV-VP22, pSFV-EGFP and pSF by respectively constructing the HA, VP22, EGFP gene and VP22/ HA fusion gene to the downstream of the pSFV vector 26S sub-genome; V-VP22/ HA plasmid. The constructed plasmid was transcribed in vitro and packaged into VRP-HA, VRP-VP22, VRP-EGFP, VRP in BHK-21 cells. -VP22/ HA replicon. After each replicon of the package was infected with BHK-21 cells, EGFP expression was observed under a fluorescence microscope, and HA, VP22, and VP were detected by RT-PCR and indirect immunofluorescence staining (IFA). 22/ HA protein expression, while Annexin V/ PI double The apoptosis of the cells was detected by the staining method. The VRP-HA and the VRP-VP22/ HA replicon vaccine and the corresponding VRP-VP22 replicon, the VRP-EGFP replicon and the normal saline control were used to immunize 6-8-week-old BALB/ c female mice. After the first and third weeks of the injection, the immunization was enhanced, and after 2 weeks of booster immunization, the patient was under the anesthesia of ether. The expression of IL-4 and CD8 ~ + T cells in CD4 ~ + T cells was detected by flow cytometry. The data were analyzed by SPSS 13.0, and the mean number (M) and standard deviation (SD) and the percentage of the cells expressing the cytokines were analyzed by means of the approximate normal method and one-factor analysis of variance (ANOVA). ), The results showed that the HA gene of A/ Anhui/1/2005 candidate strain was compared with that of China and China by using the SNK method. The relationship between the human avian influenza virus isolates in Indonesia and other countries is most recent, and the candidate strain is similar to that of Thailand and Vietnam The relationship between the isolates of avian influenza virus was relatively close to each other. The relative composition of each subline found that the number of co-system isolates in the A/ Anhua/1/2005 candidate strain accounted for 70.0% of the total isolates (162/232, when the genetic distance was 0.0245), when the human avian influenza in countries such as Thailand and Vietnam were included When the isolated strain of the virus was isolated (the genetic distance was 0.0175), the number of homologous isolates in the subseries was the total. 91.8% (213/232) of the number of isolates (213/232). The homology of the nucleotide and amino acids of the HA gene of the human isolated strain of the 14 strains of the H5N1 avian influenza virus in China was analyzed, and the homology of the nucleotide and the amino acid of the HA gene was found to be 94. The vaccine candidate A/ Anhua/1/2005 (ABD28180) has 26 antigen epitopes, one of the most virulent strains of the domestic isolates and the majority of the epitope. And sequencing the replicon vector of the constructed expression target gene to display the sequence of HA, VP22, EGFP gene and VP22/ HA fusion gene in the pSFV replicon vector and the theoretical sequence. The transcription products of pSFV-HA, pSFV-VP22, pSFV-EGFP, pSFV-VP22-HA and pSFV helper vector were found to be about 9500 bp,8700 bp,8500 bp,1040, respectively. The results of direct fluorescence microscopy, RT-PCR and indirect immunofluorescence showed that the infected cells of each replicon express their corresponding target genes; moreover, the results of indirect immunofluorescence test showed that the BHK-21 cell ratio of VRP-VP22/ HA was higher than that of V. RP-HA replicon sense The stained BHK-21 cells produced a stronger, more intense fluorescence intensity. The apoptosis test found that the BHs of the replicon infections After 6-8-week-old BALB/ c female mice with two doses of VRP-HA and VRP-VP22/ HA, both VRP-HA and VRP-VP22/ HA replicon vaccines were immunized with two doses of 10-5TU and 10-6TU for 6-8-week-old BALB/ c female mice, and the high-level expression of IL-4, CD8 + T cells and high water was found in the spleen CD4 ~ + T cells of mice immunized with VRP-HA and VRP-VP22/ HA replicon vaccine. The levels of IL-6-VRP-HA,10-5-and 10-6-VRP-VP22/ HA were significantly higher than that of VRP-VP22, VR in the control group (VRP-VP22, VRP-EGFP and NS). P-EGFP and NS control group, the difference was significant (p0.01), while the VRP-HA vaccine of 10-5TU was free of use. There was no significant difference in the expression of IL-4 and IFN-2 in the epidemic group, and the 10-6-VRP-VP22/ HA replicon vaccine immunized group was higher than 10-5-VRP-VP22/ HA replicon. VACCINE Group, the latter was higher than that of the 10-6-VRP-HA replicon vaccine group. Conclusion The HA gene of A/ Anhua/1/2005 strain was constructed. The VRP-HA, pSFV-VP22, pSFV-EGFP and pSFV-VP22-HA plasmids were constructed correctly and the VRP-HA, VRP-VP22, V were successfully packaged. Both the RP-EGFP and the VRP-VP22/ HA replicon, each of which can induce apoptosis. The VRP-HA and the VRP-VP22/ HA replicon vaccines are capable of inducing IL-4, IFN-derived cytokine expression, and presenting an agent
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
本文编号：2499755
[Abstract]:Purpose In this paper, the immunogenicity and representativeness of the HA gene antigen of A/ Anhua/1/2005 strain are compared and analyzed by means of bioinformatics, and the whole replicon has the characteristics of self-replication and high-efficiency expression of the foreign antigen and the transduction function of the protein transduction domain VP22 protein. The invention has the advantages that the human avian influenza replicon vaccine is constructed and packaged with the aim of enhancing the immune response induced by the avian influenza replicon vaccine, in particular the specific CTL reaction mediated by the antigen-specific CD8 + T lymphocyte, and laying an experiment for the development of a high-efficiency, safe and general-purpose avian influenza vaccine clinical application. The foundation. Materials and methods First, the HA gene of all human isolates of influenza A virus (H5N1 subtype) was retrieved from the influenza virus database in 2008. Sequence and amino acid sequence. A/ Anhua/1/2005 and others are analyzed using the phylogenetic tree The genetic relationship among the strains was determined by the DNA STAR, DNMAN software and the related on-line analysis protein. Next, the full length coding sequence of the HA gene and the VP22 and the EGFP gene of the human isolated strain of the avian influenza virus (H5N1) is amplified, and the fusion gene (VP22/ H) of the VP22 and the HA is amplified by using a shear overlap extension PCR (SOE-PCR) technology. A) constructing pSFV-HA, pSFV-VP22, pSFV-EGFP and pSF by respectively constructing the HA, VP22, EGFP gene and VP22/ HA fusion gene to the downstream of the pSFV vector 26S sub-genome; V-VP22/ HA plasmid. The constructed plasmid was transcribed in vitro and packaged into VRP-HA, VRP-VP22, VRP-EGFP, VRP in BHK-21 cells. -VP22/ HA replicon. After each replicon of the package was infected with BHK-21 cells, EGFP expression was observed under a fluorescence microscope, and HA, VP22, and VP were detected by RT-PCR and indirect immunofluorescence staining (IFA). 22/ HA protein expression, while Annexin V/ PI double The apoptosis of the cells was detected by the staining method. The VRP-HA and the VRP-VP22/ HA replicon vaccine and the corresponding VRP-VP22 replicon, the VRP-EGFP replicon and the normal saline control were used to immunize 6-8-week-old BALB/ c female mice. After the first and third weeks of the injection, the immunization was enhanced, and after 2 weeks of booster immunization, the patient was under the anesthesia of ether. The expression of IL-4 and CD8 ~ + T cells in CD4 ~ + T cells was detected by flow cytometry. The data were analyzed by SPSS 13.0, and the mean number (M) and standard deviation (SD) and the percentage of the cells expressing the cytokines were analyzed by means of the approximate normal method and one-factor analysis of variance (ANOVA). ), The results showed that the HA gene of A/ Anhui/1/2005 candidate strain was compared with that of China and China by using the SNK method. The relationship between the human avian influenza virus isolates in Indonesia and other countries is most recent, and the candidate strain is similar to that of Thailand and Vietnam The relationship between the isolates of avian influenza virus was relatively close to each other. The relative composition of each subline found that the number of co-system isolates in the A/ Anhua/1/2005 candidate strain accounted for 70.0% of the total isolates (162/232, when the genetic distance was 0.0245), when the human avian influenza in countries such as Thailand and Vietnam were included When the isolated strain of the virus was isolated (the genetic distance was 0.0175), the number of homologous isolates in the subseries was the total. 91.8% (213/232) of the number of isolates (213/232). The homology of the nucleotide and amino acids of the HA gene of the human isolated strain of the 14 strains of the H5N1 avian influenza virus in China was analyzed, and the homology of the nucleotide and the amino acid of the HA gene was found to be 94. The vaccine candidate A/ Anhua/1/2005 (ABD28180) has 26 antigen epitopes, one of the most virulent strains of the domestic isolates and the majority of the epitope. And sequencing the replicon vector of the constructed expression target gene to display the sequence of HA, VP22, EGFP gene and VP22/ HA fusion gene in the pSFV replicon vector and the theoretical sequence. The transcription products of pSFV-HA, pSFV-VP22, pSFV-EGFP, pSFV-VP22-HA and pSFV helper vector were found to be about 9500 bp,8700 bp,8500 bp,1040, respectively. The results of direct fluorescence microscopy, RT-PCR and indirect immunofluorescence showed that the infected cells of each replicon express their corresponding target genes; moreover, the results of indirect immunofluorescence test showed that the BHK-21 cell ratio of VRP-VP22/ HA was higher than that of V. RP-HA replicon sense The stained BHK-21 cells produced a stronger, more intense fluorescence intensity. The apoptosis test found that the BHs of the replicon infections After 6-8-week-old BALB/ c female mice with two doses of VRP-HA and VRP-VP22/ HA, both VRP-HA and VRP-VP22/ HA replicon vaccines were immunized with two doses of 10-5TU and 10-6TU for 6-8-week-old BALB/ c female mice, and the high-level expression of IL-4, CD8 + T cells and high water was found in the spleen CD4 ~ + T cells of mice immunized with VRP-HA and VRP-VP22/ HA replicon vaccine. The levels of IL-6-VRP-HA,10-5-and 10-6-VRP-VP22/ HA were significantly higher than that of VRP-VP22, VR in the control group (VRP-VP22, VRP-EGFP and NS). P-EGFP and NS control group, the difference was significant (p0.01), while the VRP-HA vaccine of 10-5TU was free of use. There was no significant difference in the expression of IL-4 and IFN-2 in the epidemic group, and the 10-6-VRP-VP22/ HA replicon vaccine immunized group was higher than 10-5-VRP-VP22/ HA replicon. VACCINE Group, the latter was higher than that of the 10-6-VRP-HA replicon vaccine group. Conclusion The HA gene of A/ Anhua/1/2005 strain was constructed. The VRP-HA, pSFV-VP22, pSFV-EGFP and pSFV-VP22-HA plasmids were constructed correctly and the VRP-HA, VRP-VP22, V were successfully packaged. Both the RP-EGFP and the VRP-VP22/ HA replicon, each of which can induce apoptosis. The VRP-HA and the VRP-VP22/ HA replicon vaccines are capable of inducing IL-4, IFN-derived cytokine expression, and presenting an agent
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392
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